Alpha Subunit Of Go Research Articles

Mice with GNAO1 R209H Movement Disorder Variant Display Hyperlocomotion Alleviated by Risperidone.

Neurodevelopmental disorder with involuntary movements (Online Mendelian Inheritance in Man: 617493) is a severe, early onset neurologic condition characterized by a delay in psychomotor development, hypotonia, and hyperkinetic involuntary movements. Heterozygous de novo mutations in the GNAO1 gene cause neurodevelopmental disorder with involuntary movements. Gα o, the gene product of GNAO1, is the alpha subunit of Go, a member of the heterotrimeric Gi/o family of G proteins. Go is found abundantly throughout the brain, but the pathophysiological mechanisms linking Gα o functions to clinical manifestations of GNAO1-related disorders are still poorly understood. One of the most common mutant alleles among the GNAO1 encephalopathies is the c.626G>A or p.Arg209His (R209H) mutation. We developed heterozygous knock-in Gnao1 +/R209H mutant mice using CRISPR/Cas9 methodology to assess whether a mouse model could replicate aspects of the neurodevelopmental disorder with involuntary movements clinical pattern. Mice carrying the R209H mutation exhibited increased locomotor activity and a modest gait abnormality at 8-12 weeks. In contrast to mice carrying other mutations in Gnao1, the Gnao1 +/R209H mice did not show enhanced seizure susceptibility. Levels of protein expression in multiple brain regions were unchanged from wild-type (WT) mice, but the nucleotide exchange rate of mutant R209H Gα o was 6.2× faster than WT. The atypical neuroleptic risperidone has shown efficacy in a patient with the R209H mutation. It also alleviated the hyperlocomotion phenotype observed in our mouse model but suppressed locomotion in WT mice as well. In this study, we show that Gnao1 +/R209H mice mirror elements of the patient phenotype and respond to an approved pharmacological agent. SIGNIFICANCE STATEMENT: Children with de novo mutations in the GNAO1 gene may present with movement disorders with limited effective therapeutic options. The most common mutant variant seen in children with GNAO1-associated movement disorder is R209H. Here we show, using a novel Gnao1 +/R209H mouse, that there is a clear behavioral phenotype that is suppressed by risperidone. However, risperidone also affects wild-type mouse activity, so its effects are not selective for the GNAO1-associated movement disorder.

The alpha subunit of Go modulates cell proliferation and differentiation through interactions with Necdin.

BackgroundHeterotrimeric GTP-binding proteins (G-proteins) play an important role in mediating signal transduction generated by neurotransmitters or hormones. Go, a member of the Gi/Go subfamily, is the most abundant G-protein found in the brain. Recently, the alpha subunit of Go (Gαo) was characterized as an inducer of neuronal differentiation. However, its underlying molecular mechanisms have remained unclear to date, since the downstream effectors of Gαo are ambiguous.ResultsA neurally differentiated embryonal carcinoma-derived protein (Necdin) was isolated as an interacting partner for Gαo from a mouse brain cDNA library using yeast two-hybrid screening. Interactions between the proteins were confirmed with several affinity binding assays, both in vitro and in vivo. Necdin interacted directly and preferentially with activated Gαo, compared to wild-type protein. Interestingly, Gαo did not interact with Gαi, despite high sequence homology between the two proteins. We subsequently analyzed whether Gαo modulates the cellular activities of Necdin. Notably, expression of Gαo significantly augmented Necdin-mediated cellular responses, such as proliferation and differentiation. Moreover, activation of type 1 cannabinoid receptor (CB1R), a Gi/oα-coupled receptor, augmented cell growth suppression, which was mediated by Gαo and Necdin in U87MG cells containing CB1R, Gαo, and Necdin as normal components.ConclusionsThese results collectively suggest that Necdin is a candidate downstream effector for Gαo. Our findings provide novel insights into the cellular roles of Gαo and its coupled receptor.

