G protein salpha 60A

EVOLUTIONARY HOMOLOGS (part 2/2)

G protein salphas target adenylyl cyclase

The alpha subunit (Gsalpha) of the stimulatory heterotrimeric guanosine triphosphate binding protein [G protein) activates all isoforms of mammalian adenylyl cyclase. Adenylyl cyclase (Type V) and its subdomains, which interact with Gsalpha, promote inactivation of the G protein by increasing its guanosine triphosphatase (GTPase) activity. Adenylyl cyclase and its subdomains also augment the receptor-mediated activation of heterotrimeric Gs and thereby facilitate the rapid onset of signaling. These findings demonstrate that adenylyl cyclase functions as a GTPase activating protein (GAP) for the monomeric Gsalpha and enhances the GTP/GDP exchange factor (GEF) activity of receptors (Scholich, 1999).

Hormonal signals activate trimeric G proteins by substituting GTP for GDP bound to the G protein alpha subunit (Galpha), thereby generating two potential signaling molecules, Galpha-GTP and free Gbetagamma. A dominant negative Galpha mutation was created. A mutant alpha subunit is described that is designed to inhibit receptor-mediated hormonal activation of Gs, the stimulatory regulator of adenylyl cyclase. To construct this mutant, three separate mutations chosen because they impair alphas function in complementary ways were introduced into the alpha subunit (alphas) of Gs: the A366S mutant reduces affinity of alphas for binding GDP, whereas the G226A and E268A mutations impair the protein's ability to bind GTP and to assume an active conformation. The triple mutant robustly inhibits (by up to 80%) Gs-dependent hormonal stimulation of adenylyl cyclase in cultured cells. Inhibition is selective in that it does not affect cellular responses to expression of a constitutively active alphas mutant (alphas-R201C) or to agonists for receptors that activate Gq or Gi. This alphas triple mutant and cognate Galpha mutants should provide specific tools for dissection of G protein-mediated signals in cultured cells and transgenic animals (Iiri, 1999).

Circadian functions of the suprachiasmatic nuclei (SCN) are influenced by cyclic AMP (cAMP). Adenylyl cyclase type II (AC-II) is a cAMP-generating enzyme that, in the context of activation by Gsalpha, is further stimulated by protein kinase C or G protein betagamma subunits. Using in situ hybridization a biphasic variation in AC-II mRNA was found within the rat SCN during the light-dark cycle (peaks at Zeitgeber time 6 and 18) and also in constant darkness (peaks at circadian time 2 and 14). The cingulate cortex shows no such variation. These findings suggest that circadian changes in AC-II expression may be pertinent to the rhythmic functions of the SCN (Cagampang, 1998).

The neurochemical basis was investigated for predominance of stimulatory mu-opioid signaling in guinea pig longitudinal muscle/myenteric plexus (LMMP) preparations after chronic in vivo morphine exposure. In a dose dependent manner, recombinant Gsalpha (rGsalpha) stimulates adenylyl cyclase (AC) activity in LMMP membranes obtained from opioid naive as well as tolerant LMMP tissue. However, the magnitude of the increase is significantly greater in the latter than in the former. The Gbetagamma blocking peptide QEHA essentially abolishes stimulation by rGsalpha in LMMP membranes obtained from both opioid naive and tolerant animals. Interestingly, after partial blockade by lower QEHA concentrations, the incremental AC stimulation by rGsalpha in tolerant LMMP membranes is no longer observed, indicating augmented Gbetagamma stimulatory responsiveness. Concomitant changes in the content of AC isoform protein are consistent with these biochemical observations. After chronic systemic morphine, AC protein is augmented significantly (56%). This increment is most likely to be composed of AC isoforms that are stimulated by Gbetagamma. This is the first demonstration in a complex mammalian tissue that persistent activation of opioid receptors results in augmented Gbetagamma/Gsalpha AC stimulatory interactiveness (Chakrabarti, 1998).

