G protein oalpha 47A


Signaling downstream of Go alpha proteins

Potassium channels in neurons are linked by guanine nucleotide binding (G) proteins to numerous neurotransmitter receptors. The ability of Go, the predominant G protein in the brain, to stimulate potassium channels was tested in cell-free membrane patches of hippocampal pyramidal neurons. Four distinct types of potassium channels, which are otherwise quiescent, are activated by both isolated brain G0 and recombinant Go alpha. Hence brain Go can couple diverse brain potassium channels to neurotransmitter receptors (Van Dongen, 1988).

Receptors stimulating phospholipase C do so through heterotrimeric GTP-binding proteins to produce two second messengers, inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol. In spite of the detailed understanding of phospholipase C structure and phosphatidyl inositol signalling, the identity of the GTP-binding protein involved is so far unknown. To address this issue, Xenopus oocytes were used in which muscarinic receptors couple to phospholipase C through a pertussis toxin-sensitive GTP-binding protein. In this cell, InsP3 mobilizes intracellular Ca2+ to evoke a Cl- current. The magnitude of this Cl- current is proportional to the amount of InsP3 in the cell, and therefore can be used as an assay for InsP3 production. The activated alpha-subunit of the GTP-binding protein GO, when directly injected into oocytes, evokes a Cl- current by mobilizing Ca2+ from intracellular InsP3-sensitive stores. Holo-GO, when injected into oocytes, can specifically enhance the muscarinic receptor-stimulated Cl- current. These data indicate that GO can serve as the signal transducer of the receptor-regulated phospholipase C in Xenopus oocytes (Moriarty, 1990).

The inhibition of voltage-dependent Ca2+ channels in secretory cells by plasma membrane receptors is mediated by pertussis toxin-sensitive G proteins. Multiple forms of G proteins have been described, differing principally in their alpha subunits, but it has not been possible to establish which G-protein subtype mediates inhibition by a specific receptor. By intranuclear injection of antisense oligonucleotides into rat pituitary GH3 cells, the essential role of the Go-type G proteins in Ca(2+)-channel inhibition is established: the subtypes Go1 and Go2 mediate inhibition through the muscarinic and somatostatin receptors, respectively (Kleuss, 1991).

Mitogen-activated protein kinase (MAPK) is activated in response to both receptor tyrosine kinases and G-protein-coupled receptors. Recently, Gi-coupled receptors, such as the alpha 2A adrenergic receptor, have been shown to mediate Ras-dependent MAPK activation via a pathway requiring G-protein beta gamma subunits (G beta gamma) and many of the same intermediates involved in receptor tyrosine kinase signaling. In contrast, Gq-coupled receptors, such as the M1 muscarinic acetylcholine receptor (M1AChR), activate MAPK via a pathway that is Ras-independent but requires the activity of protein kinase C (PKC). In Chinese hamster ovary cells, the M1AChR and platelet-activating factor receptor (PAFR) mediate MAPK activation via the alpha-subunit of the G(o) protein. G(o)-mediated MAPK activation is sensitive to treatment with pertussis toxin but insensitive to inhibition by a G beta gamma-sequestering peptide (beta ARK1ct). M1AChR and PAFR catalyze G(o) alpha-subunit GTP exchange, and MAPK activation can be partially rescued by a pertussis toxin-insensitive mutant of G(o) alpha but not by similar mutants of Gi. G(o)-mediated MAPK activation is insensitive to inhibition by a dominant negative mutant of Ras (N17Ras) but is completely blocked by cellular depletion of PKC. Thus, M1AChR and PAFR, which have previously been shown to couple to Gq, are also coupled to G(o) to activate a novel PKC-dependent mitogenic signaling pathway (Van Biesen, 1996).