The alpha subunit of Go modulates cell proliferation and differentiation through interactions with Necdin

BackgroundHeterotrimeric GTP-binding proteins (G-proteins) play an important role in mediating signal transduction generated by neurotransmitters or hormones. Go, a member of the Gi/Go subfamily, is the most abundant G-protein found in the brain. Recently, the alpha subunit of Go (Gαo) was characterized as an inducer of neuronal differentiation. However, its underlying molecular mechanisms have remained unclear to date, since the downstream effectors of Gαo are ambiguous.ResultsA neurally differentiated embryonal carcinoma-derived protein (Necdin) was isolated as an interacting partner for Gαo from a mouse brain cDNA library using yeast two-hybrid screening. Interactions between the proteins were confirmed with several affinity binding assays, both in vitro and in vivo. Necdin interacted directly and preferentially with activated Gαo, compared to wild-type protein. Interestingly, Gαo did not interact with Gαi, despite high sequence homology between the two proteins. We subsequently analyzed whether Gαo modulates the cellular activities of Necdin. Notably, expression of Gαo significantly augmented Necdin-mediated cellular responses, such as proliferation and differentiation. Moreover, activation of type 1 cannabinoid receptor (CB1R), a Gi/oα-coupled receptor, augmented cell growth suppression, which was mediated by Gαo and Necdin in U87MG cells containing CB1R, Gαo, and Necdin as normal components.ConclusionsThese results collectively suggest that Necdin is a candidate downstream effector for Gαo. Our findings provide novel insights into the cellular roles of Gαo and its coupled receptor.

The GTPase domain of Galphao contributes to the functional interaction of Galphao with the promyelocytic leukemia zinc finger protein

Go, one of the most abundant heterotrimeric G proteins in the brain, is classified as a member of the Gi/Go family based on its homology to Gi proteins. Recently, we identified promyelocytic leukemia zinc finger protein (PLZF) as a candidate downstream effector for the alpha subunit of Go (Gαo). Activated Gαo interacts with PLZF and augments its function as a repressor of transcription and cell growth. G protein-coupled receptor-mediated Gαo activation also enhanced PLZF function. In this study, we determined that the GTPase domain of Gαo contributes to Gαo:PLZF interaction. We also showed that the Gαo GTPase domain is important in modulating the function of PLZF. This data indicates that the GTPase domain of Gαo may be necessary for the functional interaction of Gαo with PLZF.

Rit contributes to neurite outgrowth triggered by the alpha subunit of Go

Heterotrimeric GTP-binding protein transduce signals initiated by a variety of hormones and neurotransmitters. Go, a member of the Go/Gi family, is the most abundant heterotrimeric GTP-binding protein in nervous tissues and has been implicated in neuronal differentiation. The mechanism by which Go modulates neuronal differentiation has not been, however, fully elucidated. Here, we identified small GTPase Rit as an interacting partner of the alpha-subunit of Go (Goalpha). The biochemical characterizations of Goalpha::Rit interaction revealed that Rit is a candidate downstream effector for Goalpha. Furthermore, dominant negative Rit inhibited Goalpha-induced neurite outgrowth and Erk phosphorylation in Neuro2a cells. These results suggest that Rit may be involved in the signaling pathway for Goalpha-mediated neuronal differentiation.

The alpha subunit of Go interacts with promyelocytic leukemia zinc finger protein and modulates its functions

Heterotrimeric GTP-binding proteins (G proteins) mediate signal transduction generated by neurotransmitters and hormones. Go, a member of the Go/Gi family, is the most abundant heterotrimeric G protein in the brain. Most mechanistic analyses on Go activation demonstrate that its action is mediated by the Gβγ dimer; downstream effectors for its α subunit (Goα) have not been clearly defined. Here, we employ the yeast two-hybrid system to screen for Goα-interacting partners in a cDNA library from human fetal brain. The transcription factor promyelocytic leukemia zinc finger protein (PLZF) specifically bound to Goα. Interactions between PLZF and Goα were confirmed using in vitro and in vivo affinity binding assays. Activated Goα interacted directly with PLZF, and enhanced its function as a transcriptional and cell growth suppressor. Notably, PLZF activity was additionally promoted by the Go/iα-coupled cannabinoid receptor (CB) in HL60 cells endogenously expressing CB and PLZF. These results collectively suggest that Goα modulates the function of PLZF via direct interactions. Our novel findings provide insights into the diverse cellular roles of Goα and its coupled receptor.

Diabetic neuropathy: inhibitory G protein dysfunction involves PKC-dependent phosphorylation of Goalpha.

