Tracing the evolution of the heterotrimeric G protein α subunit in Metazoa.

BACKGROUND: Heterotrimeric G proteins are fundamental signaling proteins composed of three subunits, Gα and a Gßγ dimer. The role of Gα as a molecular switch is critical for transmitting and amplifying intracellular signaling cascades initiated by an activated G protein Coupled Receptor (GPCR). Despite their biochemical and therapeutic importance, the study of G protein evolution has been limited to the scope of a few model organisms. Furthermore, of the five primary Gα subfamilies, the underlying gene structure of only two families has been thoroughly investigated outside of Mammalia evolution. Therefore our understanding of Gα emergence and evolution across phylogeny remains incomplete. RESULTS: We have computationally identified the presence and absence of every Gα gene (GNA-) across all major branches of Deuterostomia and evaluated the conservation of the underlying exon-intron structures across these phylogenetic groups. We provide evidence of mutually exclusive exon inclusion through alternative splicing in specific lineages. Variations of splice site conservation and isoforms were found for several paralogs which coincide with conserved, putative motifs of DNA-/RNA-binding proteins. In addition to our curated gene annotations, within Primates, we identified 15 retrotranspositions, many of which have undergone pseudogenization. Most importantly, we find numerous deviations from previous findings regarding the presence and absence of individual GNA- genes, nuanced differences in phyla-specific gene copy numbers, novel paralog duplications and subsequent intron gain and loss events. CONCLUSIONS: Our curated annotations allow us to draw more accurate inferences regarding the emergence of all Gα family members across Metazoa and to present a new, updated theory of Gα evolution. Leveraging this, our results are critical for gaining new insights into the co-evolution of the Gα subunit and its many protein binding partners, especially therapeutically relevant G protein - GPCR signaling pathways which radiated in Vertebrata evolution.

Dynamics of mouse rod phototransduction and its sensitivity to variation of key parameters.

The deep understanding of the biochemical and biophysical basis of visual transduction, makes it ideal for systems-level analysis. A sensitivity analysis is presented for a self-consistent set of parameters involved in mouse phototransduction. The organising framework is a spatio-temporal mathematical model, which includes the geometry of the rod outer segment (ROS), the layered array of the discs, the incisures, the biochemistry of the activation/deactivation cascade and the biophysics of the diffusion of the second messengers in the cytoplasm and the closing of the cyclic guanosine monophosphate (cGMP) gated cationic channels. These modules include essentially all the relevant geometrical, biochemical and biophysical parameters. The parameters are selected from within experimental ranges, to obey basic first principles such as conservation of mass and energy fluxes. By means of the model they are compared to a large set of experimental data, providing a strikingly close match. Following isomerisation of a single rhodopsin R * (single photon response), the sensitivity analysis was carried out on the photo-response, measured both in terms of number of effector molecules produced, and photocurrent suppression, at peak time and the activation and recovery phases of the cascade. The current suppression is found to be very sensitive to variations of the catalytic activities, Hill's coefficients and hydrolysis rates and the geometry of the ROS, including size and shape of the incisures. The activated effector phosphodiesterase (PDE *) is very sensitive to variations of catalytic activity of G-protein activation and the average lifetimes of activated rhodopsin R * and PDE *; however, they are insensitive to geometry and variations of the transduction parameters. Thus the system is separated into two functional modules, activation/deactivation and transduction, each confined in different geometrical domains, communicating through the hydrolysis of cGMP by PDE *, and each sensitive to variations of parameters only in its own module.

Direct modulation of phospholipase D activity by Gbetagamma.

