Gq Alpha Protein Research Articles

Mechanisms of action of emodepside

The research of the class of cyclic octadepsipeptides started at the beginning of the 1990s. PF1022A, the starting material of emodepside, is a natural secondary metabolite of the fungus Mycelia sterilia, which belongs to the microflora of the leaves of Camellia japonica. PF1022A consists of four N-methyl-L-leucins, two D-Iactic acids and two D-phenyllactic acids, which build up a cyclic octadepsipeptide with an alternating L-D-L-configuration. Emodepside is a semisynthetic derivative of PF1022A, which contains a morpholine attached in para position at each of both D-phenyllactic acids. Emodepside is efficacious against a variety of gastrointestinal nematodes. Emodepside binds to a presynaptic latrophilin receptor in nematodes. The following presynaptic signal transduction occurs via activation of Gqalpha protein and phospholipase-Cbeta, which leads to mobilization of diacylglycerol (DAG). DAG then activates UNC-13 and synaptobrevin, two proteins which play an important role in presynaptic vesicle-functioning. This finally leads to the release of a currently unidentified transmitter. The transmitter (or modulator) exerts its effects at the postsynaptic membrane and induces a flaccid paralysis of the pharynx and the somatic musculature in nematodes.

A point mutation uncouples transducin-alpha from the photoreceptor RGS and effector proteins.

A novel gain-of-function mutation, R243Q, has been recently identified in the Candida elegans Gqalpha protein EGL-30. The position corresponding to Arg243 in EGL-30 is absolutely conserved among heterotrimeric G proteins. This mutation appears to be the first gain-of-function mutation in the switch III region of Galpha subunits. To investigate consequences of the R-->Q mutation we introduced the corresponding R238Q mutation into transducin-like Gtalpha* subunit. The mutant retained intact interactions with Gtbetagamma and rhodopsin but exhibited a twofold reduction in the kcat value for guanosine 5'-triphosphate (GTP) hydrolysis. The GTPase activity of R238Q was not accelerated by the RGS domain of the visual GTPase-activating protein, RGS9-1. In addition, R238Q displayed a significant impairment in the effector function. Our data and the crystal structures of transducin suggest that the major reason for the reduced intrinsic GTPase activity of R238Q and the lack of RGS9 function is the break of the conserved ionic contact between Arg238 and Glu39, which apparently stabilizes the transitional state for GTP hydrolysis. We hypothesize that the R243Q mutation in EGL-30 severs the ionic interaction of Arg243 with Glu43, leading to a defective inactivation of the mutant by the C. elegans RGS protein EAT-16.

Effects of ACE inhibition and angiotensin II type 1 receptor blockade on cardiac function and G proteins in rats with chronic heart failure

1. Inhibition of the renin-angiotensin system (RAS) improves symptoms and prognosis in heart failure. The experimental basis for these benefits remains unclear. We examined the effects of inhibition of ACE or blockade of angiotensin II type 1 (AT1) receptor on the haemodynamics, cardiac G-proteins, and collagen synthesis of rats with coronary artery ligation (CAL), a model in which chronic heart failure (CHF) is induced. 2. Rats were orally treated with the ACE inhibitor trandolapril (3 mg kg(-1) day(-1)) or the AT1 receptor blocker L-158809 (1 mg kg(-1) day(-1)) from the 2nd to 8th week after CAL. CAL resulted in decreases in the left ventricular systolic pressure and its positive and negative dP/dt, an increase in the left ventricular end-diastolic pressure, and the rightward shift of the left ventricular pressure-volume curve. Long-term treatment with either drug improved these signs of CHF to a similar degree. 3. Cardiac Gsalpha and Gqalpha protein levels decreased, whereas the level of Gialpha protein increased in the animals with CHF. Long-term treatment with trandolapril or L-158809 attenuated the increase in the level of cardiac Gialpha protein of the animals with CHF without affecting Gsalpha and Gqalpha protein levels. Cardiac collagen content of the failing heart increased, whose increase was blocked by treatment with either drug. 4. Exogenous angiotensin I stimulated collagen synthesis in cultured cardiac fibroblasts, whose stimulation was attenuated by either drug. 5. These results suggest that blockade of the RAS, at either the receptor level or the synthetic enzyme level, may attenuate the cardiac fibrosis that occurs after CAL and thus affect the remodelling of the failing heart.