We examined the hypothesis that decreased inhibitory G protein function in diabetic neuropathy is associated with increased protein kinase C (PKC)-dependent phosphorylation of the Goalpha subunit. Streptozotocin-induced diabetic rats were studied between 4 and 8 weeks after onset of diabetes and compared with aged-matched healthy animals as controls. Opioid-mediated inhibition of forskolin-stimulated cyclic AMP was significantly less in dorsal root ganglia (DRGs) from diabetic rats compared with controls. Activation of PKC in DRGs from control rats was associated with a significant decrease in opioid-mediated inhibition of forskolin-stimulated cyclic AMP that was similar to the decrease in inhibition observed in DRGs from diabetic rats. Both basal and PKC-mediated labeling of Goalpha with 32Pi was significantly less in DRGs from diabetic rats, supporting increased endogenous PKC-dependent phosphorylation of Goalpha. Probing of immunoprecipitated Goalpha with an anti-phospho-serine/threonine specific antibody revealed a significant increase in baseline phosphorylation in diabetic DRGs. Activation of PKC produced a significant increase in phosphorylation in control DRGs but no significant increase in Goalpha in diabetic DRGs. Phosphorylation of PKC-alpha was increased, PKC-betaII was unchanged and PKC-delta decreased in diabetic DRGs. These results suggest that diminished inhibitory G protein function observed in DRGs neurons from diabetic rats involves an isoform-specific PKC-dependent pathway.

Involvement of G(q/11) in signal transduction in the mammalian vomeronasal organ.

Social behaviors of most mammals are profoundly affected by pheromones. Pheromones are detected by G-protein coupled receptors in the vomeronasal organ (VNO). To investigate the role of G alpha(q/11) in vomeronasal signal transduction pathways, microvillar membranes from murine VNO were prepared. Incubation of such membranes from prepubertal females with adult male urine results in an increase in production of inositol-(1,4,5)-trisphosphate (IP(3)). This stimulation is mimicked by GTP gamma S, blocked by GDP beta S and is tissue specific. Furthermore, use of bacterial toxins such as pertussis that lead to ADP-ribosylation of the G-protein alpha subunits of G(o) and G(i2) do not block the increase in IP(3) levels but U-73122, a PLC inhibitor, blocks the production of IP(3). Studies with monospecific antibodies revealed the presence of three G-proteins, G alpha(o), G alpha(i2) and G alpha(q/11)-related protein, in vomeronasal neurons, concentrated on their microvilli. Our observations indicate that pheromones in male urine act on vomeronasal neurons in the female VNO via a receptor-mediated, G alpha(q/11)-protein-dependent increase in IP(3) levels.

The association of heterotrimeric GTP-binding protein (Go) with microtubules.

The heterotrimeric GTP-binding regulatory proteins (G proteins) play an important role in the regulation of membrane signal transduction. Recently, we identified the association of Go protein with mitotic spindles. Here we have investigated the relationship between Go protein and microtubules. We used temperature-dependent reversible assembly and taxol methods to purify microtubules from bovine brains. Goalpha and Gbeta proteins were identified in the microtubular fraction by both methods. The Goalpha subunit in the microtubular fraction could be ADP ribosylated by pertussis toxin. Co-immunoprecipitation data also revealed that Go protein can interact with microtubules. Exogenous Go protein could be incorporated into the assembled microtubular fraction, and 5 microg/ml (60 nM) of Go protein inhibited 40% of microtubule assembly. Western blot analysis of Goalpha-1 and Goalpha-2 in microtubular fractions showed that only Goalpha-1 is associated with microtubules. We conclude that the Goalpha-1betagamma proteins are associated with microtubules and may play some role in regulating the assembly and disassembly of microtubules.

Neurite outgrowth induced by cyclic AMP can be modulated by the alpha subunit of Go.

