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PubMed1998

An animal model of autism: behavioural studies in the GS guinea-pig.

Autism is a human behavioural pathology marked by major difficulties in abnormal socialization, language comprehension and stereotypic motor patterns. These behavioural abnormalities have been associated with corticocerebral and cerebellar abnormalities in autistic patients, particularly in vermal folia VI and VII. Progress in understanding this disease has been hindered by the absence of a non-primate animal model. GS guinea-pigs are a partially inbred, non-ataxic guinea-pig strain with cerebellar and corticocerebral abnormalities similar to those reported to exist in human patients with autism. In order to determine if GS guinea-pigs represent an animal model of autism, their behaviour was compared with that of Hartley strain guinea-pigs. GS animals learned a motor task significantly more rapidly than Hartley guinea-pigs, but performed it in a more stereotypic manner and were less influenced by environmental stimuli than Hartleys. GS animals exhibited significantly less exploratory behaviour in a novel environment and were significantly less responsive to 50-95 dBA pure tones than Hartley guinea-pigs. In a social interaction assay, GS guinea-pigs interacted significantly less frequently with each other or with Hartley guinea-pigs than Hartleys did under the same conditions. GS behaviour thus exhibits autistic-like behaviour patterns: motor stereotypy, lack of exploration and response to environment and poor social interaction. Coupled with the neuropathological findings, this abnormal behaviour suggests that GS guinea-pigs could be a useful animal model of autism.

Acoustic Stimulation↗

PubMed1995

Selective coupling of prostaglandin E receptor EP3D to Gi and Gs through interaction of alpha-carboxylic acid of agonist and arginine residue of seventh transmembrane domain.

Prostaglandin (PG) E receptor EP3D is coupled to both Gi and Gs. To examine the roles of the interaction of alpha-carboxylic acid of PGE2 and its putative binding site, the arginine residue in the seventh transmembrane domain of EP3D, in receptor-G protein coupling, we have mutated the arginine residue to the noncharged glutamine. PGE2 with a negatively charged alpha-carboxylic acid and sulprostone, an EP3 agonist with a noncharged modified alpha-carboxylic acid, inhibited the forskolin-stimulated adenylate cyclase activity via Gi activation in the EP3D receptor in the same concentration-dependent manner. In contrast, the adenylate cyclase stimulation via Gs activation by sulprostone was much lower than that by PGE2. On the other hand, both PGE2 and sulprostone showed potent Gi activity but failed to show Gs activity in the mutant receptor. EP3D receptor showed a high affinity binding for PGE2 in the form coupled to either Gi or Gs. Although the mutant receptor showed high affinity binding when coupled to Gi, it lost high affinity binding in the condition of Gs coupling. Furthermore, sulprostone bound to the Gi-coupled EP3D receptor with higher affinity than the Gs-coupled receptor. Among various EP3 agonists, alpha-carboxylic acid-unmodified agonists showed both Gi and Gs activities, but the modified agonists showed only Gi activity. These findings suggest that the interaction between the alpha-carboxylic acid of PGE2 and the arginine residue of the receptor regulates the selectivity of the G protein coupling.

Abortifacient Agents, Nonsteroidal↗

PubMed1997

Localization of unmyelinated axons in rat skin and mucocutaneous tissue utilizing the isolectin GS-I-B4.

