P

Binding of hormone induces a conformational change in receptor

Activated receptor binds to Ga subunit

Ga U

GT P

Binding induces conformational change in Ga; bound GDP dissociates and is replaced by GTP; Ga dissociates from Gpy

Binding induces conformational change in Ga; bound GDP dissociates and is replaced by GTP; Ga dissociates from Gpy

Hormone dissociates from receptor; Ga binds to effector, activating it

Hormone dissociates from receptor; Ga binds to effector, activating it

5| Hydrolysis of GTP to GDP causes G from effector and reassociate with G

to dissociate

▲ EXPERIMENTAL FIGURE 13-12 Receptor-mediated activation of coupled G proteins occurs within a few seconds of ligand binding in living cells. The amoeba Dictyostelium discoideum was transfected with genes encoding two fusion proteins: a Ga fused to cyan fluorescent protein (CFP), a mutant form of green fluorescent protein (GFP), and a Gp fused to another GFP variant, yellow fluorescent protein (YFP). CFP normally fluoresces 490-nm light; YFP 527-nm light. (a) When CFP and YFP are nearby, as in the resting G„ Gp7 complex, fluorescence energy transfer can occur between CFP and YFP (left). As a result, irradiation of resting cells with 440-nm light (which directly excites CFP but not YFP) causes emission of 527-nm (yellow) light, char acteristic of YFP However, if ligand binding leads to dissociation of the Ga and Gp7 subunits, then fluorescence energy transfer cannot occur. In this case, irradiation of cells at 440 nm causes emission of 490-nm light (cyan) characteristic of CFP (right). (b) Plot of the emission of yellow light (527 nm) from a single transfected amoeba cell before and after addition of cyclic AMP (arrows), the extracellular ligand for the GPCR in these cells. The drop in fluorescence, which results from the dissociation of the Ga-CFP fusion protein from the Gp7-YFP fusion protein, occurs within seconds of cAMP addition. [Adapted from C. Janetopoulos et al., 2001, Science 291:2408.]

nist (e.g., isoproterenol) to the receptor changes its conformation, causing it to bind to the Ga subunit in such a way that GDP is displaced from Ga and GTP becomes bound. Thus the activated ligand-bound receptor functions as a GEF for the Ga subunit (see Figure 3-29).

Once the exchange of nucleotides has occurred, the Ga-GTP complex dissociates from the Gpy subunit, but both remain anchored in the membrane. In most cases, Ga-GTP then interacts with and activates an associated effector protein, as depicted in Figure 13-11. This activation is shortlived, however, because GTP bound to Ga is hydrolyzed to GDP in seconds, catalyzed by a GTPase enzyme that is an intrinsic part of the Ga subunit. The resulting Ga-GDP quickly reassociates with Gpy, thus terminating effector activation. In many cases, a protein termed RGS (regulator of G protein signaling) accelerates GTP hydrolysis by the Ga subunit, reducing the time during which the effector remains activated.

Early evidence supporting the model shown in Figure 13-11 came from studies with compounds that can bind to Ga subunits as well as GTP does, but cannot be hydrolyzed by the intrinsic GTPase. In these compounds the P-O-P phosphodiester linkage connecting the p and y phosphates of GTP is replaced by a nonhydrolyzable P-CH2-P or P-NH-P linkage. Addition of such a GTP analog to a plasmamembrane preparation in the presence of the natural ligand or an agonist for a particular receptor results in a much longer-lived activation of the associated effector protein than occurs with GTP. That is because once the GDP bound to Ga is displaced by the nonhydrolyzable GTP analog, it remains permanently bound to Ga. Because this complex is as functional as the normal Ga-GTP complex in activating the effector protein, the effector remains permanently active.

The GPCR-mediated dissociation of trimeric G proteins recently has been detected in living cells. These studies have exploited the phenomenon of fluorescence energy transfer, which can change the wavelength of emitted fluorescence when two fluorescent proteins interact. Figure 13-12 shows how this experimental approach has demonstrated the dissociation of the Ga-Gp7 complex within a few seconds of ligand addition, providing further evidence for the model of G protein cycling. This general experimental protocol can be used to follow the formation and dissociation of other protein-protein complexes in living cells.