A regulator of G Protein signaling, RGS3, inhibits gonadotropin-releasing hormone (GnRH)-stimulated luteinizing hormone (LH) secretion

BackgroundLuteinizing hormone secreted by the anterior pituitary gland regulates gonadal function. Luteinizing hormone secretion is regulated both by alterations in gonadotrope responsiveness to hypothalamic gonadotropin releasing hormone and by alterations in gonadotropin releasing hormone secretion. The mechanisms that determine gonadotrope responsiveness are unknown but may involve regulators of G protein signaling (RGSs). These proteins act by antagonizing or abbreviating interaction of Gα proteins with effectors such as phospholipase Cβ. Previously, we reported that gonadotropin releasing hormone-stimulated second messenger inositol trisphosphate production was inhibited when RGS3 and gonadotropin releasing hormone receptor cDNAs were co-transfected into the COS cell line. Here, we present evidence for RGS3 inhibition of gonadotropin releasing hormone-induced luteinizing hormone secretion from cultured rat pituitary cells.ResultsA truncated version of RGS3 (RGS3T = RGS3 314–519) inhibited gonadotropin releasing hormone-stimulated inositol trisphosphate production more potently than did RSG3 in gonadotropin releasing hormone receptor-bearing COS cells. An RSG3/glutathione-S-transferase fusion protein bound more 35S-Gqα than any other member of the G protein family tested. Adenoviral-mediated RGS3 gene transfer in pituitary gonadotropes inhibited gonadotropin releasing hormone-stimulated luteinizing hormone secretion in a dose-related fashion. Adeno-RGS3 also inhibited gonadotropin releasing hormone stimulated 3H-inositol phosphate accumulation, consistent with a molecular site of action at the Gqα protein.ConclusionsRGS3 inhibits gonadotropin releasing hormone-stimulated second messenger production (inositol trisphosphate) as well as luteinizing hormone secretion from rat pituitary gonadotropes apparently by binding and suppressing the transduction properties of Gqα protein function. A version of RGS3 that is amino-terminally truncated is even more potent than intact RGS3 at inhibiting gonadotropin releasing hormone-stimulated inositol trisphosphate production.

Cholera toxin mediated regulation of the expression of Gq alpha and G11 alpha GTP binding proteins.

Previously it has been shown that persistent activation of the stimulatory adenylyl cyclase pathway with cholera toxin (CT) downregulates the Gs alpha polypeptide (80%) in a cAMP-independent manner in C6 glioma cells (Shah, 1997). This study was conducted to examine the short and long term effects of CT on the regulation of pertussis toxin-sensitive and -insensitive G proteins and their transcripts in C6 glioma cells. Treatment of C6 cells with CT (100 ng/ml) up to 16 h had no effect on either Gi or Gq/11 alpha proteins. However, prolonged exposure (24-48 h) caused increased expression of Gi (20-30%) and Gq/11 alpha proteins (40%). Urea gradient gels, which can separate Gq alpha and G11 alpha proteins, revealed that prolonged CT treatment increased the expression of both of these G proteins. The CT-mediated enhanced expression of Gq alpha and G11 alpha proteins was accompanied by increased mRNA levels of these proteins as determined by RT/PCR. Cyclic-AMP elevating agents like forskolin (10 microM) and db-cAMP (1 mM) mimicked the effect of CT on Gi but not Gq/11 alpha proteins. These studies show long term cAMP-dependent regulation of Gi and cAMP-independent expression of Gq/11 alpha proteins in C6 glioma cells.

Signaling Mechanisms Underlying Muscarinic Receptor-mediated Increase in Contraction Rate in Cultured Heart Cells

We have investigated the mechanisms by which stimulation of cardiac muscarinic receptors result in paradoxical stimulatory effects on cardiac function, using cultured neonatal rat ventricular myocytes as a model system. Application of low concentrations of carbachol (CCh) (EC50 = 35 nM) produced an atropine-sensitive decrease in spontaneous contraction rate, while, in cells pretreated with pertussis toxin, higher concentrations of CCh (EC50 = 26 microM) elicited an atropine-sensitive increase in contraction rate. Oxotremorine, an m2 muscarinic acetylcholine receptor (mAChR) agonist, mimicked the negative but not the positive chronotropic response to CCh. Reverse transcription followed by polymerase chain reaction carried out on mRNA obtained from single cells indicated that ventricular myocytes express mRNA for the m1, m2, and, possibly, m4 mAChRs. The presence of m1 and m2 mAChR protein on the surface membranes of the cultured ventricular myocytes was confirmed by immunofluorescence. The CCh-induced positive chronotropic response was significantly inhibited by fluorescein-tagged antisense oligonucleotides directed against the m1, but not the m2 and m4, mAChR subtypes. The response was also inhibited by antisense oligonucleotides against Gqalpha protein. Finally, inhibition of CCh-induced phosphoinositide hydrolysis with 500 microM neomycin or 5 microM U73122 completely abolished the CCh-induced positive chronotropic response. These results are consistent with the stimulatory effects of mAChR activation on the rate of contractions in cultured ventricular myocytes being mediated through the m1 mAChR coupled through Gq to phospholipase C-induced phosphoinositide hydrolysis.

