# How can a ligand be an integral membrane protein?

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My background is in mathematics, and not biology, so please bear with me. I am currently working on a project involving the effects of Epidermal growth factor treatment (EGF) on cell migration. I am reading a review of EGF signaling (Epidermal growth factor receptor targeting in cancer: A review of trends and strategies by Chetan Yewale, et. al.), and it states that "Various ligands can activate EGFR… These ligands are expressed as integral membrane proteins." This statement makes absolutely no sense to me, and makes me question my understanding of signal transduction. I think of ligands as freely floating molecules that may eventually come into contact with the cell membrane and attach to some receptor. But a ligand expressed as an integral membrane protein? This seems contradictory to my understanding of ligands, which (I thought) are released from the cell in order to signal with cells (be it the same, neighbor, or distant cells). Integral membrane protein ligands would only be useful for autocrine signaling, which I don't think is true of EGF.

In biology ligand is a very broad term. Everything is called a ligand that has a receptor for it, regardless whether it is free or membrane-bound. There is very much sense in membrane bound ligands, because many cells in our body are capable of actively moving around (for example T-cells). Cells can use signal transduction by direct cell-to-cell contact - like in activation of T-cells, or cytotoxic T-cell killing. This wiki page covers the basics quite well. Also, a quite thorough wiki page on ligands.

From the comments under the question by @WYSIWYG:

These kind of ligands act via juxtacrine signalling because they cannot diffuse. Ephrin is another example.

A perfectly reasonable definition of a ligand from Wikipedia:

In biochemistry and pharmacology, a ligand is a substance that forms a complex with a biomolecule to serve a biological purpose.

A ligand can be anything, so long as it binds to a biomolecule. Often, the ligand is a small molecule or peptide, and the thing that it binds to is a protein. On the other hand, both ligand and binding partner can certainly be proteins (or one could be a Mg$$^{2+}$$ ion and the other an RNA molecule, etc.). Ligand is a very broad term, and is often used in other biochemical areas aside from signaling. For example, the things that an enzyme binds to (substrates, allosteric regulators, etc) can be called its ligands.

Also, the things you describe in your post:

This seems contradictory to my understanding of ligands, which (I thought) are released from the cell in order to signal with cells (be it the same, neighbor, or distant cells).

are hormones, which is a much more specific category of thing than is ligand.

# Edit

There seem to be some 'ahem'… doubters as to the broadness of the definition of ligand, so, time for some examples from the literature:

From the biochemistry textbook Berg, 7e (emphasis added):

The final step is affinity chromatography with the use of a ligand specific for the target enzyme.

From the paper Electrostatic steering and ionic tethering in enzyme-ligand binding: Insights from simulations, Wade et al (emphasis added):

To bind at an enzyme's active site, a ligand must diffuse or be transported to the enzyme's surface, and, if the binding site is buried, the ligand must diffuse through the protein to reach it.

From the paper Geometries of functional group interactions in enzyme-ligand complexes: Guides for receptor modelling, Tintelnot et al (emphasis added):

There are many more examples of arginine-carboxyl interactions that appear to have key functional roles in enzymes, including some in which an arginine-like structure in the ligand interacts with a carboxyl group from the binding site (i.e., the reverse of that described above).

It is true that the one particular compound that natively binds to an enzyme's active site is often called a substrate, in part to differentiate it from allosteric regulators and other ligands that can also bind to the enzyme. Thus, the set of all of an enzyme's substrates can be thought of as a subset of the enzyme's ligands. Again, ligand is an extremely broad concept that simply refers to something which binds.

# Edit 2

Fun fact! The modern word ligand comes from the latin verb ligāre, meaning "to bind". More specifically, ligand derives from the gerundive form of ligāre, ligandus, which translates to something like "(that which is) to be bound". Not that this has much bearing on how it's used in the modern scientific literature. I just thought it was cool.

Most ligands are synthesized as the same way as receptors integrated in membrane: synthesized on rough-ER, pulled into the lumen of ER. If proteins synthesized on rough-ER have a hydrophobic amino acid sequence, the hydrophobic region could stay in membrane because inside of membrane is hydrophobic and has affinity to hydrophobic amino acid sequences. Soluble ligands do not have such a region in the sequences, but membrane integrated ligands have it.

As I mentioned above there are ligands integrated on membrane: Fas, TNF, Notch ligand family, etc. As you mention in the question, they are not soluble so that ligand-receptor signaling occurs by cell to cell contact.

You can see various types of membrane integrated ligands from the URLs below.

http://www.nature.com/onc/journal/v27/n38/full/onc2008229a.html http://www.sanfordburnham.org/talent/Pages/CarlWare.aspx http://www.sci-online.org/article/view/3946/4855 http://www.bio.davidson.edu/courses/immunology/students/spring2003/swails/fas.html

## Structural biology of endogenous membrane protein assemblies in native nanodiscs

Asymmetric biological lipid bilayers can be solubilized intact using styrene-co-maleic anhydride copolymers.

Styrene and stilbene monomer subunits insert into membranes to stabilize nanodiscs.

Maleic anhydride-derived subunits engender nanodisc solubility and functionality.

Structures of in vivo lipid:protein:ligand assemblies are resolvable in native nanodiscs.

Recent progress has led to the concept of memteins as key units of membrane function.

## Cell Biology 09: Signal Transduction

Signaling is how cells and organisms get inputs from their environment, and how cells in multicellular organisms communicate with each other. In terms of differentiation, signaling in embryonic development allows establishment separate developmental lineages such as endoderm vs. mesoderm vs. ectoderm and in adult organisms for proper differentiation of stem cells. In terms of environmental response, signaling includes everything from migration of cells in response to growth factors up to fight or flight responses to environmental threats to the organism.

Signaling molecules are synthesized in signaling cells and then released to affect other receiving cells by binding to a target receptor. In some cases that receptor affects a second messenger inside the recipient cell.

Signal transduction involves the following steps:

• Synthesis of signaling molecule by signaling cell (e.g. hormones by pituitary gland)
• Release of signaling molecule (e.g. into blood or extracellular matrix)
• Transport to receiving cell (e.g. in blood)
• Binding to receptor
• Initiation of intracellular signal transduction
• Resultant changes to cellular functions functions (e.g. activating enzymes would be a fast response, changing gene expression would be a slower response)
• Feedback regulation: removal of signaling molecule or disabling of receptor (e.g. via endocytosis)

Most signaling molecules are too large and/or hydrophobic to get through the cell membrane, hence the need for protein receptors on the receiving cell’s membrane. Receptors are usually integral membrane proteins and the binding site is usually located in the strictly extracellular portion but is sometimes in the membrane spanning domain. Binding of the signaling molecules (aka first messengers) to these cell surface receptors leads to an increase or decrease in concentration of intracellular signaling molecules (second messengers) which bind other proteins to modify their activity.

Rapid responses to environmental signals usually go through the nervous system and travel via hormones (insulin, epinephrine, dopamine for instance) synthesized in places such as the pancreas, pituitary glands, hypothalamus or other neurons. Signaling molecules are synthesized in the cytosol, trafficked through the secretory pathway and then held inside the cell until a signal indicates to exocytose them. Such signals usually lead to ‘short term responses’ unless of course the cell is exposed to the signal for an extended period of time.

Here are the different types of signaling:

: molecules synthesized and released by signaling cells travel through blood and act at a distance (e.g. hormones) : when cells respond to substances that they release themselves (e.g. interleukin-1) : cells respond to substances released by nearby cells (e.g. neurotransmitters, growth factors). These sometimes form a concentration gradient resulting in a gradient of the degree of cellular response. : intracellular signaling, not covered in this lecture

Sometimes membrane-bound signals on one cell bind to receptors on adjacent cells to trigger differentiation. Here the membrane proteins themselves are the ligands. Sometimes the membrane proteins are cleaved and become solubilized and then may even act at a distance.

experimental techniques

One experimental technique for finding a protein that acts as a receptor for a particular signaling molecule is affinity chromatography:

• Conjugate the ligand (signaling molecule) to a bead
• Expose beads to cellular extract
• Wash away anything that didn’t bind to a bead
• Now add a lot more signaling molecule. The receptor’s affinity for the ligand is not absolute, they associate and dissociate frequently, so some receptor will dissociate from the bead-bound ligands and associate with the free ligand.
• Collect and purify the ligand/receptor complexes
• Determine identity of receptor peptide (e.g. through mass spec?)
• Identify the gene coding for that peptide
• Express that gene on a cell type that you know does not normally express it, and test if that causes that cell type to bind the ligand.

Similarly, molecular cloning can be used. Simply take a cell type that doesn’t bind the ligand and screen a library of cDNAs to find those which cause the cell to bind the ligand.

Alternately, if you know the impact of cell A’s signal on cell B, you can use mutagenesis screens. For instance if you know that cell A causes cell B to become an R7 neuron instead of a cone cell, you can screen for mutants that become a cone cell. Those mutants might have the receptor for that particular differentiation signal disabled. But other mutations in the relevant pathway can create false positives, so this is a tricky technique.

One way to test the function of G protein coupled receptors (introduced below) is via Fluorescence Resonance Energy Transfer (FRET) which lets you get a readout of the proximity of two different proteins.

G protein coupled receptors

The largest class of receptors is G protein coupled receptors (GPCRs). There are

900 of them. Activation of these receptors alters gene expression and can lead to differentiation. The mechanism is often that the ligand binding has GEF activity: it causes a conformational change in the GPCR that exchanges GDP for GTP. A separate GAP will later act as an ‘inactivator’, causing the G protein to hydrolize its GTP, leaving it bound to GDP and thus in an inactive state.

Epinephrine is a hormone that binds to a GPCR. In the liver it can cause an increase in cAMP levels leading to glycogen breakdown and glucose release in muscle it increases intracellular Ca 2+ and promotes muscle contraction.

All GPCRs are 7-pass transmembrane proteins with the N terminus extracellular and C terminus cytoplasmic, thus having 4 extracellular and 4 cytosolic domains (called E1-4, C1-4 respectively). They associate with trimeric G proteins (see below), usually via their C2, C3 and C4 domains. The exoplasmic surface consists mainly of hydrophobic amino acids. The cytosolic amino acids vary.