The alpha-D-galactose specific isolectin I-B4 from Griffonia simplicifolia (GS-I-B4) labels CNS microglia and certain peripheral neurons, including a subpopulation of small dark, type B dorsal root ganglion cells, some post-ganglionic sympathetic axons, and nearly all peripheral gustatory axons. The innervation patterns of GS-I-B4 reactive sensory ganglion cells are unknown for many peripheral target tissues, including their probable primary target, the skin. The present study describes the distribution of GS-I-B4 reactive axons in hairy and glabrous hindpaw skin and in the glans penis of rats, using both single and double-labelling histochemical techniques. Neuronal processes were identified using (1) histochemistry with horseradish peroxidase conjugated GS-I-B4 or (2) immunohistochemistry against PGP 9.5 to identify all axons, and biotinylated lectin histochemistry with avidin-FITC to identify the subpopulation of GS-I-B4 reactive axons. GS-I-B4 strongly labelled unmyelinated cutaneous sensory afferents, as well as some sympathetic efferents and visceral afferents. lectin reactive axons were seen to innervate the upper hair shaft epidermis in hairy skin, and were abundant in the shallow dermis in hairy and glabrous skin and glans penis. Lectin reactive axons were also abundant in the lamina propria and distal urethral epithelium of the penis. These results provide new evidence for the cutaneous sensory role of GS-I-B4 reactive primary afferents, as well as evidence to support the contention that the lectin is a specific marker for a subpopulation of unmyelinated axons and not simply a marker for the myelination state of an axon.

Animals↗

PubMed1990

The carboxy-terminal domain of Gs alpha is necessary for anchorage of the activated form in the plasma membrane.

GTP-binding proteins which participate in signal transduction share a common heterotrimeric structure of the alpha beta gamma-type. In the activated state, the alpha subunit dissociates from the beta gamma complex but remains anchored in the membrane. The alpha subunits of several GTP-binding proteins, such as Go and Gi, are myristoylated at the amino terminus (Buss, J. E., S. M. Mumby, P. J. Casey, A. G. Gilman, and B. M. Sefton. 1987. Proc. Natl. Acad. Sci. USA. 84:7493-7497). This hydrophobic modification is crucial for their membrane attachment. The absence of fatty acid on the alpha subunit of Gs (Gs alpha), the protein involved in adenylate cyclase activation, suggests a different mode of anchorage. To characterize the anchoring domain of Gs alpha, we used a reconstitution model in which posttranslational addition of in vitro-translated Gs alpha to cyc- membranes (obtained from a mutant of S49 cell line which does not express Gs alpha) restores the coupling between the beta-adrenergic receptor and adenylate cyclase. The consequence of deletions generated by proteolytic removal of amino acid sequences or introduced by genetic removal of coding sequences was determined by analyzing membrane association of the proteolyzed or mutated alpha chains. Proteolytic removal of a 9-kD amino-terminal domain or genetic deletion of 28 amino-terminal amino acids did not modify the anchorage of Gs alpha whereas proteolytic removal of a 1-kD carboxyterminal domain abolished membrane interaction. Thus, in contrast to the myristoylated alpha subunits which are tethered through their amino terminus, the carboxy-terminal residues of Gs alpha are required for association of this protein with the membrane.

Adenosine Diphosphate Ribose↗

PubMed2006

Deep anterior lamellar keratoplasty for keratoconus.

PURPOSE: To investigate surgical outcomes after deep anterior lamellar keratoplasty (DALKP) in eyes with keratoconus. METHODS: Retrospective review of 127 eyes of 118 keratoconus patients who underwent DALKP at King Khaled Eye Specialist Hospital, Riyadh, Saudi Arabia between January 1, 1998 and November 30, 2001. The mean age of all patients was 25.9 years (range 11-70 years). The mean follow-up for all patients was 10.9 months (range 4-61 months). Baring of Descemet's membrane (DM) during DALKP was achieved in 47 (37%) eyes; some stroma was left during operation in the remaining 80 (63%) eyes. Snellen visual acuity, intraoperative complications, and postoperative graft status were assessed. RESULTS: At the last follow-up visit, 74% of all eyes were able to achieve a best-corrected visual acuity of 20/50 or better. This level of improvement was more statistically significant with baring of DM (P = 0.005). Intraoperative perforation of DM occurred in 16 (13%) cases and was statistically significant in eyes with deep corneal scars (P = 0.012). However, this perforation did not seem to have an impact on the final visual acuity (P = 0.48). Main graft-related complications included graft-host vascularization (7/127), stromal graft rejection (4/127), graft infection (1/127), and persistent epithelial defect (1/127). CONCLUSIONS: Deep lamellar keratoplasty provides a safer and successful alternative to penetrating keratoplasty for keratoconus patients but remains a challenging procedure. Endothelial graft rejection is absent after DALKP. However, stromal graft rejection, although very rare, does occur.