Although abundant Go has been found in nervous tissues and it has been implicated in neuronal differentiation, the mechanism of how Go modulates neuronal differentiation has not been defined. Here, we report that the alpha subunit of Go (alphao) modulates neurite outgrowth by interfering with the signaling pathway initiated by cyclic AMP (cAMP). In F11 cells, cAMP induced neurite outgrowth and activated cAMP-responsive element binding protein (CREB). Specific inhibition of cAMP-dependent protein kinase reduced both CREB activity and neurite outgrowth (NOG). Interestingly, cAMP reduced phosphorylation of extracellular signal-regulated kinase (Erk). Neither a dominant negative form nor an active form of Ras altered neurite outgrowth. Expression of alphao (alphao(wt)) decreased the average length of neurites but increased the number of neurites per cell. An active mutant, alphaoQ205L, which lost GTPase activity and thus could not bind to Gbetagamma, gave similar results, suggesting that the effect of alphao is not mediated through Gbetagamma. Expression of ao(wt) or alphaoQ205L also prohibited CREB activation. Thus, activation of Erk may not be essential for neuronal differentiation in F11 cells and alphao may cause changes in NOG by inhibiting CREB activation.

Interaction of the Xanthine Nucleotide Binding Goα Mutant with G Protein-coupled Receptors

We constructed a double mutant version of the alpha subunit of Go that was regulated by xanthine nucleotides instead of guanine nucleotides (GoalphaX). We investigated the interaction between GoalphaX and G protein-coupled receptors in vitro. First, we found that the activated m2 muscarinic cholinergic receptor (MAChR) could facilitate the exchange of XTPgammaS for XDP in the GoalphaXbetagamma heterotrimer. Second, the GoalphaXbetagamma complex was able to induce the high affinity ligand-binding state in the N-formyl peptide receptor (NFPR). These experiments demonstrated that GoalphaX was able to interact effectively with G protein-coupled receptors. Third, we found that the empty form of GoalphaX, lacking a bound nucleotide and betagamma, formed a stable complex with the m2 muscarinic cholingeric receptor associated with the plasma membrane. Finally, we investigated the interaction of GoalphaX with receptor in COS-7 cells. The empty form of GoalphaX bound tightly to the receptor and was not activated because XTP was not available intracellularly. We tested the ability of GoalphaX to inhibit the activities of several different G protein-coupled receptors in transfected COS-7 cells and found that GoalphaX specifically inhibited Go-coupled receptors. Thus the modified G proteins may act as dominant-negative mutants to trap and inactivate specific subsets of receptors.

Alpha subunit of Go localizes in the dendritic tips of ON bipolar cells

The metabotropic glutamate receptor (mGluR6), expressed by rod bipolar cells and ON cone bipolar cells, activates a trimeric guanine nucleotide-binding protein (G-protein) that ultimately closes a cation channel. The G-protein remains unidentified, but the alpha subunit of Go (Go(alpha)) has been suggested as a candidate because it is present in rod bipolar cells. However, the precise subcellular distribution of Go within the rod bipolar cell, and its distribution among cone bipolar cells was not determined. This information is important in assessing the hypothesis that Go couple mGluR6 to its effector. Here I report the distribution of Go (alpha subunit) by immunostaining in several mammalian retinas. The overall distribution is conserved across mammalian species: strongest in the dendrites of ON bipolar cells, moderate in their somas, weak in their axons, and absent from their terminals. Go(alpha) is also present in some amacrine somas and processes. In monkey fovea, where rods and rod bipolar cells are absent, Go(alpha) is present in about half of the bipolar somas which occupy the upper tiers of the bipolar layer, and are therefore identified as ON cone bipolar cells. Ultrastructurally, in monkey and cat, Go(alpha) is present in the dendritic tips of rod bipolar cells and ON cone bipolar cells, which are identified by their invaginating contacts. It is absent from OFF cone bipolar dendrites, which are identified by their flat contacts. It is also absent from axons entering the inner plexiform layer, and their terminals. In the primary dendrites, stain for Go(alpha) mainly associates with the plasma membrane, but in the dendritic tips it is also present in the cytosol. Apparently, Go(alpha) is expressed by the same bipolar cells that also express mGluR6, and is concentrated at the same subcellular location. Thus, Go(alpha) could serve to couple mGluR6 to later stages of its signaling cascade.