Broadly, there are two classes of G proteins involved in signaling:

1. Monomeric G proteins such as Ras are just a single G protein which acts as an on/off switch according to its GTP binding (promoted by GEFs) and GTP hydrolysis (promoted by GAPs). Some of these are involved in differentiation, apoptosis, etc. and so are frequently mutated in cancers.
2. Trimeric G proteins are complexes of three proteins (predictably called alpha, beta and gamma subunits) which are also bound to a GPCR for a total of four proteins. The alpha and gamma subunits are both membrane bound via lipids. The beta and gamma subunits always stay together (as ‘the G beta gamma subunit’). Without the extracellular ligand binding to the GPCR, the alpha subunit is bound to GDP and also bound in a complex with beta and gamma (the ‘off’ position). The ligand acts as a GEF, causing alpha to exchange GDP for GTP, whereupon it dissociates from beta and gamma and drifts away to bind to a different effector complex, causing downstream signaling

The cycle of activation and deactivation of trimeric G proteins and GPCRs is shown in this Wikimedia Commons graphic by repapetilto:

The most famous G protein coupled receptor pathways are the ones in which the effector protein that Gα binds to is adenylate cyclase (AC). AC is an enzyme which catalyzes the conversion of ATP to cAMP. Various Gα may either activate or inactivate AC, but either way, the upshot is that the GPCR acts via AC to use cAMP as a second messenger effecting downstream changes in the cell, often (always?) via binding to and activation of the Protein Kinases A (PKAs).

Here are some examples of GPCR pathways:

(PGE1) is a first messenger which binds a GPCR, inhibiting AC and thus reducing cAMP, which leads to vasodilation through downstream pathways. in heart muscle. There are several kinds of these, all of which activate a G protein. Some signal via cAMP, others open or close K + and other channels. released in response to low blood sugar signals for an increase in intracellular cAMP, causing PKA to signal for less glycogen production and more glycogen degradation, thus freeing up stored energy in the cell in the form of glucose 1-phosphate. (PLC) is a phosphodiesterase, which cleaves phosphatidylinositol 4,5 bisphosphate into two molecules, IP3 and DAG, which each act as second messengers in the IP3/DAG pathway. Cytosolic IP3 binds to a Ca 2+ channel in the ER membrane, releasing Ca 2+ stored in the ER out into the cytosol. Protein kinase C (PKC) is then activated by binding to both DAG and Ca 2+ , and once active it will bind and phosphorylate various substrates. Ca 2+ will also cause all kinds of other downstream changes such as activating calmodulin.

Here is a video which summarizes many of the concepts of G protein signaling:

amplification of signals

Many signals are hugely amplified in the receiving cell. For instance one epinephrine molecule can activate a few ACs, each of which produces many cAMPs, each of which binds to one PKA, each of which phoshorylates many enzymes, each of which produces many end products. This sort of cascade can amplify a signal 100- to 1000-fold.

On the other hand, cells have many feedback mechanisms for downregulating or turning off incoming signals. For instance, PKAs may phosphorylate and inactivate the receptor, the cell may endocytose the receptor following a β-arrestin signal, GAPs may cause Galpha to hydrolyze ATP and return to the G protein in the ‘off’ position, or a type of phosphodiesterase (which is regulated by phosphorylation by PKA) may break down cAMP into AMP.

relevance to PrP

One of the theories as to PrP’s native function is that it’s involved in signal transduction. An oft-cited paper is Mouillet-Richard 2000 (ft), who implicated PrP C as a receptor in a signaling cascade involving Fyn (gene: FYN) and Caveolin-1 (gene: CAV1). The possible relevance of this to prion disease is not yet totally clear, but last year some fascinating new evidence came out for how this might play a role in Alzheimer’s [Larson 2012]. You’ll recall from the PrP/Aβ post that PrP has been shown to bind Aβ oligomers [Lauren 2009] and, though it’s still extremely controversial, this binding has been proposed to be necessary for Aβ toxicity, thus implicating PrP as an indispensible intermediary in Alzheimer’s pathogenesis. Meanwhile, there’s also been evidence (which Larson reviews) that Aβ activates Fyn, and that this activation is required for some aspects of Alzheimer’s pathology. Fyn is a Src tyrosine kinase, meaning that when active it phosphorylates Y residues on other proteins.

Larson’s contribution was to show that when Aβ binds PrP C , PrP C then complexes with Fyn, activating its kinase activity and causing it to phosphorylate Tau. If true, this would seem to position PrP at the long-missing causal link between the two big features of Alzheimer’s pathology: Aβ oligomers and hyperphosphorylated Tau tangles. This conclusion is sure to be controversial, and in recognition of the considerable controversy already surrounding PrP/Aβ connection, the authors were very explicit about exactly what proteins they used, obtained from where and purified how, and then what they measured and how. Such was the exhortation of a strangely anonymous opinion piece, “State of Aggregation” in Nature Neuroscience [No Authors Listed 2011] which stated that:

It is critical that studies examining the functional consequences of aggregated proteins clearly identify the exact source and aggregation state of the protein and critically discuss the implications of their approach.

Hopefully such openness will lead to some clear answers about PrP and Aβ in the near future. Larson’s evidence does look quite compelling, and may merit some further treatment on this blog after a second read.

Eric Vallabh Minikel is on a lifelong quest to prevent prion disease. He is a scientist based at the Broad Institute of MIT and Harvard.

## 9.1 Signaling Molecules and Cellular Receptors

There are two kinds of communication in the world of living cells. Communication between cells is called intercellular signaling , and communication within a cell is called intracellular signaling . An easy way to remember the distinction is by understanding the Latin origin of the prefixes: inter- means "between" (for example, intersecting lines are those that cross each other) and intra- means "inside" (like intravenous).

Chemical signals are released by signaling cells in the form of small, usually volatile or soluble molecules called ligands. A ligand is a molecule that binds another specific molecule, in some cases, delivering a signal in the process. Ligands can thus be thought of as signaling molecules. Ligands interact with proteins in target cells , which are cells that are affected by chemical signals these proteins are also called receptors . Ligands and receptors exist in several varieties however, a specific ligand will have a specific receptor that typically binds only that ligand.

### Forms of Signaling

There are four categories of chemical signaling found in multicellular organisms: paracrine signaling, endocrine signaling, autocrine signaling, and direct signaling across gap junctions (Figure 9.2). The main difference between the different categories of signaling is the distance that the signal travels through the organism to reach the target cell. Not all cells are affected by the same signals.

#### Paracrine Signaling

Signals that act locally between cells that are close together are called paracrine signals . Paracrine signals move by diffusion through the extracellular matrix. These types of signals usually elicit quick responses that last only a short amount of time. In order to keep the response localized, paracrine ligand molecules are normally quickly degraded by enzymes or removed by neighboring cells. Removing the signals will reestablish the concentration gradient for the signal, allowing them to quickly diffuse through the intracellular space if released again.

One example of paracrine signaling is the transfer of signals across synapses between nerve cells. A nerve cell consists of a cell body, several short, branched extensions called dendrites that receive stimuli, and a long extension called an axon, which transmits signals to other nerve cells or muscle cells. The junction between nerve cells where signal transmission occurs is called a synapse. A synaptic signal is a chemical signal that travels between nerve cells. Signals within the nerve cells are propagated by fast-moving electrical impulses. When these impulses reach the end of the axon, the signal continues on to a dendrite of the next cell by the release of chemical ligands called neurotransmitters by the presynaptic cell (the cell emitting the signal). The neurotransmitters are transported across the very small distances between nerve cells, which are called chemical synapses (Figure 9.3). The small distance between nerve cells allows the signal to travel quickly this enables an immediate response, such as, Take your hand off the stove!

When the neurotransmitter binds the receptor on the surface of the postsynaptic cell, the electrochemical potential of the target cell changes, and the next electrical impulse is launched. The neurotransmitters that are released into the chemical synapse are degraded quickly or get reabsorbed by the presynaptic cell so that the recipient nerve cell can recover quickly and be prepared to respond rapidly to the next synaptic signal.

#### Endocrine Signaling

Signals from distant cells are called endocrine signals , and they originate from endocrine cells . (In the body, many endocrine cells are located in endocrine glands, such as the thyroid gland, the hypothalamus, and the pituitary gland.) These types of signals usually produce a slower response but have a longer-lasting effect. The ligands released in endocrine signaling are called hormones, signaling molecules that are produced in one part of the body but affect other body regions some distance away.

Hormones travel the large distances between endocrine cells and their target cells via the bloodstream, which is a relatively slow way to move throughout the body. Because of their form of transport, hormones get diluted and are present in low concentrations when they act on their target cells. This is different from paracrine signaling, in which local concentrations of ligands can be very high.

#### Autocrine Signaling

Autocrine signals are produced by signaling cells that can also bind to the ligand that is released. This means the signaling cell and the target cell can be the same or a similar cell (the prefix auto- means self, a reminder that the signaling cell sends a signal to itself). This type of signaling often occurs during the early development of an organism to ensure that cells develop into the correct tissues and take on the proper function. Autocrine signaling also regulates pain sensation and inflammatory responses. Further, if a cell is infected with a virus, the cell can signal itself to undergo programmed cell death, killing the virus in the process. In some cases, neighboring cells of the same type are also influenced by the released ligand. In embryological development, this process of stimulating a group of neighboring cells may help to direct the differentiation of identical cells into the same cell type, thus ensuring the proper developmental outcome.

#### Direct Signaling Across Gap Junctions

Gap junctions in animals and plasmodesmata in plants are connections between the plasma membranes of neighboring cells. These water-filled channels allow small signaling molecules, called intracellular mediators , to diffuse between the two cells. Small molecules, such as calcium ions (Ca 2+ ), are able to move between cells, but large molecules like proteins and DNA cannot fit through the channels. The specificity of the channels ensures that the cells remain independent but can quickly and easily transmit signals. The transfer of signaling molecules communicates the current state of the cell that is directly next to the target cell this allows a group of cells to coordinate their response to a signal that only one of them may have received. In plants, plasmodesmata are ubiquitous, making the entire plant into a giant, communication network.