Phospholipase D-mediated hydrolysis of phosphatidylcholine is stimulated by protein kinase C and the monomeric G proteins Arf, RhoA, Cdc42, and Rac1, resulting in complex regulation of this enzyme. Using purified proteins, we have identified a novel inhibitor of phospholipase D activity, Gbetagamma subunits of heterotrimeric G proteins. G protein-coupled receptor activation alters affinity between Galpha and Gbetagamma subunits, allowing subsequent interaction with distinct effectors. Gbeta1gamma1 inhibited phospholipase D1 and phospholipase D2 activity, and both Gbeta1gamma1 and Gbeta1gamma2 inhibited stimulated phospholipase D1 activity in a dosedependent manner in reconstitution assays. Reconstitution assays suggest this interaction occurs through the amino terminus of phospholipase D, because Gbeta1gamma1 is unable to inhibit an amino-terminally truncated phospholipase D construct, PLD1.d311, which like full-length phospholipase D isoforms, requires phosphatidylinositol-4,5-bisphosphate for activity. Furthermore, a truncated protein consisting of the amino-terminal region of phospholipase D containing the phox/pleckstrin homology domains was found to interact with Gbeta1gamma1, unlike the PLD1.d311 recombinant protein, which lacks this domain. In vivo, expressed recombinant Gbeta1gamma2 was also found to inhibit phospholipase D activity under basal and stimulated conditions in MDA-MB-231 cells, which natively express both phospholipase D1 and phospholipase D2. These data demonstrate that Gbetagamma directly regulates phospholipase D activity in vitro and suggest a novel mechanism to negatively regulate phospholipase D signaling in vivo.

Mathematical and computational modelling of spatio-temporal signalling in rod phototransduction.

Rod photoreceptors are activated by light through activation of a cascade that includes the G protein-coupled receptor rhodopsin, the G protein transducin, its effector cyclic guanosine monophosphate (cGMP) phosphodiesterase and the second messengers cGMP and Ca2+. Signalling is localised to the particular rod outer segment disc, which is activated by absorption of a single photon. Modelling of this cascade has previously been performed mostly by assumption of a well-stirred cytoplasm. We recently published the first fully spatially resolved model that captures the local nature of light activation. The model reduces the complex geometry of the cell to a simpler one using the mathematical theories of homogenisation and concentrated capacity. The model shows that, upon activation of a single rhodopsin, changes of the second messengers cGMP and Ca2+ are local about the particular activated disc. In the current work, the homogenised model is computationally compared with the full, non-homogenised one, set in the original geometry of the rod outer segment. It is found to have an accuracy of 0.03% compared with the full model in computing the integral response and a 5200-fold reduction in computation time. The model can reconstruct the radial time-profiles of cGMP and Ca2+ in the interdiscal spaces adjacent to the activated discs. Cellular electrical responses are localised near the activation sites, and multiple photons sufficiently far apart produce essentially independent responses. This leads to a computational analysis of the notion and estimate of 'spread' and the optimum distribution of activated sites that maximises the response. Biological insights arising from the spatio-temporal model include a quantification of how variability in the response to dim light is affected by the distance between the outer segment discs capturing photons. The model is thus a simulation tool for biologists to predict the effect of various factors influencing the timing, spread and control mechanisms of this G protein-coupled, receptor-mediated cascade. It permits ease of simulation experiments across a range of conditions, for example, clamping the concentration of calcium, with results matching analogous experimental results. In addition, the model accommodates differing geometries of rod outer segments from different vertebrate species. Thus it represents a building block towards a predictive model of visual transduction.

Mathematical model of the spatio-temporal dynamics of second messengers in visual transduction.

A model describing the role of transversal and longitudinal diffusion of cGMP and Ca(2+) in signaling in the rod outer segment of vertebrates is developed. Utilizing a novel notion of surface-volume reaction and the mathematical theories of homogenization and concentrated capacity, the diffusion of cGMP and Ca(2+) in the inter-disc spaces is shown to be reducible to a one-parameter family of diffusion processes taking place on a single rod cross section; whereas the diffusion in the outer shell is shown to be reducible to a diffusion on a cylindrical surface. Moreover, the exterior flux of the former serves as a source term for the latter, alleviating the assumption of a well-stirred cytosol. A previous model of visual transduction that assumes a well-stirred rod outer segment cytosol (and thus contains no spatial information) can be recovered from this model by imposing a "bulk" assumption. The model shows that upon activation of a single rhodopsin, cGMP changes are local, and exhibit both a longitudinal and a transversal component. Consequently, membrane current is also highly localized. The spatial spread of the single photon response along the longitudinal axis of the outer segment is predicted to be 3-5 microm, consistent with experimental data. This approach represents a tool to analyze point-wise signaling dynamics without requiring averaging over the entire cell by global Michaelis-Menten kinetics.

An intramolecular contact in Galpha transducin that participates in maintaining its intrinsic GDP release rate.

