G protein oalpha 47A


Interaction of Go alpha proteins with G beta subunits

The gamma subunit composition of the major bovine brain Go and Gi proteins (GOA, GOB, GOC, Gi1, and Gi2) was characterized using antibodies against specific gamma isoforms. Each of the purified G protein heterotrimers contain a heterogeneous population of gamma subunits, and the profiles of the gamma subunits found with Gi1, Gi2, and GOA are similar. In contrast, each GO isoform has a distinct pattern of associated gamma subunits. These differences are surprising given that all three alpha O isoforms are thought to share a common amino-terminal sequence important for the binding of beta gamma dimers and that the alpha OA and alpha OC proteins may come from the same alpha O1 mRNA. The free alpha OA and alpha OC subunits have unique elution behaviors during MonoQ chromatography, compatible with differences in their post-translational processing. These results indicate that both the alpha and gamma subunit compositions of heterotrimers define the structure of an intact G protein. Furthermore, the exact subunit composition of G protein heterotrimers may depend upon regulated expression of different subunit isoforms or upon cellular processing of alpha subunits (Wilcox, 1995).

The beta-subunit has marked effects on the biophysical and pharmacological properties of voltage-dependent calcium channels. In the present study, an examination was carried out of the ability of the GABAB agonist (-) -baclofen to inhibit calcium channel currents in cultured rat dorsal root ganglion neurones following depletion of beta-subunit immunoreactivity, 108-116 h after microinjection of a beta-subunit antisense oligonucleotide. Although the calcium channel current is markedly reduced in amplitude following beta-subunit depletion, the residual current (comprising both N- and L-type calcium channel currents) shows an enhanced response to application of (-) -baclofen. Therefore, it is possible that there is normally competition between activated G protein G(o) and the calcium channel beta-subunit for binding to the calcium channel alpha 1-subunit; and this competition shifts in favour of the binding of activated G(o) following depletion of the beta-subunit, resulting in increased inhibition. This hypothesis is supported by evidence that an antibody against the calcium channel beta-subunit completely abolishes stimulation of the GTPase activity of G(o) by the dihydropyridine agonist S-(-) -Bay K 8644 in brain membranes. This stimulation of GTPase is thought to result from an interaction of G(o) alpha-subunit (G alpha o) with its calcium channel effector which may operate as a GTPase-activating protein. These data suggest that the calcium channel beta-subunit when complexed with the beta 1-subunit normally inhibits its association with activated G(o). It may function as a GTPase-activating protein to reduce the ability of activated G(o) to associate with the calcium channel, and thus limit the efficacy of agonists such as (-) -baclofen (Campbell, 1999).

The distribution and properties in brain of the alpha subunits of the major bovine brain Go isoforms, GoA, GoB and GoC, were characterized. The alpha(o)A and alpha(o)B isoforms arise from alternative splicing of RNAs from a single alpha(o) gene, whereas alpha(o)C is a deamidated form of alpha(o)A. All three Go isoforms purify from brain with different populations of betagamma dimers. This variable subunit composition of Go heterotrimers is likely a consequence of their functional differences. This study examined the biochemical properties of the alpha(o) isoforms to see if these properties explain the variable betagamma composition of their heterotrimers. The brain distribution of alpha(o)B differs substantially from that of alpha(o)A and alpha(o)C, as does its guanine nucleotide binding properties. The unique subunit composition of GoB can be explained by its expression in different brain regions. The alpha(o)A and alpha(o)C show slight differences in guanine nucleotide binding properties but no preference for particular betagamma dimers when reassociated with a heterogeneous betagamma pool. The alpha(o)C protein occurs in a constant ratio to alpha(o)A throughout the brain, but is a much larger percent of total brain alpha(o) than previously thought, approximately 35%. These results suggest that alpha(o)A is a precursor of alpha(o)C and that the association of G(o)alpha subunits with different betagamma dimers reflects the function of an adaptive, G-protein signaling mechanism in brain (McIntire, 1999).