### Types of Receptors

Receptors are protein molecules in the target cell or on its surface that bind ligand. There are two types of receptors, internal receptors and cell-surface receptors.

#### Internal receptors

Internal receptors , also known as intracellular or cytoplasmic receptors, are found in the cytoplasm of the cell and respond to hydrophobic ligand molecules that are able to travel across the plasma membrane. Once inside the cell, many of these molecules bind to proteins that act as regulators of mRNA synthesis (transcription) to mediate gene expression. Gene expression is the cellular process of transforming the information in a cell's DNA into a sequence of amino acids, which ultimately forms a protein. When the ligand binds to the internal receptor, a conformational change is triggered that exposes a DNA-binding site on the protein. The ligand-receptor complex moves into the nucleus, then binds to specific regulatory regions of the chromosomal DNA and promotes the initiation of transcription (Figure 9.4). Transcription is the process of copying the information in a cells DNA into a special form of RNA called messenger RNA (mRNA) the cell uses information in the mRNA (which moves out into the cytoplasm and associates with ribosomes) to link specific amino acids in the correct order, producing a protein. Internal receptors can directly influence gene expression without having to pass the signal on to other receptors or messengers.

#### Cell-Surface Receptors

Cell-surface receptors , also known as transmembrane receptors, are cell surface, membrane-anchored (integral) proteins that bind to external ligand molecules. This type of receptor spans the plasma membrane and performs signal transduction, in which an extracellular signal is converted into an intercellular signal. Ligands that interact with cell-surface receptors do not have to enter the cell that they affect. Cell-surface receptors are also called cell-specific proteins or markers because they are specific to individual cell types.

Because cell-surface receptor proteins are fundamental to normal cell functioning, it should come as no surprise that a malfunction in any one of these proteins could have severe consequences. Errors in the protein structures of certain receptor molecules have been shown to play a role in hypertension (high blood pressure), asthma, heart disease, and cancer.

Each cell-surface receptor has three main components: an external ligand-binding domain, a hydrophobic membrane-spanning region, and an intracellular domain inside the cell. The ligand-binding domain is also called the extracellular domain . The size and extent of each of these domains vary widely, depending on the type of receptor.

### Evolution Connection

#### How Viruses Recognize a Host

Unlike living cells, many viruses do not have a plasma membrane or any of the structures necessary to sustain life. Some viruses are simply composed of an inert protein shell containing DNA or RNA. To reproduce, viruses must invade a living cell, which serves as a host, and then take over the hosts cellular apparatus. But how does a virus recognize its host?

Viruses often bind to cell-surface receptors on the host cell. For example, the virus that causes human influenza (flu) binds specifically to receptors on membranes of cells of the respiratory system. Chemical differences in the cell-surface receptors among hosts mean that a virus that infects a specific species (for example, humans) cannot infect another species (for example, chickens).

However, viruses have very small amounts of DNA or RNA compared to humans, and, as a result, viral reproduction can occur rapidly. Viral reproduction invariably produces errors that can lead to changes in newly produced viruses these changes mean that the viral proteins that interact with cell-surface receptors may evolve in such a way that they can bind to receptors in a new host. Such changes happen randomly and quite often in the reproductive cycle of a virus, but the changes only matter if a virus with new binding properties comes into contact with a suitable host. In the case of influenza, this situation can occur in settings where animals and people are in close contact, such as poultry and swine farms. 1 Once a virus jumps to a new host, it can spread quickly. Scientists watch newly appearing viruses (called emerging viruses) closely in the hope that such monitoring can reduce the likelihood of global viral epidemics.

Cell-surface receptors are involved in most of the signaling in multicellular organisms. There are three general categories of cell-surface receptors: ion channel-linked receptors, G-protein-linked receptors, and enzyme-linked receptors.

Ion channel-linked receptors bind a ligand and open a channel through the membrane that allows specific ions to pass through. To form a channel, this type of cell-surface receptor has an extensive membrane-spanning region. In order to interact with the phospholipid fatty acid tails that form the center of the plasma membrane, many of the amino acids in the membrane-spanning region are hydrophobic in nature. Conversely, the amino acids that line the inside of the channel are hydrophilic to allow for the passage of water or ions. When a ligand binds to the extracellular region of the channel, there is a conformational change in the proteins structure that allows ions such as sodium, calcium, magnesium, and hydrogen to pass through (Figure 9.5).

G-protein-linked receptors bind a ligand and activate a membrane protein called a G-protein. The activated G-protein then interacts with either an ion channel or an enzyme in the membrane (Figure 9.6). All G-protein-linked receptors have seven transmembrane domains, but each receptor has its own specific extracellular domain and G-protein-binding site.

Cell signaling using G-protein-linked receptors occurs as a cyclic series of events. Before the ligand binds, the inactive G-protein can bind to a newly revealed site on the receptor specific for its binding. Once the ligand binds to the receptor, the resultant shape change activates the G-protein, which releases GDP and picks up GTP. The subunits of the G-protein then split into the α subunit and the βγ subunit. One or both of these G-protein fragments may be able to activate other proteins as a result. After a while, the GTP on the active α subunit of the G-protein is hydrolyzed to GDP and the βγ subunit is deactivated. The subunits reassociate to form the inactive G-protein and the cycle begins anew.

G-protein-linked receptors have been extensively studied and much has been learned about their roles in maintaining health. Bacteria that are pathogenic to humans can release poisons that interrupt specific G-protein-linked receptor function, leading to illnesses such as pertussis, botulism, and cholera. In cholera (Figure 9.7), for example, the water-borne bacterium Vibrio cholerae produces a toxin, choleragen, that binds to cells lining the small intestine. The toxin then enters these intestinal cells, where it modifies a G-protein that controls the opening of a chloride channel and causes it to remain continuously active, resulting in large losses of fluids from the body and potentially fatal dehydration as a result.

Enzyme-linked receptors are cell-surface receptors with intracellular domains that are associated with an enzyme. In some cases, the intracellular domain of the receptor itself is an enzyme. Other enzyme-linked receptors have a small intracellular domain that interacts directly with an enzyme. The enzyme-linked receptors normally have large extracellular and intracellular domains, but the membrane-spanning region consists of a single alpha-helical region of the peptide strand. When a ligand binds to the extracellular domain, a signal is transferred through the membrane, activating the enzyme. Activation of the enzyme sets off a chain of events within the cell that eventually leads to a response. One example of this type of enzyme-linked receptor is the tyrosine kinase receptor (Figure 9.8). A kinase is an enzyme that transfers phosphate groups from ATP to another protein. The tyrosine kinase receptor transfers phosphate groups to tyrosine molecules (tyrosine residues). First, signaling molecules bind to the extracellular domain of two nearby tyrosine kinase receptors. The two neighboring receptors then bond together, or dimerize. Phosphates are then added to tyrosine residues on the intracellular domain of the receptors (phosphorylation). The phosphorylated residues can then transmit the signal to the next messenger within the cytoplasm.

### Visual Connection

HER2 is a receptor tyrosine kinase. In 30 percent of human breast cancers, HER2 is permanently activated, resulting in unregulated cell division. Lapatinib, a drug used to treat breast cancer, inhibits HER2 receptor tyrosine kinase autophosphorylation (the process by which the receptor adds phosphates onto itself), thus reducing tumor growth by 50 percent. Besides autophosphorylation, which of the following steps would be inhibited by Lapatinib?

1. Signaling molecule binding, dimerization, and the downstream cellular response
2. Dimerization, and the downstream cellular response
3. The downstream cellular response
4. Phosphatase activity, dimerization, and the downsteam cellular response

### Signaling Molecules

Produced by signaling cells and the subsequent binding to receptors in target cells, ligands act as chemical signals that travel to the target cells to coordinate responses. The types of molecules that serve as ligands are incredibly varied and range from small proteins to small ions like calcium (Ca 2+ ).

#### Small Hydrophobic Ligands

Small hydrophobic ligands can directly diffuse through the plasma membrane and interact with internal receptors. Important members of this class of ligands are the steroid hormones. Steroids are lipids that have a hydrocarbon skeleton with four fused rings different steroids have different functional groups attached to the carbon skeleton. Steroid hormones include the female sex hormone, estradiol, which is a type of estrogen the male sex hormone, testosterone and cholesterol, which is an important structural component of biological membranes and a precursor of steriod hormones (Figure 9.9). Other hydrophobic hormones include thyroid hormones and vitamin D. In order to be soluble in blood, hydrophobic ligands must bind to carrier proteins while they are being transported through the bloodstream.

#### Water-Soluble Ligands

Water-soluble ligands are polar and therefore cannot pass through the plasma membrane unaided sometimes, they are too large to pass through the membrane at all. Instead, most water-soluble ligands bind to the extracellular domain of cell-surface receptors. This group of ligands is quite diverse and includes small molecules, peptides, and proteins.

#### Other Ligands

Nitric oxide (NO) is a gas that also acts as a ligand. It is able to diffuse directly across the plasma membrane, and one of its roles is to interact with receptors in smooth muscle and induce relaxation of the tissue. NO has a very short half-life and therefore only functions over short distances. Nitroglycerin, a treatment for heart disease, acts by triggering the release of NO, which causes blood vessels to dilate (expand), thus restoring blood flow to the heart. NO has become better known recently because the pathway that it affects is targeted by prescription medications for erectile dysfunction, such as Viagra (erection involves dilated blood vessels).

### Definition

Integral Proteins: Integral proteins are proteins that are permanently attached to the plasma membrane.

Peripheral Proteins: Peripheral proteins are proteins that are temporarily attached to the plasma membrane.

### Alternative Names

Integral Proteins: Integral proteins are called intrinsic proteins.

Peripheral Proteins: Peripheral proteins are called extrinsic proteins.

