Simultaneous coupling of alpha 2-adrenergic receptors to two G-proteins with opposing effects. Subtype-selective coupling of alpha 2C10, alpha 2C4, and alpha 2C2 adrenergic receptors to Gi and Gs.

Coupling of the three alpha 2-adrenergic receptor (alpha 2AR) subtypes to Gi and Gs was studied in membranes from transfected CHO cells. We observed that in the presence of low concentrations of the alpha 2AR agonist UK-14304, alpha 2C10 mediated inhibition of adenylyl cyclase activity, whereas at high concentrations of agonist, alpha 2C10 mediated stimulation of adenylyl cyclase activity. We considered that this biphasic response was due to the coupling of alpha 2C10 to both Gi and Gs. To isolate functional Gs and Gi coupling, cells were treated with pertussis toxin or cholera toxin in doses sufficient to fully ADP-ribosylate the respective G-proteins. Following treatment with cholera toxin, agonists elicited only alpha 2C10-mediated inhibition (approximately 50%) of adenylyl cyclase while after pertussis toxin treatment, agonists elicited only alpha 2C10-mediated stimulation (approximately 60%) of adenylyl cyclase. Incubation of membranes with antisera directed against the carboxyl-terminal portion of Gs alpha blocked this functional alpha 2AR.Gs coupling to the same extent as that found for beta 2AR.Gs coupling. In addition to functional Gs coupling, we also verified direct, agonist-dependent, physical coupling of alpha 2AR to Gs alpha. In agonist-treated membranes, an agonist-receptor-Gs alpha complex was immunoprecipitated with a specific alpha 2C10 antibody, and the Gs component identified by both western blots using Gs alpha antibody, and cholera toxin mediated ADP-ribosylation. Due to the differences in primary amino acid structure in a number of regions of the alpha 2AR subtypes, we investigated whether G-protein coupling was subtype-selective, using UK-14304 and cells with the same alpha 2AR expression levels (approximately 5 pmol/mg). Coupling to Gi was equivalent for alpha 2C10, alpha 2C4, and alpha 2C2: 53.4 +/- 8.8% versus 54.9 +/- 1.0% versus 47.6 +/- 3.5% inhibition of adenylyl cyclase, respectively. In marked contrast, distinct differences in coupling to Gs were found between the three alpha 2AR subtypes: stimulation of adenylyl cyclase was 57.9 +/- 6.3% versus 30.7 +/- 1.1% versus 21.8 +/- 1.7% for alpha 2C10, alpha 2C4, and alpha 2C2, respectively. Thus, alpha 2AR have the potential to couple physically and functionally to both Gi and Gs; for Gi coupling we found a rank order of alpha 2C10 = alpha 2C4 = alpha 2C2, while for Gs coupling, alpha 2C10 greater than alpha 2C4 greater than alpha 2C2.     

Restricting the mobility of Gs alpha: impact on receptor and effector coupling.

The alpha-subunit of the stimulatory G protein, Gs, has been shown to dissociate from the plasma membrane into the cytosol following activation by G protein-coupled receptors (GPCR) in some experimental systems. This dissociation may involve depalmitoylation of an amino-terminal cysteine residue. However, the functional significance of this dissociation is not known. To investigate the functional consequence of Gs alpha dissociation, we constructed a membrane-tethered Gs alpha (tetGs alpha), expressed it in Sf9 insect cells, and examined its ability to couple with the beta(2) adrenoceptor and to activate adenylyl cyclase. Compared to wild-type Gs alpha, tetGs alpha coupled much more efficiently to the beta 2 adrenoceptor and the D1 dopamine receptor as determined by agonist-stimulated GTP gamma S binding and GTPase activity. The high coupling efficiency was abolished when Gs )alpha was proteolytically cleaved from the membrane tether. The membrane tether did not prevent the coupling of tetGS alpha to adenylyl cyclase. These results demonstrate that regulating the mobility of Gs alpha relative to the plasma membrane, through fatty acylation or perhaps interactions with cytoskeletal proteins, could have a significant impact on receptor-G protein coupling. Furthermore, by enabling the use of more direct measures of receptor-G protein coupling (GTPase activity, GTP gamma S binding), tetGS alpha can facilitate the study for receptor-G protein interactions.     