Adolescent↗

PubMed2006

Antiviral activity, pharmacokinetics, and dose response of the HIV-1 integrase inhibitor GS-9137 (JTK-303) in treatment-naive and treatment-experienced patients.

BACKGROUND: GS-9137 is a potent low-nanomolar strand transfer inhibitor of HIV-1 integrase. METHODS: The antiviral activity, tolerability, pharmacokinetics, and pharmacodynamics of GS-9137 were evaluated in a randomized, double-blind, placebo-controlled monotherapy study in 40 HIV-1- infected patients not receiving antiretroviral therapy with an HIV-1 RNA between 10,000 and 300,000 copies/mL and a CD4 count of 200 cells/microL or greater. GS-9137 or matching placebo was administered with food for 10 days at 5 dosage regimens (200, 400, or 800 mg BID, 800 mg QD, or 50 mg+100 mg ritonavir QD; 6 active, 2 placebo per dose level). The primary end point was the maximum reduction from baseline in log10 HIV-1 RNA. RESULTS: Forty patients were enrolled, with a mean baseline viral load of 4.75 log10 copies/mL and a CD4 count of 442 cells/microL. Each GS-9137 dosing regimen exhibited significant, exposure-dependent (mean reductions, -0.98 to -1.99 log10 copies/mL) antiviral activity compared with placebo (P<0.01). Twice-daily administrations of GS-9137 at doses of 400 or 800 mg or once-daily dosing of 50 mg with ritonavir demonstrated mean reductions from baseline in HIV-1 RNA of 1.91 log10 copies/mL or greater, with all patients exhibiting 1 log10 or greater and 50% having 2 log10 or greater reductions. No patient developed evidence of integrase resistance. GS-9137 showed an adverse event profile similar to placebo, and there were no study drug discontinuations. CONCLUSIONS: GS-9137 demonstrated substantial short-term antiviral activity and was well tolerated as monotherapy, thus warranting further study.

Acquired Immunodeficiency Syndrome↗

PubMed1989

Modification of Gs-stimulated adenylate cyclase in brain membranes by low pH pretreatment: correlation with altered guanine nucleotide exchange.

Pretreatment of rat brain membranes at pH 4.5 before assay at pH 7.4 modifies the function of GTP-binding proteins (G-proteins) by eliminating Gs-stimulated adenylate cyclase activity while increasing opiate-inhibited adenylate cyclase activity. To help characterize the molecular nature of the low pH effect, we labeled Gs and Gi alpha-subunits in both control and low pH-pretreated membranes with the GTP photoaffinity analog [32P]P3 (4-azidoanilido)-P1-5'-GTP ([32P]AAGTP). When membranes were preincubated with unlabeled AAGTP, a persistent inhibitory state of adenylate cyclase was produced, which was overcome in untreated membranes with high (greater than 1 microM) concentrations of guanylyl-5'-imidodiphosphate [Gpp(NH)p]. In low pH-pretreated membranes, this inhibition could not be overcome, and stimulation by Gpp(NH)p was eliminated. Maximal inhibition of adenylate cyclase achieved by incubation with AAGTP was not altered by low pH pretreatment. Incorporation of [32P]AAGTP into Gs (42 kilodaltons) or Gi/o (40 kilodaltons) was unaffected by low pH pretreatment; however, transfer of 32P from Gi/o to Gs, which occurs with low (10 nM) concentrations of Gpp(NH)p in untreated membranes, was severely retarded in low pH-pretreated membranes. Both the potency and efficacy of Gpp(NH)p in producing exchange of [32P]AAGTP from Gi/o to Gs were markedly reduced by low pH pretreatment. These results correlate the loss of Gs-stimulated adenylate cyclase with a loss of transfer of nucleotide from Gi/o to Gs alpha-subunits and suggest that the nucleotide exchange participates in the modulation of neuronal adenylate cyclase.