Expression and subcellular localization of Go alpha proteins

The heterotrimeric G protein G0 is highly enriched in the growth cones of neuronal cells and makes up 10% of the membrane protein of growth cones from neonatal rat brain. PC12 cells, a cell line that differentiates to a neuron-like phenotype, has been used as a model with which to study the mechanism of G protein localization. First, the role of the beta gamma-subunit was investigated. The attachment of the beta gamma-subunit to the membrane depends on the isoprenylation of the gamma-subunit. The drug lovastatin blocks isoprenylation by inhibiting a key enzyme in the biosynthetic pathway. After treatment of PC12 cells with 10 microM lovastatin for 48 hours 50% of the beta gamma-subunits are cytosolic compared with 100% membrane bound beta gamma in control cells, as determined by cell fractionation, gel electrophoresis and western blot. Addition of 200 microM mevalonic acid reverses this effect. However, lovastatin affects neither the membrane attachment of alpha 0 nor its localization to the growth cones as determined by immunohistochemistry. This suggests that the localization and retention of alpha 0 are independent of the membrane attachment of the full complement of beta gamma-subunits. Second, pertussis toxin was used to block the interaction between alpha 0 and receptors. PC12 cells were treated with 0.1 microgram/ml pertussis toxin prior to and during nerve growth factor-induced differentiation. In vitro [32P]ADP-ribosylation confirmed that alpha 0 and alpha i are completely ADP-ribosylated by this treatment. The ADP-ribosylation by pertussis toxin does not interfere with neurite outgrowth. The localization of alpha 0 to the growth cones is indistinguishable from that in untreated cells. It is concluded that G protein-receptor interaction is not necessary for the distribution of alpha 0 to growth cones (Nusse, 1996).

The guanine nucleotide binding protein G0 alpha was immunolocalized in the guinea-pig vestibular system by confocal and electron microscopy. The vestibular sensory epithelia consist of the macula utriculi, macula sacculi and cristae ampullaris of the semicircular canals. Two types of hair cells are present in these epithelia. Type I hair cells are surrounded by an afferent nerve calyx that receives efferent innervation and type II hair cells are innervated directly by the afferent and efferent nerves. G0 alpha protein is observed on the inner face of the afferent calyceal membrane surrounding type I hair cells and in nerve endings in contact with type II hair cells. No labelling is found in the stereocilia and cuticular plate of type I and type II hair cells whereas the cytoplasmic matrix displays a diffuse labelling. The plasma membrane of the supporting cells show discreet labelling. A positive reaction was also observed along the plasma membrane of the vestibular ganglion neurons. Immunoblotting with affinity-purified polyclonal rabbit antibodies selective for the 39 kDa alpha subunit of G0 indicates that G0 alpha protein is present in both the vestibular ganglion. That G0 alpha labelling observed in the cytoplasm of vestibular hair cells and in nerve endings contacting hair cells suggests that G0 may be involved in the modulation of vestibular neurotransmission (Valat, 1995).