### Location

Integral Proteins: Integral proteins are embedded in the whole membrane.

Peripheral Proteins: Peripheral proteins are located on the inner or outer surface of the phospholipid bilayer.

### Interaction with the Hydrophobic Core of the Lipid Bilayer

Integral Proteins: Integral proteins highly interact with the hydrophobic core of the lipid bilayer.

Peripheral Proteins: Peripheral proteins interact less with the hydrophobic core of the lipid bilayer.

### Types of Interactions with Lipid Bilayer

Integral Proteins: Integral proteins bind to the lipid bilayer by hydrophobic, electrostatic or non-covalent interactions.

Peripheral Proteins: Peripheral proteins on the inner surface of the lipid bilayer are held by the cytoskeleton.

### Constituent of the Membrane Protein

Integral Proteins: Integral proteins constitute 70% of the total membrane proteins.

Peripheral Proteins: Peripheral proteins constitute 30% of the total membrane proteins.

### Hydrophilic/Hydrophobic

Integral Proteins: Integral proteins contain both hydrophilic and hydrophobic parts.

Peripheral Proteins: peripheral proteins contain hydrophilic parts.

### Function

Integral Proteins: Integral proteins serve as carrier proteins, channel proteins, and enzymes.

Peripheral Proteins: Peripheral proteins serve as receptors and surface antigens.

### Protein Removal

Integral Proteins: Detergents should be used to remove integral proteins from the plasma membrane.

Peripheral Proteins: Dilute salt solutions can be used to remove peripheral proteins from the plasma membrane.

### Examples

Integral Proteins: Glycophorin, rhodopsin, and NADH dehydrogenase are examples of integral proteins.

Peripheral Proteins: Mitochondrial cytochrome c and erythrocyte spectrin are examples of peripheral proteins.

### Conclusion

Integral and peripheral proteins are two types of membrane proteins in the phospholipid bilayer. Integral proteins penetrate the hydrophobic core of the lipid bilayer while peripheral proteins are attached to the intracellular or extracellular surface of the lipid bilayer. Transmembrane proteins are a type of integral proteins. The main difference between integral and peripheral proteins is the penetrance of the hydrophobic core of the lipid bilayer.

##### Reference:

1. Lodish, Harvey. “Membrane Proteins.” Molecular Cell Biology. 4th edition., U.S. National Library of Medicine, 1 Jan. 1970, Available here.
2. “Integral membrane proteins.” Integral membrane proteins, Available here.
3. “Peripheral membrane protein.” Peripheral membrane protein, Available here.

##### Image Courtesy:

1. “Transmembrane receptor” By Mouagip (talk) (CC BY-SA 3.0) via Commons Wikimedia
2. “Membrane protein” By Meng-jou wu at English Wikibooks – Transferred from en.wikibooks to Commons by Adrignola (Public Domain) via Commons Wikimedia

Lakna, a graduate in Molecular Biology & Biochemistry, is a Molecular Biologist and has a broad and keen interest in the discovery of nature related things

## Design of Functional Membrane Proteins

Leveraging the insight gained from studies of transmembrane helical peptides and proteins, functionality can be introduced in to designed systems. Membrane associated proteins have been designed to provide a “switch” that can be used to modulate the integrity of a lipid bilayer. In particular, amphiphilic α-helical peptides are known to be antimicrobial and to rupture cell membranes. In this regard, mastoparan X, a natural α-helical cell-lytic peptide has been redesigned to bind divalent cations. Upon binding of Zn(II) or Ni(II), the amphiphilic structure of the designed peptide is stabilized, which triggers the lysis of cells and vesicles (Signarvic and Degrado, 2009). The strategy demostrates the feasibility of designing proteins that can be selectively triggered to disrupt membranes.

Fusion of protein domains can yield chimeras that are useful for structural studies and can also be used to realize designed membrane proteins with targeted functionalities. One such effort yielded a pentameric ligand-gated ion channel, where each subunit comprised a prokaryotic extracellular domain and a eukaryotic transmembrane domain (Duret et al., 2011). The extracellular segment of the chimera was the proton-gated ion channel from Gloeobacter violaceus (GLIC), while the transmembrane segment was the anion-selective human 㬑 glycine receptor ( Figure 1 ). Putative mismatches at the interface between the prokaryotic and eukaryotic domains of the chimera were minimized. The site of fusion was carefully selected and specific interfacial motifs were switched from the extracellular identity (GLIC) to the transmembrane identity (㬑 glycine receptor). The chimera functions as a proton-gated ion channel, as evidenced from electrophysiological data obtained in Xenopus oocytes. Moreover, using patch-clamp experiments in baby hamster kidney (BHK) cells it was shown that the chimera displays anion selectivity identical to that of the glycine receptor. The activity of the chimera does not require posttranslational modifications typical of eukaryotic extracellular domains, and therefore the protein is good candidate for bacterial expression systems. This work provides a starting point for studies of the coupling between ligand gating and ion channel activity, as well as drug development the findings suggest that GLIC and 㬑 glycine receptors may possess highly similar structures.

Rendering of the chimera membrane protein structure based on the structure of GLIC (pdb accession code: 3EHZ). The extracellular domain (yellow) is from the prokaryotic proton-gated ion channel GLIC and the transmembrane domain (blue) is from eukaryotic anionic-selective 㬑 glycine receptor (Duret et al., 2011). Small modifications at the interface of the two domains are colored in magenta and orange. For clarity, the other subunits are colored gray.

Another example of creating functional membrane proteins was based on a natural transmembrane dimer motif. Using the structure of the transmembrane region of glycophorin A, a bis-histidine binding site was designed to bind the cofactor Fe-protoporphyrin IX (Cordova et al., 2007). Five out of 32 transmembrane residues were modified, and the resulting structure was characterized in dodecylphosphocholine (DPC) micelles ( Figure 2 ). The protein binds the cofactor with submicromolar affinity and retains the dimeric oligomerization state. Moreover, the catalytic activity of the complex was characterized by the oxidation of the organic substrate TMB (2,2′,5,5′ -tetramethyl-benzidine). TMB undergoes two successive oxidations in the presence of peroxide to produce TMB-ox, and formation of the latter indicated that the complex presents modest peroxidase activity. A single mutation (G25F) was introduced to assess aromatic-porphyrin interactions. The mutant binds heme with a lower dissociation constant (by a factor of 1/10), displays a change in the midpoint potential, and presents a decrease in peroxidase activity. The changes were ascribed to the stabilization of the Fe(III) form in the mutant. The findings illustrate the use of designed proteins to control the properties of the porphyrin cofactor within a membrane localized environment.

The designed bis-histidine binding site is depicted together with the protoporphyrin IX ligand (Cordova et al., 2007). The modified positions in the structure of glycophorin A are colored in blue.

## How can a ligand be an integral membrane protein? - Biology

Membrane Proteins, Properties of

Linda Columbus, Department of Chemistry, University of Virginia, Charlottesville, Virginia

Robert K. Nakamoto, Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia

David S. Cafiso, Department of Chemistry, University of Virginia, Charlottesville, Virginia

Membrane proteins constitute a significant fraction of the proteins that are encoded in the typical genome, and they are critical to many cellular processes, which include transport, cell signaling, and energy transduction. This importance is underscored by the fact that membrane proteins represent the major class of protein targets for pharmaceuticals that are currently in use. However, despite their importance, there are relatively few high-resolution models for membrane proteins, and little is known about their molecular function. The lack of information on membrane proteins in part reflects the difficulties in efficient expression of this class of proteins and difficulties with structural approaches, such as high-resolution nuclear magnetic resonance (NMR), that are not well suited to membrane proteins. In the field of structural biology, membrane proteins represent a significant challenge, and in recent years new structural and biochemical tools have been developed to characterize these systems. In this review, we discuss the functions and properties of membrane proteins, and we discuss the approaches and tools that yield new information on their structure and molecular function.

Membranes define the boundaries and compartments that make up the cellular matrix, and they are responsible for defining the chemical environment within the cell. Membranes also provide an interface that facilitates protein-protein interactions and other biochemical reactions necessary for cell signaling and trafficking. It is estimated that protein domains that reversibly associate with membrane interfaces represent the most abundant domains found in water-soluble proteins (1). Although the number of proteins that interact with or function at the membrane interface is large, this review will focus on membrane proteins that are sometimes termed integral membrane proteins. Integral membrane proteins are generally defined as proteins that cannot be isolated and purified without first dissolving the bilayer structure, typically with detergents. When compared with water-soluble proteins, membrane proteins have unique properties and present unique challenges in terms of their expression, isolation, and structural characterization. Inducing membrane protein expression in high yield can be more challenging than that of water-soluble proteins. Because the forces that dominate the fold of a membrane protein differ from those that stabilize water-soluble protein folds, purified membrane proteins must be studied in heterogeneous environments, such as membrane mimetic detergent micelles or reconstituted lipid bilayers. It is estimated that approximately 30% of the genome codes for membrane proteins (2), although the exact percentage is unknown. However, when compared with the total number of entries in the protein databank, membrane proteins represent less than 1% of the total number. Although X-ray crystallography remains the single most important method for generating structures of membrane proteins, several new spectroscopic methods are being employed to study membrane protein structure and dynamics.

Biological Functions and Distribution of Membrane Proteins

Classification of membrane proteins: nomenclature and architecture

Membrane proteins carry out a wide range of critical functions in cells, and they include passive and active transporters, ion channels, many classes of receptors, cellular toxins, proteins involved in membrane trafficking, and the enzymes that facilitate electron transport and oxidative phosphorylation. For example, the voltage-gated ion channels that facilitate the passive diffusion of sodium and potassium across the axonal membrane are responsible for the formation of an action potential. Active transport proteins establish ion gradients and are necessary for the uptake of nutrients into cells. Soluble hormones bind to membrane receptors, which then regulate the internal biochemistry of the cell.