Adenylyl Cyclases↗

PubMed1979

Gs, an allele of chickens for endogenous avian leukosis viral antigens, segregates with ev 3, a genetic locus that contains structural genes for virus.

Gs is an allele of chickens for the expression of endogenous avian leukosis virus-related core (gs) and envelope (chf) antigens. Progeny of a genetic cross in which Gs was segregating were analyzed for endogenous viral DNA as well as for the expression of endogenous viral antigens. Viral genetic information was identified by cleavage of embryo DNA with restriction endonucleases, electrophoretic separation of the resulting fragments, and identification of bands containing viral sequences by hybridization of the DNA to 32P-labeled viral RNA. Four different chromosomal sites of residence of endogenous viral sequences were identified by this method. These sites were the same as those previously assigned to the endogenous viral loci ev 1, ev 3, ev 4, and ev 5. ev 1 was present in all of the progeny of the cross. ev 3, ev 4, and ev5 were present in various combinations with ev 1. ev 3 cosegregated with the gs+chf+ phenotpye. Cells which did not contain ev 3 but contained ev 1, ev 4, and/or ev 5, did not express detectable levels of viral antigens. We suggest that Gs contains the structural genes for endogenous virus which reside at ev 3 and that these structural genes code for gs and chf in gs+chf+ cells.

Alleles↗

PubMed1990

Two forms of Drosophila melanogaster Gs alpha are produced by alternate splicing involving an unusual splice site.

G proteins are responsible for modulating the activity of intracellular effector systems in response to receptor activation. The stimulatory G protein Gs is responsible for activation of adenylate cyclase in response to a variety of hormonal signals. In this report, we describe the structure of the gene for the alpha subunit of Drosophila melanogaster Gs. The gene is approximately 4.5 kilobases long and is divided into nine exons. The exon-intron structure of the Drosophila gene shows substantial similarity to that of the human gene for Gs alpha. Alternate splicing of intron 7, involving either use of an unusual TG or consensus AG 3' splice site, results in transcripts which code for either a long (DGs alpha L) or short (DGs alpha S) form of Gs alpha. These subunits differ by inclusion or deletion of three amino acids and substitution of a Ser for a Gly. The two forms of Drosophila Gs alpha differ in a region where no variation in the primary sequence of vertebrate Gs alpha subunits has been observed. In vitro translation experiments demonstrated that the Drosophila subunits migrate anomalously on sodium dodecyl sulfate-polyacrylamide gels with apparent molecular weights of 51,000 and 48,000. Additional Gs alpha transcript heterogeneity reflects the use of multiple polyadenylation sites.

Amino Acid Sequence↗

PubMed1994

Parental origin of Gs alpha gene mutations in Albright's hereditary osteodystrophy.

Heterozygous mutations of the Gs alpha gene leading to reduced Gs alpha activity have been identified in patients with Albright's hereditary osteodystrophy (AHO). However, AHO may be associated with hormone resistance (pseudohypoparathyroidism type Ia, PHPIa) or a normal response (pseudo-pseudohypoparathyroidism, PPHP). As both disorders may occur within the same family, the relationship between Gs alpha genotype and phenotype remains unresolved. The AHO phenotype may be dependent upon the sex of the parent transmitting the Gs alpha mutation, perhaps through a gene imprinting mechanism. We have used an intragenic Gs alpha FokI polymorphism to determine the parental origin of Gs alpha gene mutations in sporadic and familial AHO. We now show that a de novo G-->A substitution at the exon 5 donor splice junction in a child with PPHP was paternally derived. Furthermore, in a female with PPHP, the Gs alpha abnormality was shown to be of paternal origin, while subsequent maternal processing and transmission resulted in PHPIa in two offspring. As transmission of PPHP has rarely been reported, determining parental origin of the disease allele in sporadic cases may provide insight into the mechanism of hormone resistance in AHO.