The beta2-adrenoceptor (beta2AR) activates the G-protein Gsalpha to stimulate adenylate cyclase (AC). Fusion of the beta2AR C-terminus to the N-terminus of Gsalpha (producing beta2ARGsalpha) markedly increases the efficiency of receptor/G-protein coupling, as compared with the non-fused state. This increase in coupling efficiency can be attributed to the physical proximity of receptor and G-protein. To determine the optimal length for the tether between receptor and G-protein, fusion proteins were constructed from which 26 [beta2AR(Delta26)Gsalpha] or 70 [beta2AR(Delta70)Gsalpha] residues of the beta2AR C-terminus had been deleted and the properties of these fusion proteins were compared with the beta2ARGsalpha. Compared with beta2ARGsalpha, basal and agonist-stimulated GTP hydrolysis is markedly decreased in beta2AR(Delta70)Gsalpha, whereas the effect of the deletion on binding of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) was relatively small. Surprisingly, deletions do not alter the efficiency of coupling of the beta2AR to Gsalpha as assessed by GTP[S]-sensitive high-affinity agonist binding. Moreover, basal and ligand-regulated AC activities in membranes expressing beta2AR(Delta70)Gsalpha and beta2AR(Delta26)Gsalpha are higher than in membranes expressing beta2ARGsalpha. These findings suggest that restricting the mobility of Gsalpha relative to the beta2AR results in a decrease in G-protein inactivation by GTP hydrolysis and thereby enhances activation of AC (Wenzel-Seifert, 1998).

Substitution of Arg258 within the switch 3 region of Gsalpha impairs activation and increases basal GDP release due to loss of an interaction between the helical and GTPase domains. The adjacent residue (Glu259) is strictly conserved in G protein alpha-subunits and is predicted to be important in activation. To determine the importance of Glu259, this residue was mutated to Ala (Gsalpha-E259A), Gln (Gsalpha-E259Q), Asp (Gsalpha-E259D), or Val (Gsalpha-E259V), and the properties of in vitro translation products were examined. The Gsalpha-E259V was studied because this mutation was identified in a patient with Albright hereditary osteodystrophy. Gsalpha-E259D stimulates adenylyl cyclase normally in the presence of GTPgammaS but is less efficient with beta-adrenergic agonist isoproterenol or GTP analog AlF4-. The other mutants have more severely impaired effector activation, particularly in response to AlF4-. In trypsin protection assays, GTPgammaS is a more effective activator than AlF4- for all mutants, with Gsalpha-E259D being the least severely impaired. For Gsalpha-E259D, the AlF4--induced activation defect is more pronounced at low Mg2+ concentrations. Gsalpha-E259D and Gsalpha-E259A purified from Escherichia coli have normal rates of GDP release (as assessed by the rate of GTPgammaS binding). However, for both mutants, the ability of AlF4- to decrease the rate of GTPgammaS binding is impaired, suggesting that neither binds AlF4- well. GTPgammaS binds to purified Gsalpha-E259D irreversibly in the presence of 1 mM free Mg2+, but dissociates readily at micromolar concentrations. Sucrose density gradient analysis of in vitro generated protein demonstrates that all mutants except Gsalpha-E259V bind to beta gamma at 0 degrees C and are stable at higher temperatures. In the active conformation Glu259 interacts with conserved residues in the switch 2 region that are important in maintaining both the active state and AlF4- in the guanine nucleotide binding pocket. Although both Gsalpha Arg258 and Glu259 are critical for activation, the mechanisms by which these residues affect Gsalpha protein activation are distinct (Warner, 1999).

G protein salpha targets Ca²⁺ channels

During early embryonic development, IP₃-Ca²⁺ signaling transduces ventral signaling at the time of dorsoventral axis formation. To identify molecules functioning upstream in this signal pathway, effects were measured of a panel of inhibitory antibodies against Galphaq/11, Galphas/olf, or Galphai/o/t/z. While all these antibodies show direct inhibition of their targets, their effects varied in terms of the redirection of ventral mesoderm to a dorsal fate. Anti-Galphas/olf antibody shows strong induction of dorsal fate; anti-Galphai/o/t/z antibody does so weakly, and anti-Galphaq/11 antibody is without effect. Injection of betaARK, a Gbetagamma inhibitor, mimics the dorsalizing effect of anti-Galphas/olf antibody, whereas injection of adenylyl cyclase inhibitors at a concentration that inhibits Galphas-coupled cAMP increase does not do so. The activation of Galphas-coupled receptor gives rise to Ca²⁺ transients. All these results suggest that activation of the Galphas-coupled receptor relays dorsoventral signal to Gbetaggamma, which then stimulates PLCbeta and then the IP₃-Ca²⁺ system. This signaling pathway may play a crucial role in transducing ventral signals (Kume, 2000).