Receptor mediated stimulation of the G protein-alpha subunit leads to exchange of GDP for GTP, activating the protein. Spontaneous GDP release from Galpha can also lead to the active state, if GTP in solution binds the nucleotide binding pocket. The purpose of this study is to evaluate the molecular determinants for maintaining the spontaneous GDP release rates between two Galpha subunits. Galpha(t) has a low rate of nucleotide release, compared to Galpha(i1). Galpha(t/i1) chimeras were used to explore the molecular basis for this behavior. The C-terminal alpha4-helix, the N-terminal 56 residues and the Switch I/II regions of Galpha(t) were shown to affect the low spontaneous GDP release rate in Galpha(t). A specific molecular contact between Asp26 and Asn191 was found in Galpha(t) that is not present in Galpha(i1). In two chimeras disrupting this interaction produced an increased spontaneous GDP release; restoring the contact present in Galpha(t) into these chimeras decreased the GDP release rate by half as compared to the original chimeras. Similarly, introduction of this contact in wild-type Galpha(i1) decreased the GDP release rate of Galpha(i1) by half. Differences in GDP release rates may reflect physiological roles these proteins play in living systems.

Modulation of the G protein regulator phosducin by Ca2+/calmodulin-dependent protein kinase II phosphorylation and 14-3-3 protein binding.

Phototransduction is a canonical G protein-mediated cascade of retinal photoreceptor cells that transforms photons into neural responses. Phosducin (Pd) is a Gbetagamma-binding protein that is highly expressed in photoreceptors. Pd is phosphorylated in dark-adapted retina and is dephosphorylated in response to light. Dephosphorylated Pd binds Gbetagamma with high affinity and inhibits the interaction of Gbetagamma with Galpha or other effectors, whereas phosphorylated Pd does not. These results have led to the hypothesis that Pd down-regulates the light response. Consequently, it is important to understand the mechanisms of regulation of Pd phosphorylation. We have previously shown that phosphorylation of Pd by cAMP-dependent protein kinase moderately inhibits its association with Gbetagamma. In this study, we report that Pd was rapidly phosphorylated by Ca(2+)/calmodulin-dependent kinase II, resulting in 100-fold greater inhibition of Gbetagamma binding than cAMP-dependent protein kinase phosphorylation. Furthermore, Pd phosphorylation by Ca(2+)/calmodulin-dependent kinase II at Ser-54 and Ser-73 led to binding of the phosphoserine-binding protein 14-3-3. Importantly, in vivo decreases in Ca(2+) concentration blocked the interaction of Pd with 14-3-3, indicating that Ca(2+) controls the phosphorylation state of Ser-54 and Ser-73 in vivo. These results are consistent with a role for Pd in Ca(2+)-dependent light adaptation processes in photoreceptor cells and also suggest other possible physiological functions.

Effect of Li+ upon the Mg2+-dependent activation of recombinant Gialpha1.

Although lithium salts have been used in the treatment and prophylaxis of manic-depressive or bipolar patients for 50 years, the mechanism of the pharmacologic action of Li+ is unknown. Based on activity studies of inhibitory and stimulatory guanine-binding (G) proteins in rat cortical membranes, it was proposed that Li+ inhibition of G-proteins may account for its pharmacologic action. We used the purified alpha subunit of the recombinant inhibitory G-protein, rGialpha1, and found that Li+ at therapeutic levels significantly inhibited the formation of the GDP.AlF4-.rGialpha1 complex. Because our studies were conducted with a purified, metal-reconstituted G-protein rather than with cell membrane suspensions, our Li+ inhibition results lend additional support to the G-protein hypothesis for Li+ action.

G protein betagamma subunit-mediated presynaptic inhibition: regulation of exocytotic fusion downstream of Ca2+ entry.

The nervous system can modulate neurotransmitter release by neurotransmitter activation of heterotrimeric GTP-binding protein (G protein)-coupled receptors. We found that microinjection of G protein betagamma subunits (Gbetagamma) mimics serotonin's inhibitory effect on neurotransmission. Release of free Gbetagamma was critical for this effect because a Gbetagamma scavenger blocked serotonin's effect. Gbetagamma had no effect on fast, action potential-evoked intracellular Ca2+ release that triggered neurotransmission. Inhibition of neurotransmitter release by serotonin was still seen after blockade of all classical Gbetagamma effector pathways. Thus, Gbetagamma blocked neurotransmitter release downstream of Ca2+ entry and may directly target the exocytotic fusion machinery at the presynaptic terminal.