Desensitization of norepinephrine receptor function is associated with G protein uncoupling in the rat aorta.

Infusion of norepinephrine (NE) in rats results in desensitization of NE-mediated aortic contraction and a reduction (55% at 1 and 10 microM) in NE-stimulated vascular inositol phosphate accumulations. The functional responses to angiotensin II (ANG II) were also reduced in the tissues of NE-infused animals. alpha 1-Adrenoceptor number determined by 2-[beta-(4-hydroxy-3-[125I]iodophenyl)-ethylaminomethyl]-tetralone ([125I]HEAT) binding and levels of G alpha or G beta proteins measured by immunoblot analyses were not changed in the aortic membranes of NE-infused animals. To determine whether desensitization is associated with receptor-G protein uncoupling, agonist-stimulated palmitoylation of G alpha proteins was measured. NE infusion decreased phenylephrine (1 microM)-stimulated [3H]palmitate incorporation into Gq alpha, Gs alpha, and Gi alpha proteins and ANG II (10 microM)-stimulated palmitoylation of Gq alpha and Gi alpha in aortic membranes. Phenylephrine- and ANG II-stimulated guanosine 5'-O-(3-[35S]thiotriphosphate) binding to Gq alpha was also decreased in the aortas of NE-infused animals. These results show that an infusion of NE causes heterologous desensitization of the contractile and inositol phosphate accumulation responses in the rat aorta and that these changes are mediated by an uncoupling of receptors from their G proteins.

Potential role for a regulator of G protein signaling (RGS3) in gonadotropin-releasing hormone (GnRH) stimulated desensitization.

The cellular and molecular mechanisms of gonadotrope desensitization are unknown but transduction of the GnRH signal is known to involve sequentially the GnRH receptor, Gq alpha protein, phospholipase C beta-1, inositol-1,4,5-trisphosphate (IP3), and intracellular Ca+2 release. Here, we report the results of studies of a new family of proteins known as regulators of G protein signaling (RGS) that recently have been implicated in desensitization of several ligand induced processes. Using DNA-mediated transfection, we co-expressed the GnRH receptor and RGS1,2,3, or 4 in COS-1 cells. Control cells and those expressing RGS1,2, and 4 produced five fold increases in IP3 levels during the 30 sec after treatment with GnRH. In contrast, RGS3 expression suppressed by 75% the GnRH-induced IP3 responses. RGS3 was shown to bind Gq alpha protein in a model in vitro system: recombinant RGS3-glutathione-S-transferase (GST) fusion protein bound five-fold more 35S-met labeled Gq alpha protein than did with GST alone, suggesting that the mechanism of RGS3 action is attenuation of Gq alpha protein activation of phospholipase C. RGS3 mRNA and protein were observed to be expressed endogenously in the gonadotropic alpha T3-1 cell line. These results suggest a potential role for RGS3 in modulating the LH secretory responsiveness of the pituitary gonadotrope to GnRH.

Functional Importance of the Amino Terminus of Gqα

Gq alpha is palmitoylated at residues Cys9 and Cys10. Removal of palmitate from purified Gq alpha with palmitoylthioesterase in vitro failed to alter interactions of Gq alpha with phospholipase C-beta 1, the G protein beta gamma subunit complex, or m1 muscarinic cholinergic receptors. Mutants C9A, C10A, C9A/C10A, C9S/C10S, and truncated Gq alpha (removal of residues 1-6) were synthesized in Sf9 cells and purified. Loss of both Cys residues or truncation prevented palmitoylation of Gq alpha. However, truncated Gq alpha and the single Cys mutants activated phospholipase C-beta 1 normally, while the double Cys mutants were poor activators. Loss of both Cys residues impaired but did not abolish interaction of Gq alpha with m1 receptors. These Cys residues are thus important regardless of their state of palmitoylation. When expressed in HEK-293 or Sf9 cells, all of the proteins studied associated entirely or predominantly with membranes, although a minor fraction of nonpalmitoylated Gq alpha proteins accumulated in the cytosol of HEK-293 cells. When subjected to TX-114 phase partitioning, a significant fraction of all of the proteins, including those with no palmitate, was found in the detergent-rich phase. Removal of residues 1-34 of Gq alpha caused a loss of surface hydrophobicity as evidenced by complete partitioning into the aqueous phase. The Cys residues at the amino terminus of Gq alpha are thus important for its interactions with effector and receptor, and the amino terminus conveys a hydrophobic character to the protein distinct from that contributed by palmitate.