Signaling from membrane receptors through heterotrimeric G-proteins (G alpha and G beta gamma) to intracellular effectors is a highly regulated process. Receptor activation causes exchange of GTP for GDP on G alpha and dissociation of G alpha from G beta gamma. Both subunits remain membrane-associated and interact with a series of other molecules throughout the cycle of activation. The N-terminal binding domain of G alpha subunits interacts with the membrane by several partially defined mechanisms: the anchoring of G alpha to the more hydrophobic G beta gamma subunits, the interaction of N-terminal lipids (palmitate and/or myristate) with the membrane, and attachment of amino acid regions to the membrane amino acids 11-14 of Go alpha (D[11-14]). N-terminal mutants of Go alpha with known G beta gamma-binding properties were characterized for the ability to interact with phospholipid vesicles and membranes prepared from cultured cells (acceptor membranes). In vitro analysis allows membrane interactions that are important to the activated and depalmitoylated state of G alpha to be characterized. Subcellular localization was also determined in transiently transfected COS cells. All of the mutant proteins are myristoylated, and differences in myristoylation do not account for changes in membrane binding. Disrupting the N-terminal alpha-helix of Go alpha with a proline point mutation at Arg-9 (R9P) does not affect interactions with G beta gamma on sucrose-density gradients but significantly reduces acceptor membrane binding. Deletion of amino acids 6-15 (D[6-15]; reduces G beta gamma binding) or deletion of amino acids 3-21 (D[3-21]); no detectable G beta gamma binding) further reduces acceptor membrane binding. When expressed in COS cells, R9P and D[6-15] are localized in the membrane similar to wild-type Go alpha as a result of the contribution from palmitoylation. In contrast, D[3-21] is completely soluble in COS cells, and no palmitoylation is detected. The binding of Go alpha and mutants translated in vitro to liposomes indicates that Go alpha preferentially binds to neutral phospholipids (phosphatidylcholine). R9P and D[11-14] bind to phosphatidylcholine liposomes like Go alpha, but D[6-15] exhibits no detectable binding. Taken together, these studies suggest that interactions of the N-terminus of G alpha subunits with the membrane may be affected by both membrane proteins and lipids. A detailed understanding of G alpha-membrane interactions may reveal unique mechanisms for regulating signal transduction (Busconi, 1997).

The 39-kDa Goalpha protein, the alpha subunit of a major heterotrimeric G protein of brain and neuroendocrine cells, was found to be present in human myometrium. Its strong expression in myometrium from pregnant patients as compared to nonpregnant ones has been demonstrated. This is in agreement with the high expression level of its two isoforms (alphao1 and alphao2), previously described in late pregnancy. To better ascertain the nature of different immunoreactive isoforms, transcripts of the Goalpha gene in myometrium from pregnant and nonpregnant patients were examined by reverse transcription-polymerase chain reaction (RT-PCR). In this tissue, the amplified cDNA product of a region common to both Go1alpha and Go2alpha mRNA variants was recognized as the Goalpha nucleotide sequence. Transcripts of Go1alpha and Go2alpha were identified by sequencing. A partial cDNA Go2alpha sequence is described, which differs from the Goalpha gene by two nucleotides in exon 8B. Levels of Go1alpha and Go2alpha transcripts analyzed by semi-quantitative RT-PCR are significantly higher in myometrium from pregnant than from nonpregnant patients. It is suggested that Goalpha gene expression in this tissue may contribute to modifications seen in the signaling pathways observed at the end of pregnancy (Duc-Goiran, 1999).

Rate of translation of Go alpha proteins mRNA

G-proteins couple membrane-bound receptors to intracellular effectors. Each cell has a characteristic complement of G-protein alpha, beta and gamma subunits that partly determines the cell's response to external signals. Very little is known about the mechanisms that set and maintain cellular levels of G-proteins or about potential points of regulation. The steady-state levels of mRNA and protein were assayed for two types of G-protein subunits, alpha s and alpha o, in rat brain, heart and GH3 cells, and it was found that in all these cases, it takes 9- to 20-fold more mRNA to produce a given amount of alpha s protein than to produce the same amount of alpha o protein. Such a situation could arise from a relatively rapid rate of alpha s protein degradation, requiring rapid protein synthesis to compensate, or from relatively inefficient translation of alpha s mRNA compared with alpha o mRNA. The latter appears to be the case in GH3 cells. These cells contain 94 times more mRNA for alpha s than for alpha o, yet the rate of alpha s protein synthesis is only 9 times greater than alpha o protein synthesis. The degradation rates of the two proteins are similar (13 h for alpha s and 18 h for alpha o). To begin to define the mechanism that accounts for the fact that it takes more mRNA to synthesize a given amount of alpha s than alpha o, it was asked whether there is a pool of alpha s mRNA that does not participate in protein synthesis. It as found that virtually all alpha s and alpha o mRNA is associated with ribosomes. Therefore, all the mRNA is likely to be capable of directing protein synthesis. Since the rate-limiting step in protein synthesis is usually binding of the ribosome to mRNA at initiation, these results suggest that the relatively slow rate of alpha s protein synthesis is regulated by a mechanism that acts beyond initiation at peptide elongation and/or termination (Li, 1996).