At the present time, representative structures exist for approximately 21 unique β-barrel membrane protein families and 35 polytopic α-helical protein families. This sample is a small fraction of the predicted 300-500 α-helical folds and 700-1700 families (3). Although the structural biology of membrane proteins is in its infancy, it is clear that membrane proteins display a rich variety of structures that vary greatly in size and topology (Fig. 1). Of the structures observed thus far, all are based on two fundamental architectures: the α-helical bundle (4) and the β-barrel (5, 6).

The membrane β-barrel fold is unique to the outer membrane of mitochondria, chloroplasts, and Gram-negative bacteria, and they are found to be composed of an even number of β-strands that vary in number between 8 and 22. The inter-strand hydrogen-bonding pattern within the barrel allows structures to form, which do not have unsatisfied hydrogen bonds within the membrane interior. Frequently, these strands are configured so that amino acid side chains with an aliphatic composition reside on the barrel exterior facing the membrane hydrocarbon, and as observed in membrane proteins based on helical bundles, β-barrel proteins display more aromatic side chains at regions near the membrane bilayer interface.

Membrane proteins based on transmembrane a-helices (Fig. 1) are typically localized to the plasma membrane, organelle membranes, and inner membrane of mitochondria and bacteria. Thus, these proteins not only differ in secondary structure, but also they differ in localization. Helical membrane proteins can be formed from 1 to 19 transmembrane segments. When they possess a single transmembrane pass, they are sometimes referred to as either monotopic or bitopic. When these proteins have two or more transmembrane helices, they are referred to as polytopic. The transmembrane helices of a polytopic membrane protein associate into a bundle, and to maintain unsatisfied hydrogen bonds to a minimum, these helices are usually regular. Although helical membrane proteins may be quite flexible and dynamic, elements of helical structure within the bilayer are thought to be rigid.

Figure 1. Examples of several bacterial membrane proteins. The outer membrane (OM) of Gram-negative bacteria contains exclusively β-barrel proteins, and three examples are shown: BtuB (PDB ID: 1NQF), which is the 22 β-stranded βonB-dependent active transporter for vitamin B12 the LamB or maltoporin trimer (PDB ID: 1AF6), which is the 18 β-stranded passive sugar transporter and OmpA (PDB ID: 1 BXW), which is an 8 P-stranded protein that provides structural support for the OM. Proteins in the cytoplasmic membrane (CM) are helical, and three examples are shown: the potassium channel KcsA (PDB ID: 1 BL8), which is a tetramer Sec YEG (PDB ID: 1 RH5), which forms the protein transport channel in Methanococcus and BtuCD (PDB ID: 1L7V), which is the ATP-driven transporter that imports vitamin B12 from the periplasm.

Membrane protein folding and membrane insertion

Integral membrane proteins must be stable and function in a unique and highly anisotropic environment. The aqueous facing domains of a membrane protein experience a very different environment than do the membrane facing regions of the protein, which must be stable in a low-dielectric region devoid of water (7). Because of the absence of a bulk aqueous phase, significant numbers of unsatisfied hydrogen bonds are highly unfavorable, and the folds of β-barrel or helical membrane proteins, as indicated above, must always be arranged to satisfy backbone hydrogen bonding.

In water-soluble proteins, the hydrophobic effect is believed to be a major force that drives the folding of proteins, dominating over van der Waals forces (8, 9). However, in membrane proteins, the hydrophobic effect is relatively unimportant in comparison to side-chain hydrogen bonding and van der Waals forces. Van der Waals forces in a helical bundle would be maximized by complementarity of the interacting surfaces on transmembrane helices as expected, sequence motifs, such as GxxxG, have been identified that maximize the packing of membrane helices (10-12). Interhelix hydrogen bonding is also important in driving the association of helices, and it may be an important determinant of helix association during membrane protein synthesis (13). Although both interactions play important roles, a mutational study on one helical bundle suggests that van der Waals forces make the largest contribution to the stability of the bundle (14).

The arrangement of the transmembrane segments of a membrane protein and the orientation of the protein C and N-termini are determined by two main features of a membrane protein sequence: the stretch of hydrophobic residues that ultimately span the bilayer, and the position of positively charged segments (15, 16). The hydrophobic residues (Ala, Ile, Leu, Val) have the highest frequency in helical regions that lie in the center of the bilayer, whereas the two aromatic residues Tyr and Trp are most abundant near the bilayer interface (17). The balance of charge on either side of a transmembrane segment determines the orientation of the segment, so that the more positively charged portions of a membrane protein sequence are found on the cytoplasmic side of the bilayer. This structure is sometimes referred to as the “positive-inside rule” (18). By altering hydrophobicity and charge, differing topologies can be generated, and it is even possible to find homologous proteins that have naturally evolved different topologies by modifying these features (15). Biosynthetically, the insertion of the growing polypeptide chain into the bilayer is mediated by a translocon, which is a helical membrane protein that is believed to have a lateral gate that opens to the bilayer interior. In the rough endoplasmic reticulum, this protein is Sec61. It is generally thought that the growing polypeptide chain may sample the surrounding bilayer through this gate, so that a thermodynamic equilibrium is established with the surrounding lipid (16, 19).

The lipid bilayer is not passive in determining membrane protein activity and function, and an accumulating body of evidence indicates that there is a coupling of membrane proteins to lipid bilayer properties. These properties include the effect of bilayer curvature strain (20), the role of specific lipids such as phosphoinositides, (21) and the effect of thickness on membrane protein function (22). The lipid composition, as well as the bilayer properties that result from this composition, act as allosteric regulators of membrane protein function.

Molecular function of membrane proteins

Structural methods and other genetic and biochemical studies have provided clues to the molecular function of membrane proteins. For example, in the case of the bacterial K + channel KcsA, the protein is designed to lower the energy for a potassium ion as it passes through the center of the channel. This function is facilitated both by a central cavity that is hydrated and by a helix that is positioned toward the channel pore so that its negatively charged end points toward this cavity (23). In the case of visual rhodopsin, a salt bridge that exists between Lys296 and Glu133 functions to maintain the protein in an inactive state. Disruption of this ionic interaction is responsible for the movement of helix 6, which activates the G-protein transducin (24), and mutations that disrupt this ionic interaction are responsible for the retinal diseases retinitis pigmentosa and congenital night blindness (25). Among outer membrane bacterial transporters, TonB-dependent transporters function to move rare nutrients, such as iron chelates, into the cell. They contain a large N-terminal domain of approximately 150 residues that is sometimes referred to as a “hatch.” The available high-resolution structures suggest that substantial rearrangements or an unfolding of this hatch domain takes place to allow the passage of substrates (26).

Expression, Isolation and Purification of Membrane Proteins

Difficulties in obtaining protein samples

In general, membrane proteins present challenges at every step on the way to structural determination. The synthesis and processing of these proteins is complex and often involves specific folding factors or chaperones (see References 27 and 28 for reviews). Expression in bacteria is favored because of the low cost to grow large culture volumes by fermentation, the potential for high yields (up to 10 to 100 mg of protein per liter of culture), the very fast growth rate, and the simplicity and flexibility of expression systems. However, intrinsic differences in how proteins are processed often prevent the expression of adequate amounts of protein with the proper fold, and it may not be possible to isolate and purify sufficient quantities of a membrane protein to a homogenous state. As a result, understanding the processing pathways for the specific protein of interest can be the important factor to achieve successful expression.

The biosynthetic membrane insertion of eukaryotic membrane proteins almost always takes place cotranslationally, whereas many prokaryotic proteins can be posttranslationally inserted, typically with the aid of chaperones. In eukaryotic cells, the proofreading mechanisms in the endoplasmic reticulum prevent misfolded proteins from leaving to the Golgi. Some of these mechanisms include glycosylation of the protein on its luminal domain (29). In addition, other protective mechanisms such as the yeast unfolded protein response may lead to increased degradation rates of improperly folded proteins (30).

Some proteins require specific types of processing. For example, β-barrel proteins in the outer membrane of Gram negative bacteria must pass through the cytoplasmic membrane in a linear fashion before being assembled in the outer membrane (for a review, see Reference 31). Processing of β-barrel proteins in the outer membranes of mitochondria and chloroplasts may also involve passage of the protein through the organelle outer membrane before insertion, but this process is not well understood (32). Several chaperones have been identified that help the assembly of mitochondrial and chloroplast membrane proteins. Some of these proteins are encoded in the nucleus and are imported to the proper compartment posttranslationally, whereas a few are encoded on the organelle’s own chromosome and processed from within. For these reasons, structural approaches for such proteins always rely on isolation from the native organelle.

Bacterial expression systems are often not an option for eukaryotic membrane proteins. Even if the protein is found embedded in the bacterial membrane, obtaining correctly folded protein is always a concern. Frequently, most or all expressed protein is found in an intracellular aggregate or inclusion body, and only a few examples have been refolded to their native state (33). In many cases, the apparent toxicity of the expressed membrane protein blocks expression as well as growth. Several reasons can explain protein toxicity (see Reference 34 for a review). For example, Miroux and Walker (35) suggest that the overproduction of a single mRNA in a typically used T7 polymerase-driven transcription system resulted in the uncoupling of transcription and translation. A strain, such as C43(DE3) (Lucigen, Middleton, WI) selected to avoid the effects of toxicity, demonstrated better yields of proteins despite a slower rate of synthesis. The requirement of a slower rate of synthesis suggests that cellular machinery, which is not overexpressed, may be required for proper folding. On the other hand, mixed results are observed in providing the cell with extra in- sertional machinery. Interestingly, one of the more productive approaches has been to provide extra chaperones or foldases in the expression strain (36).