Regulation of myometrial Gi2, Gi3, and Gq expression during pregnancy. Effects of progesterone and estradiol.

The possibility that progesterone or estradiol may regulate expression of G protein in the rat myometrium during the course of pregnancy has been investigated using 1) immunoblot analysis of Gi2 alpha, Gi3 alpha, and Gq alpha subunits and 2) hybridization blot analysis of subunit mRNA. Eighteen hours after administration, estradiol had significantly increased the levels of both Gi2 alpha subunit and Gi2 alpha mRNA (by 40% and 32%, respectively). In control pregnant rats, we observed similar changes at the end of pregnancy, when myometrial concentrations of estradiol had increased, i.e., a 41% increase in immunoreactive Gi2 alpha subunit that correlated with a parallel 45% increase in mRNA levels. In contrast, levels of immunoreactive Gi3 alpha subunit and mRNA, which decreased with advancing gestation, were not influenced by estradiol or progesterone administration. Progesterone administration resulted 30 h later in a significantly decreased level of Gq alpha immunoreactivity (32%) and Gq alpha mRNA (30%). In control rats, Gq alpha protein and mRNA were also significantly lower at midpregnancy under progesterone dominance vs. term. At this stage, a twofold increase in Gq alpha subunit correlated with a 40% increase in mRNA levels. These results demonstrate that myometrial Gi2 alpha and Gq alpha subunits are physiological targets for estradiol and progesterone, respectively, in vivo. Alterations of these G protein levels are discussed in relation to their mediating effects on adenylyl cyclase activity or the phospholipase C pathway during the course of pregnancy.

Alterations of Brain Levels of Phosphoinositidase-C–Linked Gqα/G11α Proteins and Motor Function in Rats After Cardiac Arrest

Phosphoinositidase-C-linked Gq alpha and G11 alpha proteins have only recently been characterized. Second messenger systems are known to be affected by hypoxia-ischemia. However, the effects of hypoxia-ischemia on the brain levels of Gq alpha and G11 alpha proteins are not known. Therefore, in the present studies, the effects of hypoxia-ischemia on Gq alpha and G11 alpha proteins in rats were investigated with quantitative immunoblot analysis. Cardiac arrest was induced in male Sprague-Dawley rats by an intracardial injection of KCl. Resuscitation began 10 minutes afterwards. At various time points after resuscitation, animals were killed and the cerebral cortex, striatum, and cerebellum were dissected. Levels of Gq alpha and G11 alpha proteins were investigated by quantitative immunoblot analysis. At 1, 2, 4, and 6 hours after resuscitation, Gq alpha and G11 alpha protein levels remained unaltered. However, a significant reduction of these proteins was seen in the cerebral cortex and cerebellum of rats 3 and 14 days after cardiac arrest, with partial recovery by an average of 60 days. In contrast, no significant change was detected in the striatum. These observations indicate that phosphoinositidase-C-linked signal transduction pathways may be attenuated after hypoxic-ischemic insults to the brain, and that this phenomenon, together with many other factors, may contribute to the expression of motor dysfunction in rats after cardiac arrest.

Xenopus Gq alpha subunit activates the phosphatidylinositol pathway in Xenopus oocytes but does not consistently induce oocyte maturation.