Heterotrimeric G proteins, composed of G alpha and G betagamma subunits, transmit signals from cell surface receptors to cellular effector enzymes and ion channels. The G alpha(o) protein is the most abundant G alpha subtype in the nervous system, but it is also found in the heart. Its function is not completely known, although it is required for regulation of N-type Ca2+ channels in GH3 cells and also interacts with GAP43, a major protein in growth cones, suggesting a role in neuronal pathfinding. To analyze the function of G alpha(o), mice lacking both isoforms of G alpha(o) have been generated by homologous recombination. Surprisingly, the nervous system is grossly intact, despite the fact that G alpha(o) makes up 0.2-0.5% of brain particulate protein and 10% of the growth cone membrane. The G alpha(o)-/- mice do suffer tremors and occasional seizures, but there is no obvious histologic abnormality in the nervous system. In contrast, G alpha(o)-/- mice have a clear and specific defect in ion channel regulation in the heart. Normal muscarinic regulation of L-type calcium channels in ventricular myocytes is absent in the mutant mice. The L-type calcium channel responds normally to isoproterenol, but there is no evident muscarinic inhibition. Muscarinic regulation of atrial K+ channels is normal, as is the electrocardiogram. The levels of other G alpha subunits (G alphas, G alphaq, and G alphai) are unchanged in the hearts of G alpha(o)-/- mice, but the amount of G betagamma is decreased. Whichever subunit, G alpha(o) or G betagamma, carries the signal forward, these studies show that muscarinic inhibition of L-type Ca2+ channels requires coupling of the muscarinic receptor to G alpha(o). Other cardiac G alpha subunits cannot substitute (Valenzuela, 1997).

Chemoelectrical signal transduction in olfactory neurons appears to involve intracellular reaction cascades mediated by heterotrimeric GTP-binding proteins. In this study attempts were made to identify the G protein subtype(s) in olfactory cilia that are activated by the primary (odorant) signal. Antibodies directed against the alpha subunits of distinct G protein subtypes interfer specifically with second messenger reponses elicited by defined subsets of odorants; odor-induced cAMP-formation is attenuated by Galphas antibodies, whereas Galphao antibodies block odor-induced inositol 1,4, 5-trisphosphate (IP3) formation. Activation-dependent photolabeling of Galpha subunits with [alpha-32P]GTP azidoanilide followed by immunoprecipitation using subtype-specific antibodies enables identification of particular individual G protein subtypes that are activated upon stimulation of isolated olfactory cilia by chemically distinct odorants. For example, odorants that elicite a cAMP response result in labeling of a Galphas-like protein, whereas odorants that elicite an IP3 response lead to the labeling of a Galphao-like protein. Since odorant-induced IP3 formation is also blocked by Gbeta antibodies, activation of olfactory phospholipase C might be mediated by betagamma subunits of a Go-like G protein. These results indicate that different subsets of odorants selectively trigger distinct reaction cascades and provide evidence for dual transduction pathways in olfactory signaling (Schandar, 1998).

Heterotrimeric G-proteins, composed of alpha and betagamma subunits, transmit signals from cell-surface receptors to cellular effectors and ion channels. Cellular responses to receptor agonists depend on not only the type and amount of G-protein subunits expressed but also the ratio of alpha and betagamma subunits. Thus far, little is known about how the amounts of alpha and betagamma subunits are coordinated. Targeted disruption of the alpha(o) gene leads to loss of both isoforms of alpha(o), the most abundant alpha subunit in the brain. Loss of alpha(o) protein in the brain is accompanied by a reduction of beta protein to 32% of wild type. Sucrose density gradient experiments show that all of the betagamma remaining in the brains of alpha(o)-/- mice sediments as a heterotrimer, with no detectable free alpha or betagamma subunits. Thus, the level of the remaining betagamma subunits matches that of the remaining alpha subunits. Protein levels of alpha subunits other than alpha(o) are unchanged, suggesting that they are controlled independently. Coordination of betagamma to alpha occurs posttranscriptionally because the mRNA level of the predominant beta1 subtype in the brains of alpha(o)-/- mice was unchanged. Adenylyl cyclase can be positively or negatively regulated by betagamma. Because the level of other alpha subunits is unchanged and alpha(o) itself has little or no effect on adenylyl cyclase, it was possible to examine how a large change in the level of betagamma affects this enzyme. Surprisingly, no differences were detected in the adenylyl cyclase activity between brain membranes from wild-type and alpha(o)-/- mice. It is proposed that alpha(o) and its associated betagamma are sequestered in a distinct pool of membranes that does not contribute to the regulation of adenylyl cyclase (Mende, 1998).