Effect of mutation of Go alpha proteins

The G protein Go is highly expressed in neurons and mediates effects of a group of rhodopsin-like receptors that includes the opioid, alpha2-adrenergic, M2 muscarinic, and somatostatin receptors. In vitro, Go is also activated by growth cone-associated protein of Mr 43,000 (GAP43) and the Alzheimer amyloid precursor protein, but it is not known whether this occurs in intact cells. To learn about the roles that Go may play in intact cells and whole body homeostasis, the gene encoding the alpha subunits of Go was disrupted in embryonic stem cells and Go-deficient mice were produced. Mice with a disrupted alphao gene (alphao-/- mice) live but have an average half-life of only about 7 weeks. No Goalpha is detectable in homogenates of alphao-/- mice by ADP-ribosylation with pertussis toxin. At the cellular level, inhibition of cardiac adenylyl cyclase by carbachol (50-55% at saturation) is unaffected, but inhibition of Ca2+ channel currents by opioid receptor agonist in dorsal root ganglion cells is decreased by 30%, and in 25% of the alphao-/- cells examined, the Ca2+ channel is activated at voltages that are 13.3 +/- 1.7 mV lower than in their counterparts. Loss of alphao is not accompanied by appearance of significant amounts of active free betagamma dimers (prepulse test). At the level of the living animal, Go-deficient mice are hyperalgesic (hot-plate test) and display a severe motor control impairment (falling from rotarods and 1-inch wide beams). In spite of this deficiency, alphao-/- mice are hyperactive and exhibit a turning behavior that has them running in circles for hours on end, both in cages and in open-field tests. Except for one, all alphao-/- mice turned only counterclockwise. These findings indicate that Go plays a major role in motor control, in motor behavior, and in pain perception and also predict involvement of Go in Ca2+ channel regulation by an unknown mechanism (Jiang, 1998).

The goa-1 gene encoding the alpha subunit of the heterotrimeric guanosine triphosphate-binding protein (G protein) Go from Caenorhabditis elegans is expressed in most neurons, and in the muscles involved in egg laying and male mating. Reduction-of-function mutations in goa-1 cause a variety of behavioral defects including hyperactive movement, premature egg laying, and male impotence. Expression of the activated Go alpha subunit (G alpha o) in transgenic nematodes results in lethargic movement, delayed egg laying, and reduced mating efficiency. Induced expression of activated G alpha o in adults is sufficient to cause these phenotypes, indicating that G alpha o mediates behavior through its role in neuronal function and the functioning of specialized muscles (Mendel, 1995).

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+/-2% (n = 4) 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 (s20,w = 4.4 S, n = 2), 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, an examination of how a large change in the level of betagamma affects this enzyme was carried out. Surprisingly, no difference in the adenylyl cyclase activity between brain membranes from wild-type and alpha(o)-/- mice could be detected. 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).

Go alpha proteins act downstream of various 7TM receptors

In anterior pituitary cells, dopamine, acting on D2 dopamine receptors, concomitantly reduces calcium currents and increases potassium currents. These dopamine effects require the presence of intracellular GTP and are blocked by pretreatment of the cells with pertussis toxin, suggesting that one or more G protein is involved. To identify the G proteins involved in coupling D2 receptors to these currents, patch-clamp recordings were performed in the whole-cell configuration using pipettes containing affinity-purified polyclonal antibodies raised against either Go alpha, Gi3 alpha, or Gi1,2 alpha. Dialysis with Go alpha antiserum significantly reduces the inhibition of calcium currents induced by dopamine, while increase of potassium currents is markedly attenuated only by Gi3 alpha antiserum. It is therefore concluded that in pituitary cells, two different G proteins are involved in the signal transduction mechanism that links D2 receptor activation to a specific modulation of the four types of ionic channels studied here (Llédo, 1992).