In Xenopus, patterning of the body axis occurs by sequential inductive events. Maternal activation of the Wnt pathway is required for the initiation of axis formation, by creating a Nieuwkoop center and mediating the dorsalizing function of the Nieuwkoop center. The Spemann organizer of the Xenopus embryo can be subdivided into two discrete activities: trunk organizer and head organizer. The molecular mechanism of trunk organizer formation involves several factors secreted from the blastopore lip that act by repressing signaling by bone morphogenetic proteins (BMPs), which antagonize the organizer. The finding that anti-Galphas/olf antibody or betaARK injection induces trunk but not head organizer is similar to a previous result using anti-IP3R inhibitory antibodies. These results correlate with the facts that the secondary axes induced by Noggin, Chordin, and truncated BMP receptors often lack the anteriormost structures and that head induction requires simultaneous repression of BMP and Wnt signaling in Xenopus. The IP₃-Ca²⁺ signaling system may crosstalk with the BMP pathway, by means of a mechanism which still remains unknown (Kume, 2000).

There are other candidates for upstream factors that may activate the IP₃-Ca²⁺ signaling system. Zygotic activation of the Wnt pathway is suggested to be required for ventro-lateral mesoderm formation. Members of the Wnt-5a class of proteins do not induce ectopic dorsal axis duplication, unless coexpressed with certain members of the frizzled family, yet they do decrease cell adhesion and perturb morphogenetic movement during gastrulation in Xenopus embryos. There is evidence that the Wnt-5a class can function in a non-cell-autonomous manner to block the ability of members of the Wnt-1 class to induce a secondary axis. Overexpression of Xwnt-5a with rat frizzled-2 increases the frequencies of Ca²⁺ spikes in zebrafish. Activation of Galphas/olf-coupled receptor elicits Ca²⁺ transients of an interval that resembles one reported for Xwnt-5a. It will be of interest to determine whether Xwnt-5a is the endogenous upstream ligand that activates IP₃-Ca²⁺ signaling during embryonic development (Kume, 2000).

The downstream targets of the Ca²⁺ transients during early embryonic axis formation remain largely unknown. There is evidence that varying the frequency or intensity of Ca²⁺ transients can alter the physiological output. One well-known example of molecules modulated by frequency of Ca²⁺ is calmodulin-dependent kinase II, which regulates other enzymes dependent on Ca²⁺. The enzyme is activated to varying degrees depending on the frequency of Ca²⁺ oscillations. Varying the frequency or intensity of the Ca²⁺ rise can contribute to activation of different subsets of developmental genes. Examinations of the types of IP₃-Ca²⁺ signaling that activate separate sets of genes and, in turn, lead to a specific developmental program, are expected to elucidate the mechanism underlying the early developmental events (Kume, 2000).

G proteins and differentiation

Fully-differentiated mouse 3T3-L1 fibroblasts accumulate large amounts of lipid at 7-10 days after induction by insulin or by dexamethasone and a methyl xanthine. G proteins mediate transmembrane signalling from a diverse group of cell-surface receptors to effector units that include phospholipase C, adenylyl cyclase and ion channels. They are also targets of regulation themselves. 3T3-L1 fibroblasts display marked changes in levels of G protein when induced to differentiate to adipocytes. Cholera toxin, which ADP-ribosylates and activates the G protein subunit Gs alpha, blocks the induction of differentiation, whereas increasing intracellular cyclic AMP directly with the dibutyryl analogue or indirectly with pertussis toxin or forskolin does not affect differentiation. Oligodeoxynucleotides antisense to the sequence encoding Gs alpha accelerate differentiation markedly. The time course of adipogenesis declined from 7-10 days in controls to roughly 3 days in cultures treated with antisense-Gs alpha oligodeoxynucleotides, whereas oligodeoxynucleotides, antisense to Gi alpha 1, Gi alpha 3, and sense and missense to Gs alpha, had no such effect. Antisense-Gs alpha alone induced differentiation by day 7, indicating that Gs alpha activity modulates differentiation in 3T3-L1 cells, acting in a new role which is independent of increased intracellular cAMP (Wang, 1992).

