The GProtein Coupled Receptor Superfamily

The most frequently encountered cell surface receptors belong to a very large superfamily of proteins that couple with guanosine nucleotide binding proteins (G-proteins) to communicate with intracellular effector molecules. This ancient superfamily is widely expressed throughout eukaryotic phyla. G-protein-coupled receptors are crucial for sensing signals in the external environment, such as light, taste, and odor, as well as internal signals in the form of hormones, neurotrans-mitters, immune modulators, and paracrine factors (Table 2). So pervasive are the responses mediated by these receptors in this superfamily that about 30% of all effective pharmaceutical agents target actions mediated by them. Considerably more than 1000 different G-protein-coupled receptors are expressed in humans, with perhaps as many as 1000 expressed in the olfactory epithelium alone (see Chapter 54).

All G-protein-coupled receptors contain seven membrane-spanning a-helices comprised of about 25 amino acids each (Fig. 9). The single long peptide chain that constitutes the receptor threads back and forth through the membrane seven times, creating three extracellular and three intracellular loops. For this reason, these receptors are sometimes called heptahelical receptors, or serpentine receptors. The amino terminal tail is extracellular and along with the external loops may contain covalently bound carbohydrate. Outward facing components of the receptor, including parts of the a-helices, contribute to the agonist recognition and binding site. They tend to be more extensive in receptors for larger proteins and may also form all or part of the binding pocket for such agonists. The intracellular domain consists of the carboxyl-terminal peptide and the internal loops that connect the transmembrane helices. The intracellular domain also contains one or

TABLE 2 Some Examples of Molecules That Signal Through G Protein-Coupled Receptors

Agent

Target cell

Major effect on cell

Calcium

Adenosine

Epinephrine

Angiotensin II

Acetylcholine

Interleukin-8

Thyroid-stimulating hormone

Glutamate

Prostaglandin E2

Somatostatin

Cholecystokinin

Vasopressin

Parathyroid chief cell Cardiac muscle Hepatocyte

Vascular smooth muscle Cardiac node cells Lymphocytes Thyroid follicle cell Hippocampal neurons Uterine smooth muscle Pituitary somatotrope Pancreatic acinar cell Renal tubular cell

Inhibits hormone secretion

Decreases contractility

Increases glucose production

Increases contraction

Slows heart rate

Increases cell migration

Increases hormone synthesis and secretion

Induces long-term potentiation

Increases contractility

Inhibits secretion

Stimulates enzyme secretion

Increases water permeability more surfaces that interact with the G-proteins. In addition, the intracellular portion may contain amino acid sequences required for receptor internalization by endocytosis and phosphorylation sites that regulate receptor function.

G-proteins are heterotrimers comprised of alpha, beta, and gamma subunits, which are products of different genes. Lipid moieties covalently attached to the alpha and gamma subunits insert into the inner leaflet of the plasma membrane bilayer and tether the G-proteins to the membrane (Fig. 10). The alpha subunits are GTPases, enzymes that catalyze the conversion of guanosine triphosphate (GTP) to guanosine diphosphate (GDP). In the unactivated or resting state, the catalytic site in the alpha subunit is occupied by GDP. When the receptor binds to its ligand, a conformational change transmitted across the membrane allows its cytosolic domain to interact with the G-protein in a way that causes the alpha subunit to release GDP in exchange for a molecule of GTP and to dissociate from the beta/gamma subunits, which remain tightly bound

Receptor

Receptor

G-Protein subunits

FIGURE 9 G-protein-coupled receptor. The seven transmembrane alpha helices are connected by three extracellular and three intracel-lular loops of varying length. The extracellular loops may be glycosylated, and the intracellular loops and C- terminal tail may be phosphorylated. The receptor is coupled to a G-protein consisting of a GDP-binding a subunit closely bound to a ßy component. The a and y subunits are tethered to the membrane by lipid groups.

G-Protein subunits

FIGURE 9 G-protein-coupled receptor. The seven transmembrane alpha helices are connected by three extracellular and three intracel-lular loops of varying length. The extracellular loops may be glycosylated, and the intracellular loops and C- terminal tail may be phosphorylated. The receptor is coupled to a G-protein consisting of a GDP-binding a subunit closely bound to a ßy component. The a and y subunits are tethered to the membrane by lipid groups.

to each other. Though tethered to the membrane, the dissociated subunits can diffuse laterally along the inner surface of the membrane. In its GTP-bound state, the alpha subunit interacts with and modifies the activity of membrane-associated enzymes that initiate the physiological response. The liberated beta/gamma complex can also bind to cellular proteins and modify enzyme activities or membrane permeability to ions.