We cloned the Xenopus laevis form of Gq alpha subunit to study its effects on oocyte maturation. Injection of Xenopus Gq alpha mRNA into stage 6 oocytes activated the phospholipase C/phosphatidylinositol pathway. The oocyte membrane became permeable to calcium ions and was able to generate transient inward currents (T(in)), due to the opening of Ca(2+)-dependent Cl- channels. The T(in) amplitude developed over several hours and disappeared by 24 hr. Diacylglycerol levels were found to parallel the appearance and disappearance of the T(in). The concurrent decline of T(in) values and diacylglycerol was not due to a failure in the synthesis of Gq alpha protein, which was produced continuously for > 24 hr. After Xenopus Gq alpha mRNA injection, germinal vesicle breakdown (GVBD) was variable (0-100%) in stage 6 oocytes, whereas none of the stage 4 oocytes underwent GVBD. In contrast, stage 6 oocytes injected with mRNA encoding the Go alpha G protein consistently underwent GVBD but did not acquire T(in). Our results show that activation of phospholipase C is not an absolute requisite for the induction of maturation, although in oocytes of some frogs phospholipase C activation can trigger a pathway to GVBD.

Effects of surgical sympathetic denervation on G-protein levels, alpha and beta-adrenergic receptors, cAMP production and adenylate cyclase activity in the smooth muscles of rabbit iris.

The relative densities of a number of G protein subunits were quantified in membranes prepared from iris sphincter and dilator muscles of rabbits that have undergone sympathetic denervation and from contralateral innervated controls by immunoblotting with specific polyclonal antibodies against Gs alpha, Gi alpha and Gq alpha protein subunits. In addition, alpha and beta-adrenergic receptor densities, basal and isoproterenol (ISO)-stimulated cAMP production, and basal and ISO+GTP gamma S-stimulated adenylate cyclase (AC) activities were measured in the same tissues. Densitometric analysis of the immunoblot data revealed a 32% reduction in the level of Gi alpha in the denervated sphincter, a 26% increase in the level of Gi alpha in the denervated dilator, and no changes due to denervation were found in the levels of Gs alpha and Gq alpha. Sympathetic denervation had no effect on the densities of alpha- and beta-adrenergic receptors in these tissues, however, it did induce a significant decrease in the KD values of alpha-adrenergic receptors in both dilator and sphincter, and in beta-adrenergic receptors in the sphincter. The basal- and ISO-stimulated cAMP production in the sphincter was 4-fold as high as that of the dilator, and at 0.05 microM ISO the denervation supersensitivity for cAMP production in the sphincter increased by 118% as compared to 36% in the dilator. Sympathetic denervation increased by 19-47% the basal- and ISO-stimulated activity of AC in these tissues, however, it had no effect on the phospholipase C activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Agonist‐induced Transfer of the α Subunits of the Guanine‐nucleotide‐binding Regulatory Proteins Gq and G11, and of Muscarinic m1 Acetylcholine Receptors from Plasma Membranes to a Light‐vesicular Membrane Fraction

A clone of a Chinese hamster ovary (CHO) cell line expressing high levels of the human muscarinic M1 acetylcholine (Hm1) receptor undergoes a substantial agonist-specific down-regulation of both Hm1 receptors and the alpha subunits of the guanine-nucleotide-binding (G)-proteins Gq and G11 which is accompanied by a desensitization of inositol-phospholipid-specific-phospholipase-C response [Mullaney, I., Dodd, M. W., Buckley, N. J. & Milligan, G. (1993) Biochem. J. 289, 125-131]. To examine early events in this process, the effect of agonist on subcellular distribution of Gq alpha and G11 alpha and of Hm1 receptors was assessed after short-term and long-term treatment with carbachol. Short-term (30 min) incubation with carbachol (1 mM) induced a simultaneous transfer of a proportion of both Gq alpha and G11 alpha and Hm1 receptors from plasma membranes to distinct light vesicular membranes. The total number of receptors and of Gq alpha and G11 alpha in each cell remained unchanged under these conditions. A similar transfer was noted for the G-protein Gs alpha but not for intrinsic plasma membrane markers. The plasma membrane, as well as light vesicular membrane, pool of Gs alpha subunit was unaffected by further sustained incubation with carbachol (16 h), whereas Hm1 receptors and both Gq alpha and G11 alpha proteins were down-regulated to 25% and 40%, respectively, when compared with untreated cells. Such observations support the idea that down-regulation of both the Hm1 receptor and its associated inositol-phospholipid-specific-phospholipase-C-linked G-proteins is produced by two sequential steps. The first is a transfer of signal-transducing polypeptides from the plasma membrane to a non-plasma membrane light vesicle fraction. The second step is represented by an agonist-specific down-regulation pathway. Both the Hm1 receptor and Gq alpha/G11 alpha appear to follow similar sequestration and down-regulation patterns.

Glucocorticoid increases angiotensin II type 1 receptor and its gene expression.