G alpha o mediates WNT-JNK signaling through dishevelled 1 and 3, RhoA family members, and MEKK 1 and 4 in mammalian cells

In Drosophila, activation of Jun N-terminal Kinase (JNK) mediated by Frizzled and Dishevelled leads to signaling linked to planar cell polarity. A biochemical delineation of WNT-JNK planar cell polarity was sought in mammalian cells, making use of totipotent mouse F9 teratocarcinoma cells that respond to WNT3a via Frizzled-1. The canonical WNT-β-catenin signaling pathway requires both Gαo and Gαq heterotrimeric G-proteins, whereas this study shows that WNT-JNK signaling requires only Gαo protein. Gαo propagates the signal downstream through all three Dishevelled isoforms, as determined by epistasis experiments using the Dishevelled antagonist Dapper1 (DACT1). Suppression of either Dishevelled-1 or Dishevelled-3, but not Dishevelled-2, abolishes WNT3a activation of JNK. Activation of the small GTPases RhoA, Rac1 and Cdc42 operates downstream of Dishevelled, linking to the MEKK 1/MEKK 4-dependent cascade, and on to JNK activation. Chemical inhibitors of JNK (SP600125), but not p38 (SB203580), block WNT3a activation of JNK, whereas both the inhibitors attenuate the WNT3a-β-catenin pathway. These data reveal both common and unique signaling elements in WNT3a-sensitive pathways, highlighting crosstalk from WNT3a-JNK to WNT3a-β-catenin signaling (Bikkavilli, 2008).

Antagonism between Go alpha proteins and other G proteins

To elucidate the cellular role of the heterotrimeric G protein G(o), a molecular genetic approach has been taken in Caenorhabditis elegans. A screen was carried out for suppressors of activated GOA-1 (G(o)alpha) that do not simply decrease its expression. Mutations were found in only two genes, sag-1 and eat-16. Animals defective in either gene display a hyperactive phenotype similar to that of goa-1 loss-of-function mutants. Double-mutant analysis indicates that both sag-1 and eat-16 act downstream of, or parallel to, G(o)alpha and negatively regulate EGL-30 (G(q)alpha) signaling. eat-16 encodes a regulator of G protein signaling (RGS) most similar to the mammalian RGS7 and RGS9 proteins and this RGS protein inhibit endogenous mammalian G(q)/G(11) in COS-7 cells. Animals defective in both sag-1 and eat-16 are inviable, but reducing function in egl-30 restores viability, indicating that the lethality of the eat-16;sag-1 double mutant is due to excessive G(q)alpha activity. Analysis of these mutations indicates that the G(o) and G(q) pathways function antagonistically in C. elegans, and that G(o)alpha negatively regulates the G(q) pathway, possibly via EAT-16 or SAG-1. It is proposed that a major cellular role of G(o) is to antagonize signaling by G(q) (Hajdu-Cronin, 1999).

Go alpha proteins and exocytosis

The use of non-hydrolyzable analogues of GTP in permeabilized secretory cells suggests that guanine nucleotide-binding regulatory proteins (G proteins) may be involved in regulated exocytosis. Because GTP analogues are known to modulate both monomeric low molecular mass G proteins and heterotrimeric G proteins, the effect of mastoparan, an activator of heterotrimeric G proteins, was examined on secretion from intact and permeabilized chromaffin cells. In intact cells, mastoparan inhibits catecholamine secretion evoked by nicotine but had no effect on release induced by other secretagogues. In permeabilized cells, mastoparan inhibits calcium-dependent secretion providing that the pores created in the plasma membrane allow the penetration of the peptide into the cytoplasm. These results indicate that mastoparan blocks the exocytotic machinery through an intracellular target protein that may not be located just beneath the plasma membrane. Accordingly, mastoparan was able to stimulate G proteins associated with purified chromaffin granule membranes, in a range of concentration and Mg2+ requirement that is similar to its inhibitory effect on secretion. Mas 17, a mastoparan analogue inactive on purified G proteins, neither modified catecholamine secretion nor stimulated chromaffin granule G proteins. The substance P-related peptide, GPAnt-2, known to antagonize the effects of mastoparan on G(o), blocks both the inhibitory effect of mastoparan on secretion and the mastoparan-stimulated GTPase activity in chromaffin granule membranes. Moreover, specific antibodies raised against the carboxyl terminus of G(o) alpha reverses in a dose-dependent manner the inhibition by mastoparan on catecholamine release and the stimulation by mastoparan of chromaffin granule-associated G proteins. These results suggest that the secretory machinery in chromaffin cells can be blocked by activating a G(o) protein. Consistent with this finding, two other known activators of heterotrimeric G proteins, aluminum fluoride and benzalkonium chloride, inhibit calcium-evoked catecholamine secretion in streptolysin O-permeabilized chromaffin cells. It is concluded that an inhibitory G(o) protein, possibly located on the membrane of secretory granules, is involved in the final stages of exocytosis in chromaffin cells (Vitale, 1993).