The thyroliberin receptor interacts directly with a stimulatory guanine-nucleotide-binding protein in the activation of adenylyl cyclase in GH3 rat pituitary tumour cells. Evidence obtained by the use of antisense RNA inhibition and immunoblocking of the stimulatory guanine-nucleotide-binding protein.

The thyroliberin receptor in GH3 pituitary tumour cells is known to couple to phospholipase C via a guanine-nucleotide-binding protein (G protein). Thyroliberin is postulated also to activate adenylyl cyclase, via the stimulatory G protein (Gs). In order to study this coupling, we constructed an antisense RNA expression vector that contained part of the Gs alpha-subunit cDNA clone (Gs alpha) in an inverted orientation relative to the mouse metallothionein promoter. The cDNA fragment included part of the coding region and all of the 3' non-translated region. Transient expression of Gs alpha antisense RNA in GH3 cells resulted in the specific decrease of Gs alpha mRNA levels, followed by decreased Gs alpha protein levels. Thyroliberin-elicited adenylyl cyclase activation in membrane preparations showed a reduction of up to 85%, whereas phospholipase C stimulation remained unaffected. Activation of adenylyl cyclase by vasoactive intestinal peptide was reduced by 30-40%. Investigation of the effects of thyroliberin and vasoactive intestinal peptide on adenylyl cyclase in GH3 cell membranes pretreated with antisera against Gs alpha and Gi-1 alpha/Gi-2 alpha support the results obtained by the use of the antisense technique. We conclude that thyroliberin has a bifunctional effect on GH3 cells, in activating adenylyl cyclase via Gs or a Gs-like protein in addition to the coupling to phospholipase C.     

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.     

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.     

Coupling of a mutated form of the human beta 2-adrenergic receptor to Gi and Gs. Requirement for multiple cytoplasmic domains in the coupling process.

We constructed five genes encoding mutant human beta 2-adrenergic receptor sequence (beta 2AR) which contained 12-22 amino acid substitutions with corresponding sequence from the human alpha 2AAR in order to assess the receptor domains involved in Gs versus Gi recognition and coupling. Mutant beta 2AR with substitutions in the N (S1)- and C-terminal (S2) portions of the third intracellular loop, the proximal cytoplasmic tail (S3), and two combinations thereof (S2,3 and S1,2,3), were stably expressed in Chinese hamster fibrobasts (CHW-1102), as were the human beta 2AR and alpha 2AAR at comparable receptor levels. All mutant receptors with S2 substitutions (i.e. S2, S2,3, S1,2,3) were significantly (approximately 85%) uncoupled from Gs. Upon exposure to pertussis toxin, which uncouples receptors from Gi, S1,2,3 exhibited a 526 +/- 99% increase in agonist-stimulated adenylylcyclase activity compared with a 59 +/- 13% increase with the wild type receptor. This enhanced ability of S1,2,3 to interact with Gs following pertussis toxin treatment indicates that, in the absence of toxin exposure, substantial coupling occurs between the mutant receptor and Gi. Mutant beta 2AR bearing only one or two alpha 2AAR-substituted sequences showed no such enhancement. Forskolin-stimulated enzyme activities were increased by pertussis toxin treatment to similar degrees in all clones examined, indicating that the observed effects are confined to the receptor-mediated pathway. In the absence of GTP, competition binding experiments with S1,2,3, beta 2AR and alpha 2AAR revealed that approximately 40-50% of the receptors formed a high affinity binding state for agonist. Pertussis toxin treatment markedly reduced this to approximately 19% with S1,2,3, while having no effect on beta 2AR and completely eliminating high affinity agonist binding to alpha 2AAR. These results suggest that S1,2,3 interacts with Gi as well as Gs, and that receptor:G protein coupling requires the concerted participation of multiple cytoplasmic receptor domains.     