Despite the severe limitations to membrane protein expression, successful approaches have been developed. Three general strategies are used by investigators to obtain sufficient amounts of protein for structural studies. First, protein can be obtained from a native source. Many membrane protein structures have been obtained from protein purified from tissue or organelles for example, rhodopsin, the sarcoplasmic reticulum calcium pump, and the electron transport complexes from mitochondria. Obviously, this approach only works in situations where the protein of interest expresses at naturally high levels. Furthermore, genetic manipulation of the protein primary structure, for example to add a purification tag, is usually not possible. Second, large numbers of bacterial orthologs of the protein of interest can be screened. With the availability of chromosomal DNA or cDNA libraries from a wide range of species, investigators can clone several homologs and test for high level expression and folding in an Escherichia coli expression strain. The hope is that one or more of the genes will be amenable to stable expression, purification, and structural determination. This approach has been used successfully in many cases such as various transporters, aquaporins, mechanosensitive channels, and potassium channels (37). Finally, the eukaryotic protein can be produced in a eukaryotic cell. This approach has become increasingly feasible as synthetic media have brought the expense of large-scale growth into the realm of the academic laboratory. The cells of choice are insect cells, either those from the fall army worm, Spodoptera frugiperda (Sf9 or 21) or the cabbage looper Trichoplusia ni (High Five Invitrogen, Carlsbad, CA). Mammalian cells, such as CHO or COS, have been used in a few cases, as have the yeast Saccharomyces cerevisiae or Pichia pastoris (from Invitrogen). This approach assumes that the eukaryotic cells can recognize the processing signals intrinsic to the protein sequence and that the proper chaperones and foldases will be present. Although this scenario is more likely, it is certainly not true in all cases. As mentioned above, other factors may be necessary for proper folding. Furthermore, a protein that is part of a complex may not express in a stable or properly folded manner in the absence of its partners or assembly factors.

Although the optimization of growth conditions for eukaryotic cells is limited, the growth conditions for bacterial systems can be varied widely, and they have a large influence on the expression of the protein target. Many “tricks” investigators use are anecdotal and may be specific to their protein of interest. Several publications detail the approaches and often many different conditions must be tested (see References 33 and 38 for extensive reviews). We will not attempt a thorough listing of such methods, but a few are worth noting. First, medium strength promoters are used to slow down synthesis rate. As mentioned above, the cell may respond to an overwhelming synthesis of a specific protein by activating degradation systems. Second, a lower culture temperature is used, as low as 15° C, with expression induced for many hours to days. Third, using a defined minimal medium may also result in accumulation of considerable amounts of protein, as the cells grow much more slowly. Development of expression protocols in defined medium is always advantageous for structural approaches because the investigator hopefully will need to generate seleno-methionine derivatives for MAD or SAD phasing crystallographic data, or isotopically 2 H, 15 N, 13 C labeled samples for heteronuclear NMR.

Without exception, isolation and purification of an integral membrane protein must involve detergents to solubilize the membrane and membrane-associated proteins. Although many detergents may be tested, the prudent investigator will generally carry out the initial membrane solubilization in a readily available and less expensive nonionic detergent. In our experience, almost all proteins will solubilize in one of the following: n-decyl-β-D-maltopyranoside, N,N-dimethyldodecylamine-N-oxide (LDAO or DAO-12), n-dodecylphosphocholine (Fos-choline 12 or DPC), n-octyl-β-D-glucopyanoside (octyl glucoside), or tetraethylene glycol monoctyl ether (C8E4). Solubilization is tested by adding the detergent solutions to a small amount of membrane preparation followed by an incubation period, usually at room temperature, and ultracentrifugation. The presence of the protein in the membrane pellet versus a detergent-protein micelle in solution is determined by Coomassie-stained SDS-PAGE gel or an immunoblot using a specific antibody or an antibody against the affinity tag.

Purification can usually follow the same approaches as soluble proteins except that all procedures are performed in the presence of the detergent. The function of commonly used affinity tags is usually not affected by detergent however, membrane proteins are often at relatively low concentrations, and the affinity chromatography step should sometimes be followed by another form of chromatography, such as ion exchange and/or gel filtration. High-resolution gel filtration chromatography can also provide an indication of the oligomeric state of the protein. A general protocol for the purification of membrane proteins is shown in Fig. 2.

To characterize the protein preparation, the critical parameters are purity, which is estimated by SDS-PAGE, the fold and evaluated by secondary structure content via circular dichroism spectroscopy, as well as the oligomeric state determined by gel filtration, light scattering, or sedimentation velocity (39-41). Furthermore, the homogeneity of the protein can be evaluated by mass spectrometry. Detergents will normally interfere with mass spectrometry, but MALDI TOF approaches have been developed to avoid such problems (42). As discussed in the next section, a range of detergents must be explored for protein structural determination, and as a result, the characteristics of the protein in different detergents must be determined (see Fig. 2).

If possible, the function of the protein should be assessed. Unfortunately, detergent solublization can often make it difficult to assay protein activity, as in the case of an ion channel however, other parameters may be assessed in such instances. Ligand binding affinity, enzymatic activity, or association with another protein may provide a convincing assessment of protein function. Other biochemical techniques will also indicate folding and homogeneity, such as limited protease digestion, reactivity with reagents such as sulfhydryl reactive compounds, or chemical cross-linking patterns. Spectroscopic approaches have also been used. The dynamics of specific regions on the protein can be evaluated by nitroxide spin labeling and electron paramagnetic spectroscopy. If the protein can be metabolically labeled with 2 H, 15 N, then a relatively quick heteronuclear single quantum coherence NMR experiment can provide critical information on the homogeneity and fold of the protein (39).

Figure 2. A flow chart that outlines the general strategies in preparing membrane proteins for structural studies.

Structural Characterization of Membrane Proteins

High-resolution membrane protein structures are determined predominantly using X-ray crystallography. Although notoriously difficult to crystallize, several methods have been applied successfully to crystallize membrane proteins either by manipulating the detergent/lipid components or by altering the protein component. Most membrane protein structures have been determined using detergent solubilized protein in which the entire protein detergent complex is crystallized. Often, the best-quality crystals of a membrane protein may be obtained only in one or a few detergents, and extensive screening based on detergent properties is required (41).

Thirty-three detergents have been used to crystallize membrane proteins, and three of those detergents have been used to determine three NMR structures (43). Some detergents are fold-specific. For example, C8E4 is predominantly used for β-barrel proteins whereas DDM has been mostly successful for α-helical membrane proteins. The four detergents that have been used to crystallize most membrane proteins are octyl-glucoside, lauryl dimethyl amine oxide, C8E4, and dodecyl maltoside. These four detergents vary in alkyl chain length and shape and size of the head group however, they are all neutral. This commonality is very significant. For three-dimensional protein crystals to form, protein molecules need to contact each other to form crystal contacts that are essential to propagate the lattice. It is likely that charged or even zwitterionic repulsive forces would hinder the association of the protein detergent complexes, which is a process that must occur at early stages of crystal nucleation.

Membrane protein crystals have significantly more solvent (64%) content than soluble proteins [47% (44)] presumably because of the detergent in the crystal. The organization of the detergent in the membrane protein crystal has been investigated in a select few cases and is different in each case. In the LH2 crystal, the detergent forms a belt around the hydrophobic surface of the protein consistent with the dimension of the OG detergent molecule (45). Similar to LH2, the OG detergents form a belt around the hydrophobic surface of phospholipase A in the crystal however, the belts fuse to form a continuous three-dimensional network throughout the crystal (46). The continuous density of detergent was also observed in crystals of porin and two photoreaction centers. Snijder et al. (46) suggest the possibility that organic amphiphilic additives in the crystal screen could facilitate this fusion however, no experimental data correlate the detergent structure observed in the crystal and the physical properties of the detergent/amphiphile mixed micelle. In addition to the detergent used for solubilizing the membrane protein, the native (or synthetic) lipid concentration may have profound effects on diffraction quality as in the instance of the crystallization of LacY (47), cytocrome b6f (48), Ca 2+ -ATPase (49), and GlpT (50).

Lipid cubic (51) and sponge (52) phases, as well as bicelles (53), are alternatives to detergents that have been applied successfully to membrane protein crystallization. In these instances, the protein is embedded in a lipid bilayer environment, which is considered more natural compared with the detergents that form micellar phases. In the recent high-resolution crystal structure of the human β2 adrenergic G-protein-coupled receptor, lipid cubic phase was used with necessary cholesterol and 1,4-butandiol additives (54). The cholesterol and lipid molecules were important in facilitating protein-protein contacts in the crystal.

In addition to the influence of the detergent on crystallization, the introduction of additional protein components has proven to be successful. In several cases, an antibody fragment was cocrystallized with the membrane protein to provide essential crystal contacts for crystallization (55-57). The protein itself can be engineered with an insertion to provide crystal contacts as demonstrated by the recent success of human β2 adrenergic G-protein-coupled receptor structure, in which a lysozyme molecule was engineered into one of the loops (58).

Despite these advances, Oberai et al. (3) estimate that if no acceleration of membrane protein structure determination occurs, then it will take more than three decades to determine at least one structural representative of 90% of the α-helical membrane protein sequence families (3).

Solution nuclear magnetic resonance spectroscopy

Although solution NMR techniques do not require crystallization, a molecular weight limit does exist [33 kD is the largest to date (59)], and optimization of detergent conditions has proven to be difficult (60). Several NMR structures of β-barrel outer membrane proteins have been determined (59, 61-63) however, a solution structure of a polytopic α-helical membrane protein remains a challenge. Several research groups are making great strides with the first step to NMR structure determination, the sequential chemical shift assignment achieved for two polytopic membrane proteins diacylglycerol kinase (64) and the potassium channel KcsA (65). NMR structure determination of β-barrel proteins has been more successful than a-helical membrane proteins primarily because the nuclear Overhauser effects (NOEs) between amide protons are across strands (i.e., between secondary elements), rather than within the secondary element as in α-helices, which provides valuable structural constraints. The lack of NOE data is currently being overcome with measurements from paramagnetic relaxation (66) and residual dipolar coupling experiments (67).

In addition to the limited distance restraints, the preparation of membrane protein samples has proven to be a major challenge to high-resolution NMR. Similar to X-ray crystallography, the selection of detergent strongly influences the quality of NMR spectra. No single detergent is well suited for NMR studies of membrane proteins solubility, dynamics, the hydrophobic surface area of the protein, and other physical properties differ for each protein detergent complex, and the proper combination still needs to be determined empirically through extensive screening (39, 68).