G alpha minigenes expressing C-terminal peptides serve as specific inhibitors of thrombin-mediated endothelial activation.

The C termini of G protein alpha subunits are critical for binding to their cognate receptors, and peptides corresponding to the C terminus can serve as competitive inhibitors of G protein-coupled receptor-G protein interactions. This interface is quite specific as a single amino acid difference annuls the ability of a G alpha(i) peptide to bind the A(1) adenosine receptor (Gilchrist, A., Mazzoni, M., Dineen, B., Dice, A., Linden, J., Dunwiddie, T., and Hamm, H. E. (1998 ) J. Biol. Chem. 273, 14912--14919). Recently, we demonstrated that a plasmid minigene vector encoding the C-terminal sequence of G alpha(i) could specifically inhibit downstream responses to agonist stimulation of the muscarinic M(2) receptor (Gilchrist, A., Bunemann, M., Li, A., Hosey, M. M., and H. E. Hamm (1999) J. Biol. Chem. 274, 6610--6616). To selectively antagonize G protein signal transduction events and determine which G protein underlies a given thrombin-induced response, we generated minigene vectors that encode the C-terminal sequence for each family of G alpha subunits. Minigene vectors expressing G alpha C-terminal peptides (G alpha(i), G alpha(q), G alpha(12), and G alpha(13)) or the control minigene vector, which expresses the G alpha(i) peptide in random order (G(iR)), were systematically introduced into a human microvascular endothelial cell line. The C-terminal peptides serve as competitive inhibitors presumably by blocking the site on the G protein-coupled receptor that normally binds the G protein. Our results not only confirm that each G protein can control certain signaling events, they emphasize the specificity of the G protein-coupled receptor-G protein interface. In addition, the C-terminal G alpha minigenes appear to be a powerful tool for dissecting out the G protein that mediates a given physiological function following thrombin activation.

Structural requirements for the stabilization of metarhodopsin II by the C terminus of the alpha subunit of transducin.

The retinal receptor rhodopsin undergoes a conformational change upon light excitation to form metarhodopsin II (Meta II), which allows interaction and activation of its cognate G protein, transducin (G(t)). A C-terminal 11-amino acid peptide from transducin, G(talpha)-(340-350), has been shown to both bind and stabilize the Meta II conformation, mimicking heterotrimeric G(t). Using a combinatorial library we identified analogs of G(talpha)-(340-350) that bound light-activated rhodopsin with high affinity (Martin, E. L., Rens-Domiano, S., Schatz, P. J., and Hamm, H. E. (1996) J. Biol. Chem. 271, 361-366). We have made peptides with key substitutions either on the background of the native G(talpha)-(340-350) sequence or on the high affinity sequences and used the stabilization of Meta II as a tool to determine which amino acids are critical in G protein-rhodopsin interaction. Removal of the positive charge at the N termini by acylation or delocalization of the charge by K to R substitution enhances the affinity of the G(talpha)-(340-350) peptides for Meta II, whereas a decrease was observed following C-terminal amidation. Cys-347, a residue conserved in pertussis toxin-sensitive G proteins, was shown to interact with a hydrophobic site in Meta II. These studies provide further insight into the mechanism of interaction between the G(talpha) C terminus and light-activated rhodopsin.

A novel bifunctional phospholipase c that is regulated by Galpha 12 and stimulates the Ras/mitogen-activated protein kinase pathway.

Three families of phospholipase C (PI-PLCbeta, gamma, and delta) are known to catalyze the hydrolysis of polyphosphoinositides such as phosphatidylinositol 4,5-bisphosphate (PIP(2)) to generate the second messengers inositol 1,4,5 trisphosphate and diacylglycerol, leading to a cascade of intracellular responses that result in cell growth, cell differentiation, and gene expression. Here we describe the founding member of a novel, structurally distinct fourth family of PI-PLC. PLCepsilon not only contains conserved catalytic (X and Y) and regulatory domains (C2) common to other eukaryotic PLCs, but also contains two Ras-associating (RA) domains and a Ras guanine nucleotide exchange factor (RasGEF) motif. PLCepsilon hydrolyzes PIP(2), and this activity is stimulated selectively by a constitutively active form of the heterotrimeric G protein Galpha(12). PLCepsilon and a mutant (H1144L) incapable of hydrolyzing phosphoinositides promote formation of GTP-Ras. Thus PLCepsilon is a RasGEF. PLCepsilon, the mutant H1144L, and the isolated GEF domain activate the mitogen-activated protein kinase pathway in a manner dependent on Ras but independent of PIP(2) hydrolysis. Our findings demonstrate that PLCepsilon is a novel bifunctional enzyme that is regulated by the heterotrimeric G protein Galpha(12) and activates the small G protein Ras/mitogen-activated protein kinase signaling pathway.