Adult↗

PubMed1990

ATP-dependent efflux of GSSG and GS-conjugate from isolated rat hepatocytes.

The driving force for efflux of dinitrophenyl-glutathione (GS-DNP) and oxidized glutathione (GSSG) from freshly isolated rat hepatocytes was studied. Incubation of hepatocytes in Krebs with increasing K+ concentrations (equivalently replaced for Na+) or in Krebs with 3 mM ouabain led to a partial or complete dissipation of the plasma membrane potential, as measured by the equilibrium distribution of 36Cl-. This had no effect on the initial efflux rate of GSSG and GS-DNP. On the other hand, partial depletion of the cellular ATP content via different independent mechanisms significantly reduced the initial efflux rate of these compounds. Titration of the cellular ATP content by incubation of the cells with different concentrations of atractyloside revealed a linear relation between the cellular ATP content and the initial efflux rate of GS-DNP. The efflux of GS-DNP was also studied in hepatocytes from mutant rats with hepatobiliary transport defect (TR- rats). These rats have a hereditary canalicular secretion defect for a number of organic anions including GS-DNP. As we have shown previously, the efflux of GS-DNP from TR- rat hepatocytes is significantly slower than from normal hepatocytes (J. Clin. Invest. 84: 476-483, 1989). Depletion of the cellular ATP content in these cells had no significant effect on the residual efflux of GS-DNP. From these studies, we conclude that an ATP-dependent transport system for oxidized glutathione and glutathione conjugates is involved in the biliary transport of these compounds. The possible relation of this transport system with that described in other cell types and tissues, like erythrocytes and heart sarcolemma, is discussed.

Adenosine Triphosphate↗

PubMed1992

GTP-dependent protein(Gs) activity in preterm infants.

In a previous study, we showed a renal resistance to PTH in preterm infants during their 1st week of life. We proposed it could explain early neonatal hypocalcemia. Such renal resistance is well known in type 1 pseudohypoparathyroid patients and is explained by a defect of stimulatory GTP-dependent protein (Gs). To determine if functional immaturity in the Gs protein could be involved in PTH resistance, we studied 27 newborn babies: 7 full-term and 20 preterm babies. Biological activity of the Gs unit was determined on days 1, 3 and 10 after delivery by bioassay. No correlation was found between the Gs unit activity and either gestation or birth weight at these dates. Eight infants had hypocalcemia and their Gs unit activity did not differ from those with normocalcemia. Furthermore, we showed that the Gs unit is active from 29 weeks of gestation. We conclude that the Gs protein appears not to be involved in the pathophysiology of early renal resistance to PTH and therefore in early neonatal hypocalcemia.

Adult↗

PubMed1993

The GTP-binding regulatory proteins, Gs and G(i), are altered in erythrocyte membranes of patients with ischemic heart disease resulting from coronary atherosclerosis.

Acute ischemic heart disease is associated with alterations in the cardiac adenylate cyclase system response, although the specificity and mechanism of these events are unknown. We studied the characteristics of inhibitory (G(i)) and stimulatory (Gs) GTP-binding regulatory proteins (G proteins) of adenylate cyclase in erythrocyte membranes of patients (n = 16) with nonacute ischemic heart disease resulting from coronary atherosclerosis. Gs was measured by reconstitution with the resolved catalytic unit of adenylate cyclase and by cholera toxin-catalyzed ADP-ribosylation of a 42-kD protein; G(i) was tested as a 41-kD substrate of pertussis toxin-catalyzed ADP-ribosylation. Gs activity was decreased by 27 +/- 2% in the cholate extract and by 25 +/- 3% in the supernatant of guanosine 5'-(gamma-thio)triphosphate-treated membranes. The amount of cholera toxin substrate was decreased by 33 +/- 3%, and the pertussis toxin substrate was increased by 27 +/- 5% compared with healthy subjects (n = 10). All changes in G-protein characteristics appear to be specific relative to other erythrocyte membrane proteins and hemoglobin. Those patients who have a decreased Gs possess approximately normal Gi, and those with increased G(i) showed no change in Gs. Patients with increased G(i) (normal Gs) exhibited more severe deterioration of their coronary arteries than did patients with decreased Gs (normal G(i)) (P < .05), but these two groups did not differ significantly in serum lipids, hormones, drug therapy, historical data, or baseline assessment (P < 0.05).