Alternative promoter and 5' exon generate a novel Gs alpha mRNA

Several species of mRNA have been shown to encode the alpha subunit of the stimulatory GTP-binding regulatory protein, Gs alpha. The various Gs alpha mRNAs are generated through alternative splicing of a single precursor RNA and through the use of alternative acceptor splice sites. We now report the existence of a Gs alpha mRNA that uses a previously unidentified promoter and leading exon (termed exon 1'). In both the canine and human Gs alpha genes, exon 1' is located 2.5 kilobases 5' of exon 1. Exon 1' does not contribute an in-frame ATG, and thus its mRNA encodes a truncated form of Gs alpha. Initiation of translation is predicted to begin at an AUG in exon 2, as demonstrated both by in vitro translation and COS cell expression studies.     

Receptor-mediated adenylyl cyclase activation through XLalpha(s), the extra-large variant of the stimulatory G protein alpha-subunit

XLalpha(s), the large variant of the stimulatory G protein alpha subunit (Gsalpha), is derived from GNAS1 through the use of an alternative first exon and promoter. Gs(alpha) and XLalpha(s) have distinct amino-terminal domains, but are identical over the carboxyl-terminal portion encoded by exons 2-13. XLalpha(s) can mimic some functions of Gs(alpha), including betagamma interaction and adenylyl cyclase stimulation. However, previous attempts to demonstrate coupling of XLalpha(s) to typically Gs-coupled receptors have not been successful. We now report the generation of murine cell lines that carry homozygous disruption of Gnas exon 2, and are therefore null for endogenous XLalpha(s) and Gs(alpha) (Gnas(E2-/E2-)). Gnas(E2-/E2-) cells transfected with plasmids encoding XLalpha(s) and different heptahelical receptors, including the beta2-adrenergic receptor and receptors for PTH, TSH, and CRF, showed agonist-mediated cAMP accumulation that was indistinguishable from that observed with cells transiently coexpressing Gs(alpha) and these receptors. Our findings thus indicate that XLalpha(s) is capable of functionally coupling to receptors that normally act via Gs(alpha).     

Characterization of the molecular mechanisms of the coupling between intracellular loops of prostacyclin receptor with the C-terminal domain of the Galphas protein in human coronary artery smooth muscle cells

The C-terminal domain of the Gs protein alpha subunit (Galphas Ct) and the first intracellular loop (iLP1) of prostacyclin receptor (IP) have been predicted to be involved in the receptor signaling mediated through the IP/Gs protein coupling by our previous NMR studies using synthetic peptides. To test whether the results of the peptide studies can be applied to the protein interaction between the IP receptor and the Gs protein in cells, a minigene technique was used to construct cDNAs that encoded either the amino acid residues of the Galphas or that of the individual intracellular loops of the IP receptor. The effects of the minigene-expressed protein fragments on cAMP production mediated by the IP/Gs coupling were evaluated through experiments that co-expressed peptides either through the Galphas Ct or the IP intracellular loops with the IP receptor in HEK293 cells. The first (iLP1) and third (iLP3) IP intracellular loops, as well as the Galphas Ct, which are important to the IP/Gs coupling-mediated signaling, were identified by the significant reduction of cAMP production when the corresponding peptides were expressed in the cells. Furthermore, the cAMP productions were significantly impaired in Galphas-knockout cells co-expressing the IP receptor with the Galphas C-terminal mutants (E392A, L393A and L394A), compared with the Galphas wild type. Blocking of the endogenous IP/Gs coupling by the minigene-expressed peptides of the Galphas CT, iLP1 and iLP3 was further observed in the human coronary artery smooth muscle cells (SMCs). These results indicate that the three residues (E392-L394) of the Galphas protein predicted from NMR peptide studies, and the IP iLP1 and iLP3 play important roles in the Galphas-mediated IP receptor signaling in the cells, which may be a general binding site for the corresponding regions of the other prostanoid receptors that couple to Gs protein.     