Endothelial cell-surface gp60 activates vesicle formation and trafficking via G(i)-coupled Src kinase signaling pathway.

We tested the hypothesis that the albumin-docking protein gp60, which is localized in caveolae, couples to the heterotrimeric GTP binding protein G(i), and thereby activates plasmalemmal vesicle formation and the directed migration of vesicles in endothelial cells (ECs). We used the water-soluble styryl pyridinium dye N-(3-triethylaminopropyl)-4-(p-dibutylaminostyryl) pyridinium dibromide (FM 1-43) to quantify vesicle trafficking by confocal and digital fluorescence microscopy. FM 1-43 and fluorescently labeled anti-gp60 antibody (Ab) were colocalized in endocytic vesicles within 5 min of gp60 activation. Vesicles migrated to the basolateral surface where they released FM 1-43, the fluid phase styryl probe. FM 1-43 fluorescence disappeared from the basolateral EC surface without the loss of anti-gp60 Ab fluorescence. Activation of cell-surface gp60 by cross-linking (using anti-gp60 Ab and secondary Ab) in EC grown on microporous filters increased transendothelial (125)I-albumin permeability without altering liquid permeability (hydraulic conductivity), thus, indicating the dissociation of hydraulic conductivity from the albumin permeability pathway. The findings that the sterol-binding agent, filipin, prevented gp60-activated vesicle formation and that caveolin-1 and gp60 were colocalized in vesicles suggest the caveolar origin of endocytic vesicles. Pertussis toxin pretreatment and expression of the dominant negative construct encoding an 11-amino acid G(alphai) carboxyl-terminal peptide inhibited endothelial (125)I-albumin endocytosis and vesicle formation induced by gp60 activation. Expression of dominant negative Src (dn-Src) and overexpression of wild-type caveolin-1 also prevented gp60-activated endocytosis. Caveolin-1 overexpression resulted in the sequestration of G(alphai) with the caveolin-1, whereas dn-Src inhibited G(alphai) binding to caveolin-1. Thus, vesicle formation induced by gp60 and migration of vesicles to the basolateral membrane requires the interaction of gp60 with caveolin-1, followed by the activation of the downstream G(i)-coupled Src kinase signaling pathway.

Functional roles of the two domains of phosducin and phosducin-like protein.

Phosducin and phosducin-like protein regulate G protein signaling pathways by binding the betagamma subunit complex (Gbetagamma) and blocking Gbetagamma association with Galpha subunits, effector enzymes, or membranes. Both proteins are composed of two structurally independent domains, each constituting approximately half of the molecule. We investigated the functional roles of the two domains of phosducin and phosducin-like protein in binding retinal G(t)betagamma. Kinetic measurements using surface plasmon resonance showed that: 1) phosducin bound G(t)betagamma with a 2. 5-fold greater affinity than phosducin-like protein; 2) phosphorylation of phosducin decreased its affinity by 3-fold, principally as a result of a decrease in k(1); and 3) most of the free energy of binding comes from the N-terminal domain with a lesser contribution from the C-terminal domain. In assays measuring the association of G(t)betagamma with G(t)alpha and light-activated rhodopsin, both N-terminal domains inhibited binding while neither of the C-terminal domains had any effect. In assays measuring membrane binding of G(t)betagamma, both the N- and C-terminal domains inhibited membrane association, but much less effectively than the full-length proteins. This inhibition could only be described by models that included a change in G(t)betagamma to a conformation that did not bind the membrane. These models yielded a free energy change of +1.5 +/- 0.25 kcal/mol for the transition from the G(t)alpha-binding to the Pd-binding conformation of G(t)betagamma.

A galpha(s) carboxyl-terminal peptide prevents G(s) activation by the A(2A) adenosine receptor.