Epinephrine Binds to Several Different G Protein-Coupled Receptors

Epinephrine is particularly important in mediating the body's response to stress, such as fright or heavy exercise, when all tissues have an increased need to catabolize glucose and fatty acids to produce ATP. These principal metabolic fuels can be supplied to the blood in seconds by the rapid breakdown of glycogen to glucose in the liver (glycogenofysis) and of tri-acylglycerols to fatty acids in adipose cells (lipolysis).

In mammals, the liberation of glucose and fatty acids can be triggered by binding of epinephrine (or norepinephrine) to p-adrenergic receptors on the surface of hepatic (liver) and adipose cells. Epinephrine bound to ^-adrenergic receptors on heart muscle cells increases the contraction rate, which increases the blood supply to the tissues. In contrast, epinephrine stimulation of ^-adrenergic receptors on smooth muscle cells of the intestine causes them to relax. Another type of epinephrine receptor, the a ¿-adrenergic receptor, is found on smooth muscle cells lining the blood vessels in the intestinal tract, skin, and kidneys. Binding of epinephrine to these receptors causes the arteries to constrict, cutting off circulation to these peripheral organs. These diverse effects of epinephrine are directed to a common end: supplying energy for the rapid movement of major locomotor muscles in response to bodily stress.

Although all epinephrine receptors are G proteincoupled receptors, the different types are coupled to different G proteins. Thus in addition to their physiological importance, these receptors are of interest because they trigger different in-tracellular signal-transduction pathways. Both subtypes of p-adrenergic receptors, termed p and p2, are coupled to a stimulatory G protein (Gs) that activates the membrane-bound enzyme adenylyl cyclase (see Table 13-1). Once activated, adenylyl cyclase catalyzes synthesis of the second messenger cAMP. That binding of epinephrine to ^-adrenergic receptors induces a rise in cAMP has been demonstrated in functional expression assays like that depicted in Figure 13-6. When cloned cDNA encoding the ^-adrenergic receptor is transfected into receptor-negative cells, the transfected cells accumulate cAMP in response to epinephrine stimulation. Similar experiments in which mutant receptors are expressed have helped to define the functions of specific amino acids in binding hormones and activating different G proteins.

The two subtypes of a-adrenergic receptors, a1 and a2, are coupled to different G proteins. The ^-adrenergic receptor is coupled to a G( protein that inhibits adenylyl cyclase, the same effector enzyme associated with ^-adrenergic receptors. In contrast, the Gq protein coupled to the a2-adrenergic receptor activates a different effector enzyme that generates different second messengers (see Section 13.5).

Some bacterial toxins contain a subunit that penetrates the plasma membrane of cells and catalyzes a chemical modification of Gsa-GTP that prevents hydrolysis of bound GTP to GDP. As a result, Gsa remains in the active state, continuously activating adenylyl cyclase in the absence of hormonal stimulation. Cholera toxin produced by the bacterium Vibrio cholera and enterotoxins produced by certain strains of E. coli act in this way on intestinal epithelial cells. The resulting excessive rise in intracellular cAMP leads to the loss of electrolytes and water into the intestinal lumen, producing the watery diarrhea characteristic of infection by these bacteria.

Bordetella pertussis, a bacterium that commonly infects the respiratory tract, is the cause of whooping cough. Pertussis toxin catalyzes a modification of Gia that prevents re lease of bound GDP, thus locking Gia in the inactive state. This inactivation of Gi leads to an increase in cAMP in epithelial cells of the airways, promoting loss of fluids and electrolytes and mucus secretion. I

Critical Functional Domains in Receptors and Coupled G Proteins Have Been Identified

As noted already, all G protein-coupled receptors contain seven transmembrane a helices and presumably have a similar three-dimensional structure. Studies with chimeric adrenergic receptors, like those outlined in Figure 13-13, suggest that the long C3 loop between a helices 5 and 6 is important for interactions between a receptor and its coupled G protein. Presumably, lig-and binding causes these helices to move relative to each other. As a result, the conformation of the C3 loop connecting these two helices changes in a way that allows the loop to bind and

Exterior I-NN3+

Cytosol roo-

a2-Adrenergic receptor (wild type)

NN3+

NN3+

roo-

^-Adrenergic receptor (wild type)

Chimeric receptor 1

roo-

Chimeric receptor 1

Chimeric receptor 2

roo-

Chimeric receptor 2

Effect on adenylyl cyclase

Inhibits (binds Gi)

Activates (binds Gs)

Activates (binds Gs)

Inhibits (binds Gi)

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