The exocytotic release of potent hormones is a tightly controlled process. Its direct regulation without the involvement of second messengers would ensure rapid signal processing. In streptolysin O-permeabilized insulin-secreting cells, a preparation allowing dialysis of cytosolic macromolecules, activation of alpha 2-adrenergic receptors caused pertussis toxin-sensitive inhibition of calcium-induced exocytosis. This inhibition was mimicked very efficiently by the use of specific receptor-mimetic peptides, indicating the involvement of Gi and, to a lesser extent, of G(o). The regulation was exerted beyond the ATP-dependent step of exocytosis. In addition, low nanomolar amounts of pre-activated Gi/G(o) directly inhibit exocytosis. As transient overexpression of constitutively active mutants of G alpha i1, G alpha i2, G alpha i3 and G alpha o2 but not of G alpha o1 reproduces this regulation, the G alpha subunit alone is sufficient to induce inhibition. These results define exocytosis as an effector for heterotrimeric G-proteins and delineate the properties of the transduction pathway (Lang, 1995).

Heterotrimeric Go proteins have recently been described as regulators of vesicular traffic. The Goalpha gene encodes, by alternative splicing, two Goalpha polypeptides, Go1alpha and Go2alpha. By immunofluorescence and electron microscopy, Go1alpha has been detected on the membrane of small intracellular vesicles in C6 glioma cells. After stable transfection of these cells, overexpression of Go1alpha but not Go2alpha was followed by a rise in the secretion of a serine protease inhibitor, protease nexin-1 (PN-1). This secretion is enhanced as a function of the amount of expressed Go1alpha. Metabolic cell labeling indicates that this increase in PN-1 secretion is not the result of an enhancement in PN-1 biosynthesis or a decrease in its uptake, but reveals a potential role of Go1alpha in the regulation of vesicular PN-1 trafficking. Furthermore, activators of Go proteins, mastoparan and a peptide derived from the amino terminus of the growth cone-associated protein GAP43, increased PN-1 secretion in parental and Go1alpha-overexpressing cells. Brefeldin A, an inhibitor of vesicular traffic, inhibits both basal and mastoparan-stimulated PN-1 secretions. These results indicate, that in C6 glioma cells, PN-1 secretion can be regulated by both Go1alpha expression and activation (Lagriffoul, 1996).

Besides having a role in signal transduction, heterotrimeric G proteins may be involved in membrane trafficking events. In chromaffin cells, Go is associated with secretory organelles, and its activation inhibits the ATP-dependent priming of exocytosis. The control exerted by the granule-bound Go on exocytosis may be related to effects on the cortical actin network through a sequence possibly involving Rho. To provide further insight into the function of Rho in exocytosis, its intracellular localization in chromaffin cells was examined. By immunoreplica analysis, immunoprecipitation, and confocal immunofluorescence, it was found that RhoA is specifically associated with the membrane of secretory chromaffin granules. Parallel subcellular fractionation experiments reveal the occurrence of a mastoparan-stimulated phosphatidylinositol 4-kinase activity in purified chromaffin granule membranes. This stimulatory effect of mastoparan is mimicked by GAP-43, an activator of the granule-associated Go, and specifically inhibited by antibodies against Galphao. In addition, Clostridium botulinum C3 exoenzyme completely blocks the activation of phosphatidylinositol 4-kinase by mastoparan. It is proposed that the control exerted by Go on peripheral actin and exocytosis is related to the activation of a downstream RhoA-dependent phosphatidylinositol 4-kinase associated with the membrane of secretory granules (Gasman, 1998).

G protein oalpha 47A Evolutionary homologs part 1/3 | part 2/3 |

G protein oalpha 47A: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.