Retinoic acid induces the differentiation of P19 mouse embryonal carcinoma cells into endoderm, and increases expression of the heterotrimeric G-protein subunits Galpha12 and Galpha13. Retinoic acid was found to induce differentiation and sustain activation of c-Jun amino-terminal kinase, but not for ERK1,2 or of p38 mitogen-activated protein kinases. Much like retinoic acid, expression of constitutively active forms of Galpha12 and Galpha13 induce differentiation and constitutive activation of c-Jun amino-terminal kinase. Expression of the dominant negative form of c-Jun amino-terminal kinase 1 blocks both the activation of c-Jun amino-terminal kinase and the induction of endodermal differentiation in the presence of retinoic acid. These data implicate c-Jun amino-terminal kinase as downstream of Galpha12 or Galpha13 and as obligate for retinoic acid-induced differentiation (Jho, 1997).

Receptors coupled to heterotrimeric G proteins can effectively stimulate growth promoting pathways in a large variety of cell types, and if persistently activated, these receptors can also behave as dominant-acting oncoproteins. Consistently, activating mutations for G proteins of the Galphas and Galphai2 families have been found in human tumors; members of the Galphaq and Galpha12 families are fully transforming when expressed in murine fibroblasts. In an effort aimed to elucidate the molecular events involved in proliferative signaling through heterotrimeric G proteins, this study has focused on gene expression regulation. Using NIH 3T3 fibroblasts expressing m1 muscarinic acetylcholine receptors as a model system, it was observed that activation of these transforming G protein-coupled receptors induces the rapid expression of a variety of early responsive genes, including the c-fos protooncogene (see Drosophila Fos). One of the c-fos promoter elements, the serum response element (SRE), plays a central regulatory role; activation of SRE-dependent transcription has been found to be regulated by several proteins, including the serum response factor and the ternary complex factor. Stimulation of m1 muscarinic acetylcholine receptors potently induces SRE-driven reporter gene activity in NIH 3T3 cells. In these cells, only the Galpha12 family of heterotrimeric G protein alpha subunits strongly induces the SRE, while Gbeta1gamma2 dimers activate SRE to a more limited extent. M1, Galpha12 and the small GTP-binding protein RhoA are components of a novel signal transduction pathway that leads to the ternary complex factor-independent transcriptional activation of the SRE and to cellular transformation (Fromm, 1997).

G protein coupled receptors, long term potentiation and behavior

Agonists of the dopamine D1/D5 receptors that are positively coupled to adenylyl cyclase specifically induce a slowly developing long-lasting potentiation of the field excitatory postsynaptic potential in the CA1 region of the hippocampus that lasts for more than 6 hr. This potentiation is blocked by a specific D1/D5 receptor antagonist and is occluded by the potentiation induced by cAMP agonists. An agonist of the D2 receptor, which is negatively coupled to adenylyl cyclase through G ialpha, does not induce potentiation. Although this slow D1/D5 agonist-induced potentiation is partially independent of N-methyl-D-aspartate receptors, it seems to share some steps with and is occluded by the late phase of long-term potentiation (LTP) produced by three repeated trains of nerve stimuli applied to the Schaffer collateral pathway. Similarly, a D1/D5 antagonist attenuates the late phase of LTP, induced by repeated trains, and the D1/D5 agonist-induced potentiation is blocked by the protein synthesis inhibitor anisomycin. These results suggest that the D1/D5 receptor may be involved in the late, protein synthesis-dependent component of LTP in the hippocampal CA1 region, either as an ancillary component or as a mediator directly contributing to the late phase (Huang, 1995).