Ligand-induced alpha2-adrenoceptor endocytosis: relationship to Gi protein activation

Most G protein-coupled receptors are desensitized by a uniform two-step mechanism: phosphorylation followed by arrestin binding and internalization. In this study we explored the time-, ligand-, and concentration dependence of alpha2-adrenoceptor internalization in human embryonal kidney (HEK-293) cells expressing alpha2A- and alpha2B-adrenoceptors. We also explored the relationship between ligand-induced receptor internalization and agonist efficacy, determined with a [35S]GTPgammaS binding assay. The results showed rapid dose-dependent internalization of both alpha2A- and alpha2B-receptors; the extent of internalization was directly proportional to agonist efficacy. The agonist UK 14,304 had a subtype-specific high efficacy at alpha2A-AR and dexmedetomidine at alpha2B-AR. Agonist-induced [35S]GTPgammaS binding was totally blocked by pretreatment with pertussis toxin (PTX) for both receptor subtypes, while only about 50% of the internalization was blocked by PTX. The results indicate that the extent of internalization of alpha2A-AR and alpha2B-AR is proportional to agonist efficacy, but only partly dependent on Gi protein coupling.     

Existence of multiple novel Gs alpha splice variants in acute leukemia patients

The alpha subunit of the stimulatory G protein, Gs alpha, is involved in stimulation of the adenylate cyclase pathway of signal transduction. In this study, we investigated the status of the Gs alpha gene in 29 acute leukemia patients and identified three novel splice variants (designated Gs alpha L-1, Gs alpha L-2, and Gs alpha L-3), possibly derived from aberrant splicing. All of the splice variants have in-frame deletions, removing the functional domain responsible for GTPase activity of Gs alpha, and would encode truncated proteins of 160(Gs alpha L-1), 90(Gs alpha L-2) and 70(Gs alpha L-3) amino acids, respectively. The data suggest that these novel products may be implicated in an as-yet-unidentified signal transduction pathway in hematopoietic cells.     

The effects of agonist stimulation and beta(2)-adrenergic receptor level on cellular distribution of gs(alpha) protein

This study examines the effects of adrenergic ligands, cholera toxin, forskolin, and varying levels of beta(2) adrenergic receptors (beta(2)AR) on the cellular distribution of Gs(alpha) subunits in CHO cells. Localization of Gs(alpha) was evaluated by confocal microscopy and beta(2)AR-mediated signalling was assessed by adenylyl cyclase (AC) activity. In cells expressing 0.2 pmol/mg protein beta(2)ARs (WT18), the localization of Gs(alpha) subunit was restricted to the plasma membrane region. Isoproterenol (ISO), cholera toxin or forskolin elicited redistribution of cellular Gs(alpha) so that Gs(alpha) appeared as intense spots throughout the plasma membrane as well as the cytoplasm. Exposure to a neutral beta(2)AR antagonist, alprenolol, prevented the ISO-stimulated Gs(alpha) translocation from peripheral to inner cytoplasm. In cells expressing high level of beta(2)ARs (8.2 pmol/mg) (WT4), basal and ISO-stimulated AC activities were significantly elevated when compared to the values detected in WT18 clone, suggesting a positive correlation between receptor expression and receptor-mediated signalling. Basal Gs(alpha) distribution in this group was similar to that observed in ISO-, cholera toxin-, or forskolin-stimulated WT18 clone. ISO, cholera toxin, or forskolin did not change the distribution of Gs(alpha) significantly when tested in WT4 clone. No difference in the cellular level of Gs(alpha) protein between WT18 and WT4 clones was detected. Alprenolol did not affect the distribution of Gs(alpha) in WT4 clone. ICI 118,551, a negative beta(2)AR antagonist, altered Gs(alpha) distribution from a dispersed basal pattern to a membrane-confined pattern. The latter appearance was similar to that observed in unstimulated WT18 clone. Taken together, these data suggest that: (1) enhanced beta(2)AR-Gs(alpha) coupling induced by agonist stimulation or by increased expression of beta(2)ARs remodel the cellular distribution of Gs(alpha); (2) the alteration in Gs(alpha) distribution induced by beta(2)AR overexpression provides evidence for agonist-independent interaction of beta(2)AR and Gs(alpha), that can be inhibited by a negative antagonist but not by a neutral antagonist; and (3) forskolin influences the activity state of Gs(alpha) that displays a Gs(alpha) distribution pattern comparable to that observed when Gs(alpha) is activated via beta(2)AR stimulation or directly by cholera toxin.     