Alpha subunit of Go localizes in the dendritic tips of ON bipolar cells

Alpha subunit of Go localizes in the dendritic tips of ON bipolar cells

Interactions of Phosducin with the Subunits of G-Proteins: BINDING TO THE α AS WELL AS THE βγ SUBUNITS

The high affinity interactions of phosducin with G-proteins involve binding of phosducin to the G-protein betagamma subunits. Here we have investigated whether phosducin interacts also with G-protein alpha subunits. Interactions of phosducin with the individual subunits of Go were measured by retaining phosducin-G-protein subunit complexes on columns containing immobilized anti-phosducin antibodies. Both the alpha and the beta subunits of trimeric Go were specifically retained by the antibodies in the presence of phosducin. This binding was almost completely abolished for both subunits following protein kinase A-mediated phosphorylation of phosducin and was reduced, more for alpha than for beta subunits, by the stable GTP analog guanosine 5'-(3-O-thio)triphosphate. Isolated alphao was also retained on the columns in the presence of phosducin but not in the presence of protein kinase A-phosphorylated phosducin. Likewise, purified G-protein betagamma subunit complexes as well as purified alpha subunits of Go and Gt were precipitated together with His6-tagged phosducin with nickel-agarose; this co-precipitation occurred concentration-dependently, with apparent affinities for phosducin of 55 nM (Gbetagamma), 110 nM (alphao), and 200 nM (alphat). In functional experiments, the steady state GTPase activity of isolated alphao was inhibited by phosducin by approximately 60% with an IC50 value of approximately 300 nM, whereas the GTPase activity of trimeric Go was inhibited by approximately 90% with an IC50 value of approximately 10 nM. Phosducin did not inhibit the GTP-hydrolytic activity of isolated alphao as measured by single-turnover assays, but it inhibited the release of GDP from alphao; the rate constant of GDP release was decreased approximately 40% by 500 nM phosducin, and the inhibition occurred with an IC50 value for phosducin of approximately 100 nM. These data suggest that phosducin binds with high affinity to G-protein betagamma subunits and with lower affinity to G-protein alpha subunits. We propose that the alpha subunit-mediated effects of phosducin might increase both the extent and the rapidity of its inhibitory effects compared with an action via the betagamma subunit complex alone.

Activators of epithelial Na+ channels inhibit cytosolic feedback control. Evidence for the existence of a G protein-coupled receptor for cytosolic Na+.

We have previously shown that epithelial Na+ channels in mouse mandibular gland duct cells are controlled by cytosolic Na+ and Cl-, acting, respectively, via Go and Gi proteins. Since we found no evidence for control of epithelial Na+ channels by extracellular Na+ ([Na+]o), our findings conflicted with the long-held belief that Na+ channel activators, such as sulfhydryl reagents, like para-chloromercuriphenylsulfonate (PCMPS), and amiloride analogues, like benzimidazolylguanidinium (BIG) and 5-N-dimethylamiloride (DMA), induce their effects by blocking an extracellular channel site which otherwise inhibits channel activity in response to increasing [Na+]o. Instead, we now show that PCMPS acts by rendering epithelial Na+ channels refractory to inhibition by activated G proteins, thereby eliminating the inhibitory effects of cytosolic Na+ and Cl- on Na+ channel activity. We also show that BIG, DMA, and amiloride itself, when applied from the cytosolic side of the plasma membrane, block feedback inhibition of Na+ channels by cytosolic Na+, while leaving inhibition by cytosolic Cl- unaffected. Since the inhibitory effects of BIG and amiloride are overcome by the inclusion of the activated alpha-subunit of Go in the pipette solution, we conclude that these agents act by blocking a previously unrecognized intracellular Na+ receptor.

Multiple neurological abnormalities in mice deficient in the G protein Go.

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, we disrupted the gene encoding the alpha subunits of Go in embryonic stem cells and derived Go-deficient mice. Mice with a disrupted alphao gene (alphao-/- mice) lived but had an average half-life of only about 7 weeks. No Goalpha was 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) was unaffected, but inhibition of Ca2+ channel currents by opioid receptor agonist in dorsal root ganglion cells was decreased by 30%, and in 25% of the alphao-/- cells examined, the Ca2+ channel was activated at voltages that were 13.3 +/- 1.7 mV lower than in their counterparts. Loss of alphao was 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.