Metabotropic glutamate receptors (mGluRs) are critically involved in the maintenance of long-term potentiation. In order to assess further the physiological role of MGluRs in LTP, freely moving rats were injected with a competitive mGluR antagonist (MCPG) and then recordings were made from granule cells of the dentate gyrus in response to stimulation of fibers of the perforant path. Normal synaptic transmission is not altered by MCPG. However, the mGluR antagonist inhibits LTP in a concentration-dependent manner. MCPG concentrations have no influence on the time course of preestablished LTP. These results corroborate recent findings that mGluRs are required for the induction of LTP in CA1 and CA3. The process of STP is found to be independent of mGluR activation (Riedel, 1995).

Mice lacking the alpha-subunit of the heterotrimeric guanine nucleotide binding protein Gq (Galphaq) are viable but suffer from ataxia with typical signs of motor discoordination. The anatomy of the cerebellum is not overtly disturbed, and excitatory synaptic transmission from parallel fibers to cerebellar Purkinje cells (PCs) and from climbing fibers (CFs) to PCs is functional. However, about 40% of adult Galphaq mutant PCs remain multiply innervated by CFs because of a defect in regression of supernumerary CFs in the third postnatal week. Evidence is provided suggesting that Galphaq is part of a signaling pathway that is involved in the elimination of multiple CF innervation during this period (Offermanns, 1997).

G proteins and transformation

The heterotrimeric G-protein, G alpha12, together with the closely-related G alpha13, are both members of the G12 class of alpha-subunits important in mediating the signaling from seven transmembrane domain-spanning receptors. Recent evidence implicating both G alpha12 and G alpha13 in the activation of signaling pathways involving members of the RHO gene family has led to an examination of the role of Rac1, RhoA and Cdc42Hs in the transforming properties of G alpha12. Asparagine 17 (Asn 17) dominant inhibitory mutants of Rac1, and to a lesser extent RhoA, block focus forming ability of the GTPase-deficient mutant of G alpha12 (G alpha12 Leu 229) in NIH3T3 cells. In turn, wild-type G alpha12 cooperates well with Rac1 Val 12 but not with the RhoA Leu 63 mutant in transforming NIH3T3 cells. Interestingly, the morphology of foci induced by G alpha12 and RhoA mutants is strikingly similar and is distinct from those displayed by Rac1 Val 12 mutant. The fact that G alpha12's ability to induce mitogenesis in NIH3T3 cells is not significantly perturbed by C3 ribosyltransferase suggests that RhoA does not play a major role in G alpha12-induced mitogenic events. Activated mutant of Rac1 stimulates the activity of the stress-induced c-Jun N-terminal kinase/stress-activated protein kinases (JNK/SAPKs). Transient co-transfection of Rac1 Val 12 mutant with the wild-type G alpha12 in COS7 cells leads to the further activation of an exogenously expressed hemagglutinin(HA)-tagged JNK. Furthermore, the cooperation between G alpha12 and Rac1 in cellular transformation is correlated with their ability to stimulate transcription from c-fos serum response element (SRE) (Tolkacheva, 1997).

Restoration of the GTPase activity and cellular interactions of Gα(o) mutants by Zn(2+) in GNAO1 encephalopathy models

De novo point mutations in GNAO1, gene encoding the major neuronal G protein Gα(o), have recently emerged in patients with pediatric encephalopathy having motor, developmental, and epileptic dysfunctions. Half of clinical cases affect codons Gly(203), Arg(209), or Glu(246); these mutations accelerate GTP uptake and inactivate GTP hydrolysis through displacement Gln(205) critical for GTP hydrolysis, resulting in constitutive GTP binding by Gα(o). However, the mutants fail to adopt the activated conformation and display aberrant interactions with signaling partners. Through high-throughput screening of approved drugs, rhis study identifie zinc pyrithione and Zn(2+) as agents restoring active conformation, GTPase activity, and cellular interactions of the encephalopathy mutants, with negligible effects on wild-type Gα(o). A Drosophila model is described of GNAO1 encephalopathy where dietary zinc restores the motor function and longevity of the mutant flies. Zinc supplements are approved for diverse human neurological conditions. This work provides insights into the molecular etiology of GNAO1 encephalopathy and defines a potential therapy for the patients (Larasati, 2022).

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