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 alpha_s, G alpha_q, and G alpha_i) 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).

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 Gα_o and Gα_q heterotrimeric G-proteins, whereas this study shows that WNT-JNK signaling requires only Gα_o protein. Gα_o 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).

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).