Molecular and immunohistochemical studies in expression of voltage-dependent Ca2+ channels in dorsal root ganglia from streptozotocin-induced diabetic mice

We have recently demonstrated that intrathecal injection of a selective P/Q-type blocker of the voltage-dependent Ca(2+) channels (VDCCs) significantly inhibited the mechanical hyperalgesia in streptozotocin (STZ)-induced diabetic mice, its antinociceptive effect being greater than in controls. In this study, to further clarify the underlying mechanism of the STZ-induced hyperalgesia, we investigated the expression level of the VDCC alpha1A and alpha1B subunits in the dorsal root ganglia (DRGs) and the dorsal spinal cord under this hyperalgesia. Real-time PCR analysis showed mRNA expression of alpha1A (P/Q-type), but not alpha1B (N-type), was significantly increased in the DRGs from the STZ-induced diabetic mice. On the other hand, gene expression of both alpha1 subunits was not changed in the dorsal part of the spinal cord. In diabetic DRG neurons, the number of large nerve cells was significantly reduced, whereas small neurons were significantly increased. Immunohistochemical study demonstrated the alpha1A-positive neurons, but not alpha1B-positive neurons, increased significantly greater in diabetic DRGs than in control in all cell size. These results indicate that an alteration in expression of P/Q-type VDCCs, especially in the small and medium-diameter primary afferent fibers, in pain pathways ascending input to the spinal cord may be involved in hypersensitivity in STZ-induced diabetes.     

Reconstitution of cyc- S49 membranes by in vitro translated Gs alpha. Membrane anchorage and functional implications

After ADP-ribosylation by cholera toxin which promotes dissociation of the subunits, the alpha-subunit of Gs (Gs alpha) remained strongly associated with plasma membranes of wild-type S49 cells, since its interaction with the membrane was insensitive to 1 M KCl. Its association with the membrane was partially disrupted by 6 M urea and totally abolished by treatment with alkali at pH greater than or equal to 11.5. In vitro translated Gs alpha could interact with plasma membranes from the cyc- mutant of S49 cells as revealed by its cosedimentation with the membrane fraction and incubation of reconstituted membranes with GTP gamma S did not alter anchorage of Gs alpha. The characteristics of the association of in vitro translated Gs alpha with cyc- membranes after GTP gamma S treatment, i.e. sensitivity to 1 M KCl, 6 M urea and alkali treatment, were very similar to those described for the ADP-ribosylated form in wild-type membranes. Restoration of the coupling between the adrenergic receptor and adenylate cyclase further confirmed the vectorial reconstitution of cyc- membranes by in vitro translated alpha-subunit of Gs.     

Protein kinase A-induced negative regulation of the corticotropin-releasing hormone R1alpha receptor-extracellularly regulated kinase signal transduction pathway: the critical role of Ser301 for signaling switch and selectivity