Beyond structure determination, solution NMR can be used to investigate backbone dynamics and protein-ligand interactions. Bax and colleagues (65, 69) have characterized backbone dynamics (69) and ion binding affinity of the tetrameric KcsA potassium channel (65). These studies added additional structural insights to the crystal structure. On the ps-ns time scale, the selectivity filter is not dynamic. In SDS micelles, the intracellular C-terminal α-helix is dynamic on the ns-ps timescale and does not associate into a tetrameric bundle. In addition to determining the PagP NMR solution structure (62), Hwang et al. (70) characterized a two state dynamic rearrangement in which the more flexible state facilitates the entry of the substrate into the central cavity of the β-barrel.

Site-directed spin labeling

Site-directed spin labeling (SDSL) is used to investigate membrane protein structure and dynamics in lipid bilayers as well as in detergents. In SDSL, a nitroxide probe is introduced to a unique site within a protein. In most cases, a cysteine residue is introduced and subsequently reacted with a sulfhydryl-reactive nitroxide reagent. The resulting nitroxide side chain is sensitive to the molecular environment, which allows the determination of secondary and tertiary structure (71), conformational dynamics (72), and site-specific dynamics (73). Unlike solution NMR, the technique does not have a molecular weight limit, and membrane proteins can be investigated in detergent solutions or lipid bilayers.

From the electron paramagnetic resonance (EPR) spectrum of the nitroxide side chain, four primary parameters are obtained: 1) solvent accessibility, 2) mobility of the R1 side chain, 3) a polarity index for its immediate environment, and 4) the distance between R1 and another paramagnetic center in the protein. Solvent accessibility of the side chain is determined from the collision frequency of the nitroxide with paramagnetic reagents in solution. The mobility, polarity, and distances are deduced from the EPR spectral line shape. For regular secondary structures, accessibility, mobility, and polarity are periodic functions of sequence position. The period and the phase of the function reveal the type of secondary structure and its orientation within the protein, respectively (71, 74). In the case of membrane proteins, the topography of the secondary structure with respect to the membrane surface can also be described (75, 76).

When a pair of spin labels is incorporated into a protein or a protein complex, the dipolar interactions between labels can be used to measure the distance and distance distribution between labels (77). If the labels are separated by 7 to 20 A, then the dipolar interactions are sufficiently strong that they can be observed and quantified by continuous wave (CW) EPR spectroscopy (78-81). If the labels are separated by a distance greater than 20 A, then the resulting weak dipolar interactions can be measured with newer pulse methods such as double electron-electron resonance (DEER) or double quantum coherence (82-84). These methods have been used to measure internitroxide distances and distance distributions out to 60 A or more. An advantage of the CW EPR measurements is that they can be performed at room temperature (78), whereas the pulse measurements, such as DEER, require that the samples be frozen, typically at liquid nitrogen temperatures or lower.

Changes in any of the SDSL parameters measured can be used to detect changes in protein conformations, and most importantly, the data can be interpreted in terms of helix rigid body motions, relative domain movement, and changes in secondary structure. Recently, SDSL distance measurements were used to map ligand-induced conformational changes in BtuB (85), LacY (86), and the NhaA antiporter (87). Figure 3 shows an example of the use of SDSL and DEER to determine structural changes that accompany ligand binding to the extracellular loops in the outer membrane transporter, BtuB (88).

Figure 3. Site-directed spin labeling can be used to provide information on structural changes in membrane proteins (72). (a) A common nitroxide side chain R1, which is produced by cysteine mutagenesis and covalent modification. (b) The OM transporter, BtuB, showing the nitroxide side-chain, R1, at positions 188 and 291 in the extracellular loops of the transporter. The substrate, vitamin B12, is shown (CPK rendering), along with the co-ligand Ca 2+ . (c) DEER signals obtained from this spin pair in the absence and presence of the substrate (vitamin B12) and the Ca 2+ co-ligand. The solid trace is a fit of the data to a single Gaussian distance distribution. Both the distance and distrance distribution (which is the standard deviation to the Gaussian) are shown. Ligand binding shortens the distance between spin pairs, which results in an ordering of the loop and narrowing of the distance distribution (88).

Solid-state nuclear magnetic spectroscopy

Solid-state NMR also can be applied to membrane proteins in lipid bilayers, and recent advancements in magic angle spinning solid-state NMR show promise for structure determination. Although the structures of small crystalline proteins (89) and membrane bound peptides (90) have been determined, the structure of a polytopic membrane protein has yet to be reported. The major necessity that is required to push the technique forward is the de novo sequential chemical shift assignment of the amino acid residues, and in the last few years, several groups have reported successful strategies (91, 92).

Beyond structure determination, solid-state NMR has been used to investigate the structures of membrane bound ligands. Several recent examples are a low-resolution structure of neurotensin bound to its G-protein coupled receptor (93), scorpion toxin bound to a chimeric potassium channel (94), retinal in both the rhodopsin and the metarhodopsin II intermediate (95), and acetylcholine bound to its receptor (96). These studies have great potential for designing and optimizing drugs-targeting membrane proteins (97).

Several biophysical tools can provide information on the structure, dynamics, and conformational changes of membrane proteins. Many of these methods are beyond the scope of this review and were not mentioned here. However, each of these approaches has strengths and weaknesses to investigate membrane protein structure and functional dynamics fully, a multitude of techniques is required. Static high-resolution structures are highly informative and provide a good starting point to generate hypotheses however, they do not provide a complete understanding of protein molecular function.

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Lipid Bilayers, Properties of

Membrane Assembly in Living Systems

Membrane Proteins, Properties of

Protein Targeting and Transport

## Abstract

Saturation transfer difference (STD) NMR is a fast and versatile method to screen compound mixtures in the presence of a receptor for binding affinity and to characterize the ligand's binding epitope. Here we demonstrate that ligand interactions with integral membrane proteins can be investigated by STD NMR if the receptor is embedded into the lipid bilayer of a liposome. The integrin αIIbβ3, also termed GPIIb-IIIa, is a platelet surface glycoprotein that plays a pivotal role in platelet aggregation and that interacts with proteins and peptides presenting the peptide recognition motif RGD. Purified human integrin αIIbβ3 was incorporated into liposomes, and the binding of RGD peptides was analyzed by STD NMR techniques. Cyclo(RGDfV) gave STD NMR effects in the presence of liposomes containing the integrin. The magnitude of the STD effect as a function of the ligand's concentration gave a value for the dissociation constant of 30−60 μM. Adding the weakly binding RGD to the solution of cyclo(RGDfV) resulted in STD effects of the stronger ligand cyclo(RGDfV) only. This demonstrates in agreement with literature that the peptide RGD is a much weaker ligand to the integrin than the peptide cyclo(RGDfV) that largely replaces the RGD peptides from the binding site. The binding epitope of the ligand cyclo(RGDfV) was characterized by STD NMR to contain sections of the d -Phe, the Val methyl groups, Arg α, β, and γ protons, one Hβ of Asp, and one Hα of Gly.

Abbreviations: integrin αIIbβ3 fibrinogen receptor, platelet glycoprotein IIb-IIIa cyclo(RGDfV), cyclo(1,5)-arginyl-glycinyl-aspartyl- d -phenylalanyl-valine DMPC, 1,2-dimyristoyl-rac-glycero-3-phosphocholine DMPG, 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] sodium salt LUV, large unilamellar vesicles PAGE, polyacrylamide gel electrophoresis RGD, arginyl-glycinyl-aspartic acid SDS, sodium dodecyl sulfate SAR, structure−activity relationship STD, saturation transfer difference trNOE, transferred nuclear Overhauser effect.

## How can a ligand be an integral membrane protein? - Biology

Biosignaling is a process in which cells receive and act on signals. Proteins participate in biosignaling in different capacities, including acting as extracellular ligands, transporters for facilitated diffusion, receptor proteins, and second messengers. The proteins involved in biosignaling can have functions in substrate binding or enzymatic activity.

Ion channels are proteins that create specific pathways for charged molecules. They are classified into three main groups, which have different mechanisms of opening, but all permit facilitated diffusion of charged particles. Facilitated diffusion, a type of passive transport, is the diffusion of molecules down a concentration gradient through a pore in the membrane created by this transmembrane protein. It is used for molecules that are impermeable to the membrane (large, polar, or charged). Facilitated diffusion allows integral membrane proteins to serve as channels for these substrates to avoid the hydrophobic fatty acid tails of the phospholipid bilayer. The three main types of ion channels are ungated, voltage-gated, and ligand-gated.

Hundreds of ion channels have been identified that function in cell signaling and cell excitability. In addition, ion channels are drug targets in the treatment of everything from heart disease (calcium channel blockers) to seizures (sodium channel blockers) to irritable bowel syndrome (acetylcholine receptor/cation channel blockers).

Ungated Channels

As their name suggests, ungated channels have no gates and are therefore unregulated. For example, all cells possess ungated potassium channels. This means there will be a net efflux of potassium ions through these channels unless potassium is at equilibrium.

Voltage-Gated Channels

In voltage-gated channels, the gate is regulated by the membrane potential change near the channel. For example, many excitable cells such as neurons possess voltage-gated sodium channels. The channels are closed under resting conditions, but membrane depolarization causes a protein conformation change that allows them to quickly open and then quickly close as the voltage increases. Voltage-gated nonspecific sodium–potassium channels are found in cells of the sinoatrial node of the heart. Here, they serve as the pacemaker current as the voltage drops, these channels open to bring the cell back to threshold and fire another action potential, as shown in Figure 3.4.

Figure 3.4. Action Potential of the Sinoatrial Node

Ligand-Gated Channels

For ligand-gated channels, the binding of a specific substance or ligand to the channel causes it to open or close. For example, neurotransmitters act at ligand-gated channels at the postsynaptic membrane. The inhibitory neurotransmitter &gamma-aminobutyric acid (GABA) binds to a chloride channel and opens it.

The activity at the neuromuscular junction and most chemical synapses relies on ligand-gated ion channels. The nervous system especially makes use of this type of gating, as well as voltage-gated ion channels, as discussed in Chapter 4 of MCAT Biology Review.