The molecular mechanisms of interaction between G(s) and the A(2A) adenosine receptor were investigated using synthetic peptides corresponding to various segments of the Galpha(s) carboxyl terminus. Synthetic peptides were tested for their ability to modulate binding of a selective radiolabeled agonist, [(3)H]2-[4-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxam idoade nosine ([(3)H]CGS21680), to A(2A) adenosine receptors in rat striatal membranes. The Galpha(s) peptides stimulated specific binding both in the presence and absence of 100 microM guanosine-5'-O-(3-thiotriphosphate) (GTPgammaS). Three peptides, Galpha(s)(378-394)C(379)A, Galpha(s)(376-394)C(379)A, and Galpha(s)(374-394)C(379)A, were the most effective. In the presence of GTPgammaS, peptide Galpha(s)(374-394)C(379)A increased specific binding in a dose-dependent fashion. However, the peptide did not stabilize the high-affinity state of the A(2A) adenosine receptor for [(3)H]CGS21680. Binding assays with a radiolabeled selective antagonist, [(3)H]5-amino-7-(2-phenylethyl)-2-(2-furyl)pyrazolo[4, 3-e]-1,2,4-triazolo[1,5-c]pyrimidine ([(3)H]SCH58261), showed that the addition of the Galpha(s) peptide modified the slope of the 5'-N-ethylcarboxamidoadenosine (NECA) competition curve, suggesting modulation of receptor affinity states. In the presence of GTPgammaS, the displacement curve was right-shifted, whereas the addition of Galpha(s)(374-394)C(379)A caused a partial left-shift. Both curves were fitted by one-site models. This same Galpha(s) peptide was also able to disrupt G(s)-coupled signal transduction as indicated by inhibition of the A(2A) receptor-stimulated adenylyl cyclase activity without affecting either basal or forskolin-stimulated enzymatic activity in the same membrane preparations. Shorter peptides from Galpha(s) and Galpha(i1/2) carboxyl termini were not effective. NMR spectroscopy showed the strong propensity of peptide Galpha(s)(374-394)C(379)A to assume a compact carboxyl-terminal alpha-helical conformation in solution. Overall, our results point out the conformation requirement of Galpha(s) carboxyl-terminal peptides to modulate agonist binding to rat A(2A) adenosine receptors and disrupt signal transduction.

Two amino acids within the alpha4 helix of Galphai1 mediate coupling with 5-hydroxytryptamine1B receptors.

We previously reported that residues 299-318 in Galphai1 participate in the selective interaction between Galphai1 and the 5-hydroxytryptamine1B (5-HT1B) receptor (Bae, H., Anderson, K., Flood, L. A., Skiba, N. P., Hamm, H. E., and Graber, S. G. (1997) J. Biol. Chem. 272, 32071-32077). The present study more precisely defines which residues within this domain are critical for 5-HT1B receptor-mediated G protein activation. A series of Galphai1/Galphat chimeras and point mutations were reconstituted with Gbetagamma and Sf9 cell membranes containing the 5-HT1B receptor. Functional coupling to 5-HT1B receptors was assessed by 1) [35S]GTPgammaS binding and 2) agonist affinity shift assays. Replacement of the alpha4 helix of Galphai1 (residues 299-308) with the corresponding sequence from Galphat produced a chimera (Chi22) that only weakly coupled to the 5-HT1B receptor. In contrast, substitution of residues within the alpha4-beta6 loop region of Galphai1 (residues 309-318) with the corresponding sequence in Galphat either permitted full 5-HT1B receptor coupling to the chimera (Chi24) or only minimally reduced coupling to the chimeric protein (Chi25). Two mutations within the alpha4 helix of Galphai1 (Q304K and E308L) reduced agonist-stimulated [35S]GTPgammaS binding, and the effects of these mutations were additive. The opposite substitutions within Chi22 (K300Q and L304E) restored 5-HT1B receptor coupling, and again the effects of the two mutations were additive. Mutations of other residues within the alpha4 helix of Galphai1 had minimal to no effect on 5-HT1B coupling behavior. These data provide evidence that alpha4 helix residues in Galphai participate in directing specific receptor interactions and suggest that Gln304 and Glu308 of Galphai1 act in concert to mediate the ability of the 5-HT1B receptor to couple specifically to inhibitory G proteins.