Activation of CRH receptors type 1 (CRH-R1) by CRH or urocortin (UCN) leads to stimulation of multiple G proteins with consequent effects on diverse signaling cascades in a tissue-specific manner. In human myometrium and human embryonic kidney (HEK)293 cells, binding of UCN to CRH-R1alpha receptors activates both the Gs and Gq, leading to activation of the adenylyl cyclase/protein kinase A (PKA) and the phospholipase C/protein kinase C and ERK1/2 signaling pathways, respectively. The overall result of these signals is often unpredictable, as these two signaling pathways can interact in many cellular systems, with either potentiation or inhibition of ERK1/2 activity. In the present studies we investigated potential signaling interactions after stimulation of CRH-R1alpha receptors in human cultured pregnant myometrial cells or HEK293 cells overexpressing recombinant CRH-R1alpha receptors. We found that the adenylyl cyclase/PKA pathway has the capacity to markedly decrease UCN-induced ERK1/2 activation, and that these effects were due in part to the ability of PKA to phosphorylate the CRH-R1alpha at position Ser(301) in the third intracellular loop. Mutant CRH-R1alpha receptors with substitutions at position Ser(301), which is the only potential PKA phosphorylation site, were resistant to PKA-dependent phosphorylation and showed altered signaling characteristics, which were dependent upon the amino acid substitution at this position.We conclude that Ser(301), which is located in the third intracellular loop of CRH-R1alpha, is critical for efficient coupling of the receptor to G proteins and to second messenger generation. Phosphorylation by PKA prevents maximal coupling of the CRH-R1alpha to Gq-protein, and thereby reduces activation of ERK 1/2.     

Peptide elongation factor eEF1A-2/S1 expression in cultured differentiated myotubes and its protective effect against caspase-3-mediated apoptosis.

Peptide elongation factor eEF1A-2/S1, which shares 92% homology with eEF1A-1/EF-1alpha, is exclusively expressed in brain, heart, and skeletal muscle. In these tissues, eEF1A-2/S1 is the only type 1A elongation factor expressed in adulthood because a transition from eEF1A-1/EF-1alpha to eEF1A-2/S1 occurs in early postnatal development. In this article, we report that the expression of eEF1A-2/S1 protein is activated upon myogenic differentiation. Furthermore, we show that upon serum deprivation-induced apoptosis, eEF1A-2/S1 protein disappears and is replaced by its homolog eEF1A-1/EF-1alpha in dying myotubes; cell death is characterized by the activation of caspase-3. In addition, we show that the continuous expression of eEF1A-2/S1 resulting from adenoviral gene transfer protects differentiated myotubes from apoptosis by delaying their death, thus suggesting a prosurvival function for eEF1A-2/S1 in skeletal muscle. In contrast, myotube death is accelerated by the introduction of the homologous gene, eEF1A-1/EF-1alpha, whereas cells transfected with antisense eEF1A-1/EF-1alpha are protected from apoptosis. These results demonstrate that the two sister genes, eEF1A-1/EF-1alpha and eEF1A-2/S1, regulate myotube survival with the former exerting prodeath activity and the latter a prosurvival effect.     

S111N mutation in the helical domain of human Gs(alpha) reduces its GDP/GTP exchange rate

G-protein alpha subunits consist of two domains: a Ras-like domain also called GTPase domain (GTPaseD), structurally homologous to monomeric G-proteins, and a more divergent domain, unique to heterotrimeric G-proteins, called helical domain (HD). G-protein activation, requires the exchange of bound GDP for GTP, and since the guanine nucleotide is buried in a deep cleft between both domains, it has been postulated that activation may involve a conformational change that will allow the opening of this cleft. Therefore, it has been proposed, that interdomain interactions are playing an important role in regulating the nucleotide exchange rate of the alpha subunit. While constructing different Gs(alpha) quimeras, we identified a Gs(alpha) random mutant, which was very inefficient in stimulating adenylyl cyclase activity. The introduced mutation corresponded to the substitution of Ser(111) for Asn (S111N), located in the carboxi terminal end of helix A of the HD, a region neither involved in AC interaction nor in the interdomain interface. In order to characterize this mutant, we expressed it in bacteria, purified it by niquel-agarose chromatography, and studied its nucleotide exchange properties. We demonstrated that the recombinant S111N Gs(alpha) was functional since it was able to undergo the characteristic conformational change upon GTP binding, detected by the acquisition of a trypsin-resistant conformation. When the biochemical properties were determined, the mutant protein exhibited a reduced GDP dissociation kinetics and as a consequence a slower GTPgammaS binding rate that was responsible for a diminished adenylyl cyclase activation when GTPgammaS was used as activator. These data provide new evidence that involves the HD as a regulator of Gs(alpha) function, in this case the alphaA helix, which is not directly involved with the nucleotide binding site nor the interdomain interface.