The Km and vmax parameters that apply to enzymes are also applicable to transporters such as ion channels in membranes. The kinetics of transport can be derived from the Michaelis–Menten and Lineweaver–Burk equations, where Km refers to the solute concentration at which the transporter is functioning at half of its maximum activity.

Membrane receptors may also display catalytic activity in response to ligand binding. These enzyme-linked receptors have three primary protein domains: a membrane-spanning domain, a ligand-binding domain, and a catalytic domain. The membrane-spanning domain anchors the receptor in the cell membrane. The ligand-binding domain is stimulated by the appropriate ligand and induces a conformational change that activates the catalytic domain. This often results in the initiation of a second messenger cascade. Receptor tyrosine kinases (RTKs) are classic examples. RTKs are composed of a monomer that dimerizes upon ligand binding. The dimer is the active form that phosphorylates additional cellular enzymes, including the receptor itself (autophosphorylation). Other classes of enzyme-linked receptors include serine/threonine-specific protein kinases and receptor tyrosine phosphatases.

KEY CONCEPT

G PROTEIN-COUPLED RECEPTORS

G protein-coupled receptors (GPCRs) are a large family of integral membrane proteins involved in signal transduction. They are characterized by their seven membrane-spanning &alpha-helices. The receptors differ in specificity of the ligand- binding area found on the extracellular surface of the cell. In order for GPCRs to transmit signals to an effector in the cell, they utilize a heterotrimeric G protein. G proteins are named for their intracellular link to guanine nucleotides (GDP and GTP). The binding of a ligand increases the affinity of the receptor for the G protein. The binding of the G protein represents a switch to the active state and affects the intracellular signaling pathway. There are several different G proteins that can result in either stimulation or inhibition of the signaling pathway. There are three main types of G proteins:

·&emspGs stimulates adenylate cyclase, which increases levels of cAMP in the cell

·&emspGi inhibits adenylate cyclase, which decreases levels of cAMP in the cell

·&emspGq activates phospholipase C, which cleaves a phospholipid from the membrane to form PIP2. PIP2 is then cleaved into DAG and IP3 IP3 can open calcium channels in the endoplasmic reticulum, increasing calcium levels in the cell

Figure 3.5. Trimeric G Protein Cycle (Gs or Gi)

Functions of heterotrimeric G proteins:

·&emspGs stimulates.

·&emspGi inhibits.

·&emsp“Mind your p's and q's”: Gq activates phospholipase C.

The three subunits that comprise the G protein are &alpha, &beta, and &gamma. In its inactive form, the &alpha subunit binds GDP and is in a complex with the &beta and &gamma subunits. When a ligand binds to the GPCR, the receptor becomes activated and, in turn, engages the corresponding G protein, as shown in Step 1 of Figure 3.5. Once GDP is replaced with GTP, the &alpha subunit is able to dissociate from the &beta and &gamma subunits (Step 2). The activated &alpha subunit alters the activity of adenylate cyclase. If the &alpha subunit is &alphas, then the enzyme is activated if the &alpha subunit is &alphai, then the enzyme is inhibited. Once GTP on the activated &alpha subunit is dephosphorylated to GDP (Step 3), the &alpha subunit will rebind to the &beta and &gamma subunits (Step 4), rendering the G protein inactive.

MCAT Concept Check 3.2:

Before you move on, assess your understanding of the material with these questions.

## DISCUSSION

HA is a negatively charged high molecular mass polysaccharide composed of repeated disaccharides of d -glucuronic acid andN-acetyl glucosamine (Goa and Benfield, 1994). It is ubiquitously expressed in the extracellular space, represents a major constituent of the ECM, and has a central role in stability of ECM (Knudson and Knudson, 1993). Numerous cellular processes including cell migration (Chen et al., 1989), adhesion (Klein et al., 1996), and proliferation (Mast et al., 1993 Wiiget al., 1996) are influenced by HA. Thus, HA has an important role in such processes as morphogenesis (Toole, 1997), wound healing (Nishida et al., 1991), inflammation (Weigelet al., 1988), and immune cell trafficking (Mohamadzadehet al., 1998), as well as in many aspects of tumor biology (Delpech et al., 1997).

Many of the effects of HA are mediated via its interaction with HA-binding proteins and receptors. The majority of the HA-binding proteins contain a common protein module termed link module, which is a structural domain of ∼100 amino acids in length (Neame and Barry, 1993 Kohda et al., 1996). Link modules have been described in several ECM molecules (link protein, aggrecan, versican, neurocan, and brevican) as well as on cell surface receptors. However, not all HA-binding proteins contain a link domain, namely, RHAMM, Cdc37, inter-α-trypsin inhibitor, plasma HA-binding protein, fibroblast HA-binding protein (reviewed by Day, 1999), and intracellular hyaluronate-binding protein (Hofmann et al., 1998). The best characterized HA cell surface receptors are CD44, RHAMM, ICAM-1, and LYVE-1 (Entwistle et al., 1996 Banerji et al., 1999), although there is some controversy as to whether ICAM-1 is a genuine HA receptor (McCourt and Gustafson, 1997).

In this work we searched for a ligand for layilin, a novel talin-binding protein localized in membrane ruffles and showed that layilin is a novel HA-binding cell surface receptor based on the following criteria: 1) layilin-Fc fusion protein binds to HA immobilized on Sepharose, and the bound material can be eluted with highly purified HA dodecasaccharides 2) HA, but not other tested GAGs, precipitates layilin in the presence of 1% CPC 3) layilin binds to HA immobilized in microtiter wells, and soluble HA binds to immobilized layilin 4) layilin-IgG stains HA on frozen tissue sections in a hyaluronidase-sensitive manner, whereas chondroitinase or heparitinase treatment of the sections did not affect the staining intensity 5) layilin-negative cells that do not bind to HA in adhesion assays become adherent after transfection with layilin. Thus, layilin is a member of the family of HA-binding proteins and can serve as an HA-binding cell adhesion receptor.

Layilin does not have obvious sequence identity with previously cloned HA receptors and does not contain a link module or a Bx7B motif (an α-helical sequence with clusters of basic amino acids), which is the HA-binding sequence in RHAMM (Yanget al., 1994), another HA receptor without a link domain. However, the extracellular domain of layilin is homologous with C-type lectins. The structural homology between the link domain and C-type lectins probably accounts for layilin's ability to bind HA (Brissett and Perkins, 1996 Kohda et al., 1996).

Although layilin and CD44, a known HA receptor, do not share any sequence homology, there is an interesting parallel between layilin and CD44. They both bind to HA via their extracellular part (link domain in CD44, lectin in layilin), they both can bind to molecules of the ERM superfamily with their intracellular parts because CD44 has been reported to bind ezrin and merlin (Sainio et al., 1997Heiska et al., 1998 Yonemura et al., 1998), and layilin can bind talin and radixin (Borowsky and Hynes, 1998 Cordero, unpublished data). For CD44, the ERM-binding site has been mapped to a positively charged 19-amino acid cluster in the juxtamembrane region of the cytoplasmic domain (Yonemura et al., 1998). However, layilin does not have an obvious positively charged amino acid cluster next to the membrane-spanning region, and we have previously reported that the shortest talin-binding motif is in the C terminus of layilin's cytoplasmic domain (Borowsky and Hynes, 1998). However, similar extracellular and intracellular binding partners suggest possible shared functions between these molecules, and therefore, it will be of interest to investigate whether layilin also has a role in the processes of leukocyte migration to inflamed sites, cell adhesion and migration, and tumor metastasis (Gunthert et al., 1991 Stamenkovic et al., 1991 DeGrendeleet al., 1996, 1997), all processes in which CD44 is known to play a role.

CD44 is known to have several isoforms due to alternative splicing (Haynes et al., 1991), although not all CD44 variants can bind HA and/or mediate lymphocyte homing (Berg et al., 1989Stamenkovic et al., 1991). Although layilin genomic clones have not yet been analyzed, it will be of interest to study whether there also exist different layilin isoforms. Based on a Northern blot analysis of CHO cell RNA (Borowsky and Hynes, 1998), there is so far no evidence for multiple layilin forms, although it should be remembered that there may be species-specific differences.

Layilin's binding to HA is not remarkably strong the affinity of layilin for HA is on the order of 10 −7 M. Based on the similarities in the binding curves (Figure 4) layilin's affinity for HA appears similar to that of CD44. In comparison with other cell adhesion mechanisms such as those involving cadherins or integrins, the detected layilin affinity for HA is fairly weak. However, there are situations in which such weak binding may be an advantage. For example, in transient interactions, strong binding can be a disadvantage, and it is tempting to speculate that layilin's function is to mediate early cell-matrix interactions followed by more stable binding mediated, for example, by integrins.

Layilin's binding to HA may be affected by several factors including layilin's interactions with the cytoskeleton. The cytoskeleton may indirectly control layilin's binding to HA, for example by controlling the distribution and clustering of layilin on the cell surface. This could lead to possible multiple and higher affinity interactions between layilin and HA, which may increase the binding avidity over that of a monovalent interaction. Such multivalent interactions, in which several molecules of CD44 bind to the same HA molecule, have been reported (Underhill and Toole, 1980 Underhill, 1992), and the importance of proper cytoskeletal connection for CD44 binding to HA has also been suggested by experiments in which cells transfected with CD44 with a truncated cytoplasmic domain bind soluble HA less well than do cells transfected with intact CD44 (Lesley et al., 1992). Hence, it will be of interest to study whether cytoskeleton can regulate layilin's distribution on the cell surface and in this way regulate layilin's binding to HA.

In conclusion, we have identified layilin as a novel receptor for HA. Layilin binds specifically to HA but not to heparin or chondroitin sulfate under the binding conditions used in this study. Layilin's binding to this ECM component suggests a role for layilin in processes in which HA is known to be involved, including cell adhesion and motility. The identification of HA as a ligand for layilin defines this talin-binding partner as a novel member of the diverse family of HA-binding proteins and should facilitate attempts to find the true biological role for layilin.