Adult↗

PubMed1995

Transcriptional effects in GH3 cells of Gs alpha mutants associated with human pituitary tumors: stimulation of adenosine 3',5'-monophosphate response element-binding protein-mediated transcription and of prolactin and growth hormone promoter activity via protein kinase A.

Somatic mutations of the alpha-subunit of Gs (Gs alpha) have been detected previously at high frequency in human PRL- and/or GH-producing pituitary tumors. To test whether these mutants are responsible for the increased production of these hormones, we used transient cotransfection assays to analyze their genomic effects in GH3 rat pituitary cells. We first show that guanosine triphosphatase (GTPase)-deficient Gs alpha subunits (mutated at amino acid 201 or 227) stimulate transcription from a reporter construct bearing the consensus cAMP response element (CRE; TGACGTCA). Using GAL4-CRE-binding protein fusion constructs, we further show that this stimulatory effects of Gs alpha on the CRE is probably mediated by the transacting factor CRE-binding protein. Then, in experiments using a reporter gene driven by the human promoters for either the PRL (position -250 to 18) or GH (position -500 to 13) genes, we show that these mutant Gs alpha subunits stimulate expression driven by either the PRL or GH promoter. Finally, we show that a dominant inhibitory mutant of cAMP-dependent kinase (protein kinase A) completely blocks the ability of these Gs alpha mutants to stimulate the activity of either the PRL or GH promoter, implying that GTPase-deficient Gs alpha subunits stimulation of the activities of these promoters is mediated entirely via the cAMP/protein kinase A pathway. Taken together, these results imply that activation of this pathway by the GTPase-deficient mutants found in human pituitary tumors stimulates the expression of PRL and GH genes. The transcriptional effects exerted via this pathway may thus provide a basis for the secretory phenotype and endocrine disorders associated with these tumors.

Cyclic AMP Response Element-Binding Protein↗

PubMed1998

Effect of an atypical adrenergic beta3-agonist, GS-332: sodium (2R)-[3-[3-[2-(3-chlorophenyl)-2-hydroxyethylamino]cyclohexyl]phenoxy] acetate, on urinary bladder function in rats.

We have developed an atypical adrenergic beta3-agonist, GS 332: Sodium (2R)-[3-[3-[2-(3 Chlorophenyl)-2-hydroxyethylamino]cyclohexyl]phenoxy]acetate, which has an unique structure compared to other beta3 agonists. In vitro study, we compared effects of GS 332 on rat urinary bladder muscle strip contractility with those of clenbuterol hydrochloride (clenbuterol), an adrenergic beta2 agonist. GS-332 relaxed isolated rat urinary bladder strips in a concentration dependent manner with 50% effective concentration (EC50) of 15.7 nM, and the relaxant activity of GS 332 was as potent as that of clenbuterol(EC50; 30.8 nM). The concentration response curve of GS 332 on isolated rat urinary bladder was competed by a specific beta3-antagonist, SR59230A in a concentration-dependent manner, in which Schild slope was 1.1 and pA2 value of SR59230A was 7.1. In vivo study, cystometory investigated in anesthetized rats demonstrated that GS 332 was more potent in increasing the urinary storage volume than clenbuterol and less potent in inhibiting the contractile force of urinary bladder at micturition reflex than clenbuterol. These data demonstrate that GS 332, a new adrenergic beta3-agonist, may be more useful to maintain continence than clenbuterol, an adrenergic beta3 adrenergic agonist.

Adrenergic beta-Agonists↗

PubMed2002

[Jumping gene gs of Lycopersicon lycopersicum (L.) Karsten ex Farw].