Recently, we demonstrated that glucocorticoid potentiates inositol triphosphate production evoked by angiotensin II in vascular smooth muscle cells. To clarify this mechanism, we investigated the effects of dexamethasone on the modulation of angiotensin II type 1 receptor and on postreceptor mechanisms in vascular smooth muscle cells. The number of angiotensin II type 1 receptors began to increase after 12 hours' incubation with dexamethasone. After 48 hours, the Bmax value reached 27 +/- 3 fmol/mg protein in dexamethasone-treated cells and 15 +/- 3 fmol/mg protein in control cells. However, binding affinity did not change. A glucocorticoid antagonist, RU 38486, completely blocked these effects of dexamethasone. Also, to elucidate the effects of dexamethasone on postreceptor mechanisms, GTP analogue-induced inositol trisphosphate production in permeabilized cells was examined. Pretreatment with 1 mumol/L dexamethasone for 48 hours did not affect these inositol trisphosphate productions. Moreover, dexamethasone had no effect on the level of Gq alpha protein. Furthermore, steady-state levels of angiotensin II type 1 receptor messenger RNA were increased 2.2 +/- 0.3-fold after 30 minutes' exposure to 1 mumol/L dexamethasone and 7.8 +/- 0.4-fold after 24 hours. We conclude that glucocorticoid induced expression of the angiotensin II type 1 receptor gene and resulted in an increase in the number of angiotensin II type 1 receptors through the glucocorticoid-specific receptor, without significant effect on postreceptor systems in vascular smooth muscle cells.

G protein coupling of human platelet V1 vascular vasopressin receptors

We used several approaches to identify the G protein coupled to V1 vascular arginine vasopressin (AVP) receptors of human platelets. In purified platelet membranes, high-affinity specific binding of [3H]AVP but not that of the V1 vascular antagonist [3H]d(CH2)5Tyr(Me)AVP was modulated by guanosine 5'-O-(3-thiotriphosphate) or sodium fluoride both in the presence and absence of MgCl2. AVP failed to modify the [alpha-32P]GTP labeling pattern or the cytosolic translocation of the 24- to 27-kDa GTP-binding proteins. AVP-stimulated GTPase activity of platelet membranes was blocked by antibodies specific for the COOH-terminal of the Gq alpha protein. AVP increased labeling of a 42-kDa platelet membrane protein by the photoreactive GTP analogue [alpha-32P]azidoanilido GTP. Immunoblotting of platelet proteins with various G protein-specific antibodies revealed that the 42-kDa protein labeled with [alpha-32P]azidoanilido GTP was immunoblotted only by antibodies specific for the alpha-subunit of GQ-11. Thus V1 vascular AVP receptors of human platelets are coupled in a divalent cation-dependent manner to a G protein belonging to the Gq-11 family.

Cytoplasmic and nuclear signaling pathways of V1-vascular vasopressin receptors.

We studied the cytoplasmic and nuclear signaling pathways of V1-vascular AVP receptors of human platelets, primary cultures of renal glomerular mesangial cells, and established cultures of the A7r5 aortic smooth muscle cell line. The immediate transmembrane signals are triggered by the formation of ligand-receptor complexes as illustrated by binding experiments with [3H]AVP (Kd = 2.50 nM), d(CH2)5Tyr(Me)AVP (Kd = 0.62 nM), the linear V1 antagonist phenylacetyl-D-Tyr(Et)-Phe-Val-Asn-Lys-Pro-[125I]Tyr-NH2 (Kd = 1.42 nM) or by fluorescence experiments with linear antagonists like phenylacetyl-D-Tyr(Et)-Phe-Gln-Asn-Lys-Pro-Arg-NH2 coupled to biotin and made fluorescent by labeling with tetramethylrhodamine-avidin. We used several approaches (radioreceptor binding, radioactive labeling, autoradiographic, enzymatic, photoaffinity labeling, and immunoblotting procedures) to identify the guanine nucleotide regulatory protein coupled to V1-vascular vasopressin receptors. AVP-stimulated GTPase activity of human platelet membranes was blocked by pretreatment with antibodies specific for the C-terminal of the newly described Gq alpha protein. In the presence of MgCl2, AVP increased labeling by the photoreactive GTP analog [alpha-32P]azidoanilido GTP of a platelet membrane protein of apparent molecular mass of 42 kDa. AVP effect was reversed by the specific V1-vascular antagonist d(CH2)5Tyr(Me)AVP and labeling was completely abolished by GTP gamma s.(ABSTRACT TRUNCATED AT 250 WORDS)