Hydrolysis of GTP to GDP restores the resting state of the alpha subunit, allowing it to reassociate with the beta/gamma subunits to reconstitute the heterotrimer. Because the hydrolysis of GTP is relatively slow, the alpha subunit may interact multiple times with effector enzymes before it returns to its resting state. In addition, because some G-proteins may be as much as 100 times as abundant as the receptors they associate with, a single liganded receptor may interact sequentially with multiple G-proteins before it dissociates from the agonist. These characteristics provide mechanisms for amplification of the signal. That is, interaction of a single signal molecule with a single receptor molecule may result in multiple signal-generating events within a cell.

Desensitization and Downregulation of G-Protein-Coupled Receptors

In addition to simple dissociation of an agonist from its receptor, signaling is often terminated by active cellular processes that may also desensitize receptors to further stimulation. G-protein-coupled receptors may be inactivated by phosphorylation of serine or threonine residues in one of their intracellular loops catalyzed by a special G-protein receptor kinase. Such phosphorylation uncouples the receptor from the a subunit and promotes binding to a cytoplasmic protein of the ^-arrestin family. ^-Arrestin was originally described in photoreceptors, where rapid arrest of the

cGSE>

FIGURE 10 Activation of G-protein-coupled receptor. (I) Resting state. (II) Hormone binding produces a conformational change in the receptor that causes (III) the a subunit to exchange ADP for GTP, dissociate from the fly subunit, and interact with its effector molecule. The fly subunit also interacts with its effector molecule. (IV) The a subunit converts GTP to GDP which allows it to reassociate with the fly subunit, and the hormone dissociates from the receptor, restoring the resting state (I).

cGSE>

FIGURE 10 Activation of G-protein-coupled receptor. (I) Resting state. (II) Hormone binding produces a conformational change in the receptor that causes (III) the a subunit to exchange ADP for GTP, dissociate from the fly subunit, and interact with its effector molecule. The fly subunit also interacts with its effector molecule. (IV) The a subunit converts GTP to GDP which allows it to reassociate with the fly subunit, and the hormone dissociates from the receptor, restoring the resting state (I).

response to light is crucial for visual acuity. Similar proteins were subsequently found in other G-protein signaling systems where binding to fl-arrestin may lead to receptor internalization and downregulation by sequestration in intracellular vesicles. Sequestered receptors may recycle to the cell surface, or, when cellular stimulation is prolonged, they may be degraded in lysosomes.

G-Proteins and Signal Transduction

At least 20 different isoforms of Ga have been identified and can be categorized into four different classes according to similarities in their amino acid sequences. Each class includes the products of several closely related genes, and, in general, each class signals through characteristic transduction pathways. Alpha subunits of the "s" (stimulatory) class (Gas) stimulate the transmembrane enzyme, adenylyl cyclase, to catalyze the synthesis of cyclic 30,50 adenosine monophosphate (cyclic AMP, or cAMP) from ATP (Fig. 11). Alpha subunits belonging to the "q" class stimulate the activity of the membrane-bound enzyme phospholipase C-beta (PLC-fl) which catalyzes hydrolysis of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate to liberate inositol 1,4,5 trisphosphate (IP3) and diacylglycerol (DAG) (Fig. 12). Alpha sub-units of the "i" (inhibitory) class (Gai) inhibit the

FIGURE 11 Cyclic adenosine monophosphate (cAMP).

activity of adenylyl cyclase and may also activate other enzymes. Beta/gamma subunits released from their association with this class of Ga subunits may interact with membrane proteins that allow passage of potassium (potassium channels), and activate PLC-^. Alpha subunits of the 12/13 class are understood less well, but appear to activate pathways that lead to gene transcription. The differences in transduction pathways activated by the different classes of G-proteins are not absolute, and many points of crossover of these pathways have been observed.

The Second Messenger Concept

For a chemical signal that is received at the cell surface to be effective its information must be transmitted to the intracellular organelles and enzymes that produce the cellular response. To reach intracellular effectors, the G-protein-coupled receptors rely on intermediate molecules called second messengers, which are formed and/or released into the cytosol in response to agonist stimulation (the first message). Second messengers activate intracellular enzymes and also amplify signals. A single

Site of Cleavage by Phospholipase C

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