Studies on tomato culture revealed for the first time two phenomena: (1) instability of gene gs (green stripes on fruits) and (2) spontaneous expression of the character determined by the gs gene without involvement of sexual reproduction in some cultivars. The instability of the gs gene was detected by the phenotypic expression of character in different plants from the same strain, racemes of the same plant, fruits of the same raceme, and parts of the same fruit. The instability of the gs gene may be determined by endogenous regulatory genetic elements causing specific changes of the character, with the degree of these changes varying for different parts of the fruit and places of its formation on the plant. The instability of the gs gene was expressed for four generations. There were no other phenotypic changes in the offsprings studied. In addition, migration of the recessive gs gene to other cultivars followed by its complete expression in a whole plant or some parts of the plant was observed. Exogenous transfer of the gs gene is suggested.

Chimera↗

PubMed1992

Evidence for the formation of a functional complex between vasoactive intestinal peptide, its receptor, and Gs in lung membranes.

The molecular weight of the vasoactive intestinal peptide (VIP) receptor in rat lung and its interaction with the stimulatory guanine nucleotide-binding protein (Gs) were assessed by covalent cross-linking, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunological techniques. Studies with two cross-linking agents indicated that the VIP receptor in this tissue is a single polypeptide of Mr = 54,000. The VIP-occupied receptor could be cross-linked to neighboring proteins after detergent solubilization; higher molecular weight complexes of Mr = 114,000 and 184,000 were formed. Immunoblotting with antisera against G-protein subunits demonstrated that both complexes contained the alpha-subunit of Gs as well as the 125I-VIP cross-linked receptor whereas only the Mr = 184,000 complex contained the beta-subunit. Pretreatment with GTP reduced the prominence of these complexes, verifying the functional nature of this receptor-Gs association. Studies with a third cross-linking agent, ethylene glycol bis(succinimidyl succinate), provided direct evidence of physically associated, ternary VIP-receptor-Gs complexes actually in the membrane milieu. That these complexes were functionally associated with shown by their inhibition by anti-Gs alpha anti-serum. Since treatment of membranes with guanosine 5'-O-(3-thiotriphosphate) resulted in the separation of the VIP-cross-linked receptor from Gs such that no cross-linking could occur, we conclude that the binding of GTP analogs induces a conformational change in Gs in the membrane milieu.

Animals↗

PubMed1991

Deletion within the amino-terminal region of Gs alpha impairs its ability to interact with beta gamma subunits and to activate adenylate cyclase.

Proteolytic experiments performed on transducin and Go alpha subunit strongly suggest that the amino-terminal residues of the alpha chain are involved in the interaction with beta gamma subunits. To test the possibility that the same region in Gs may fulfill a similar function, we introduced a deletion in the amino-terminal domain of Gs alpha. The properties of the wild type and the deleted alpha chains were characterized on in vitro translated proteins or after reconstitution of cyc- membranes by in vitro-translated alpha subunits. The mutant (delta 2-29) Gs alpha could still bind guanosine 5'-3-O-(thio)triphosphate, as revealed by its resistance to trypsin proteolysis and was still able to interact with the membrane. However, (delta 2-29) Gs alpha was not ADP-ribosylated by cholera toxin. In contrast to Gs alpha, addition of beta gamma subunits did not increase the rate of sedimentation of (delta 2-29) Gs alpha in sucrose gradients. Binding experiments on reconstituted membranes showed that the coupling to beta-adrenergic receptors was very low with (delta 2-29) Gs alpha. Finally, the mutant did not restore activation of adenylate cyclase of cyc- membranes. We propose that the primary functional defect is the loss of interaction with beta gamma subunits, which secondarily impairs beta gamma-dependent properties such as receptor coupling and cholera toxin-catalyzed ADP-ribosylation. However, it remains to be established that the lack of adenylate cyclase activation also results from this impaired interaction with beta gamma subunits.

Adenosine Diphosphate Ribose↗