Seven transmembrane receptors and their associated heterotrimeric guanine nucleotide-binding proteins (G proteins) have been proposed to play a key role in modulating the activities of neurons and muscles. The physiological function of the Caenorhabditis elegans G protein Go has been genetically characterized. Mutations in the goa-1 gene, which encodes an alpha subunit of Go (G alpha o), cause behavioral defects similar to those observed in mutants that lack the neurotransmitter serotonin (5-HT), and goa-1 mutants are partially resistant to exogenous 5-HT. Mutant animals that lack G alpha o and transgenic animals that overexpress G alpha o [goa-1(xs) animals] have reciprocal defects in locomotion, feeding, and egg laying behaviors. In normal animals, all of these behaviors are regulated by 5-HT. These results demonstrate that the level of Go activity is a critical determinant of several C. elegans behaviors and suggest that Go mediates many of the behavioral effects of 5-HT (Segalat, 1995).

Bipolar cells are retinal interneurons that receive synaptic input from photoreceptors. Glutamate, the photoreceptor transmitter, hyperpolarizes On bipolar cells by closing nonselective cation channels, an effect mediated by the metabotropic receptor mGluR6. Studies of mGluR6 transduction have suggested that this serpentine receptor couples to a phosphodiesterase (PDE) that preferentially hydrolyzes cGMP, and that cGMP directly gates the nonselective cation channel. This hypothesis was tested by dialyzing On bipolar cells with nonhydrolyzable analogs of cGMP. Whole-cell recordings were obtained from On bipolar cells in slices of larval tiger salamander retina. Surprisingly, On bipolar cells dialyzed with two such analogs respond normally to glutamate or L-2-amino-4-phosphonobutyrate (L-APB). Response amplitudes and kinetics are not significantly altered compared with cells dialyzed with cGMP alone. Comparable results were obtained with a PDE inhibitor or a nonhydorlyzable analog and PDE inhibitor together, indicating that PDE is not required for mGluR6 signal transduction. Addition of the G-protein subunit G(o)alpha to the pipette solution suppresses the cation current and occludes the glutamate response, whereas dialysis with G(i)alpha or with transducin Gbetagamma has no significant effect on either the cation current or the response. Dialysis of an antibody directed against G(o)alpha also reduces the glutamate response, indicating a functional role for endogenous G(o)alpha. These results indicate that mGluR6 may signal through G(o), rather than a transducin-like G-protein (Nawy, 1999).

Chemosensory neurons in the vomeronasal organ (VNO) detect pheromones relate to social and reproductive behavior in most terrestrial vertebrates. Current evidence indicate that the chemoelectrical transduction process is mediated by G protein-coupled second messenger cascades. In the present study, attempts were made to identify the G protein subtypes which are activated upon stimulation with urinary pheromonal components. G protein-specific antibodies were employed to interfere specifically with inositol 1,3,4-trisphosphate formation induced by urinary stimuli and to immunoprecipitate Galpha-subunits, activation dependently labeled with [alpha-32P]GTP azidoanilide. The results of both experimental approaches indicate that stimulation of female VNO membrane preparations with male urine samples induces activation of Gi as well as Go subtypes. Experiments using different fractions of urine revealed that upon stimulation with lipophilic volatile odorants, only Gi proteins were activated, whereas Go activation was elicited by alpha2u-globulin, a major urinary protein, which is a member of the lipocalin superfamily. Since each G protein subtype is stereotypically coexpressed with one of the two structurally different candidate pheromone receptors (V1R and V2R), the results provide the first experimental evidence that V1Rs coexpressed with Gi may be activated by lipophilic probably volatile odorants, whereas V2Rs coexpressed with Go seem to be specialized to interact with pheromonal components of proteinaceous nature (Krieger, 1999).

Signalling downstream of Go alpha proteins

continued: G protein oalpha 47A Evolutionary homologs part 3/3 | back to part 1/3 |

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

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