Functional Coupling of a Human Retinal Metabotropic Glutamate Receptor (hmGluR6) to Bovine Rod Transducin and Rat Go in an in Vitro Reconstitution System

The cDNA encoding hmGluR6, appended with a 15-amino acid antibody epitope (1D4), was transiently transfected in COS-7 cells. The receptor was purified from COS cell membranes using an antibody affinity column. The purified receptor was then reconstituted into lipid vesicles, and its ability to activate either transducin, the rod photoreceptor-specific GTP-binding protein, or the alpha subunit of Go was assayed in vitro using a guanosine 5'-3-O-(thio)triphosphate binding assay. Activation of both transducin and Go was observed. The rate of Go activation was 18-fold greater than the rate of transducin activation. This indicates that the coupling of mGluR6 to Go is more efficient and suggests that Go may be involved in coupling to mGluR6 in ON-bipolar cells.

Characterization of a Goalpha mutant that binds xanthine nucleotides.

Several GTP binding proteins, including EF-Tu, Ypt1, rab-5, and FtsY, and adenylosuccinate synthetase have been reported to bind xanthine nucleotides when the conserved aspartate residue in the NKXD motif was changed to asparagine. However, the corresponding single Goalpha mutant protein (D273N) did not bind either xanthine nucleotides or guanine nucleotides. Interestingly, the introduction of a second mutation to generate the Goalpha subunit D273N/Q205L switched nucleotide binding specificity to xanthine nucleotide. The double mutant protein GoalphaD273N/Q205L (GoalphaX) bound xanthine triphosphate, but not guanine triphosphate. Recombinant GoalphaX (GoalphaD273N/Q205L) formed heterotrimers with betagamma complexes only in the presence of xanthine diphosphate (XDP), and the binding to betagamma was inhibited by xanthine triphosphate (XTP). Furthermore, as a result of binding to XTP, the GoalphaX protein underwent a conformational change similar to that of the activated wild-type Goalpha. In transfected COS-7 cells, we demonstrate that the interaction between GoalphaX and betagamma occurred only when cell membranes were permeabilized to allow the uptake of xanthine diphosphate. This is the first example of a switch in nucleotide binding specificity from guanine to xanthine nucleotides in a heterotrimeric G protein alpha subunit.

Interaction of Phosducin-like Protein with G Protein βγ Subunits

Phosducin-like protein (PhLP), a widely expressed ethanol-responsive gene (Miles, M. F., Barhite, S., Sganga, M., and Elliott, M. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10831-10835), is a homologue of phosducin, a known major regulator of Gbetagamma signaling in retina and pineal gland. However, although phosducin has a well characterized role in retinal phototransduction, function of the PhLP remains unclear. In this study we examine the ability of PhLP to bind Gbetagamma dimer in vitro and in vivo. Using PhLP glutathione S-transferase fusion proteins, we show that PhLP directly binds Gbetagamma in vitro. Studies with a series of truncated PhLP fusion proteins indicate independent binding of Gbetagamma to both the amino- and C-terminal halves of PhLP. Protein-protein interactions between Gbetagamma and PhLP are inhibited by the alpha subunit of Go and Gi3, suggesting that PhLP can bind only free Gbetagamma. Finally, we show that PhLP complexes, at least partially, with Gbetagamma in vivo. Following overexpression of epitope-tagged PhLP together with Gbeta1gamma2 proteins in COS-7 cells, a PhLP-Gbetagamma complex is co-immunoprecipitated by monoclonal antibody directed against the epitope tag. Similarly, polyclonal anti-PhLP antibody co-precipitates endogenous PhLP and Gbetagamma proteins from NG108-15 cell lysates. These data are consistent with the hypothesis that PhLP is a widely expressed modulator of Gbetagamma function. Furthermore, because alternate forms of the PhLP transcript are expressed, there may be functional implications for the existence of two Gbetagamma-binding domains on PhLP.

D2L, D2S, and D3 dopamine receptors stably transfected into NG108-15 cells couple to a voltage-dependent potassium current via distinct G protein mechanisms.

The D2-like dopamine (DA) receptor family has continued to expand and now includes the D2-short (D2S) and D2-long (D2L) receptor isoforms and the D3 and D4 receptors. The D2 receptor isoforms differ in length by 29 amino acids within the third cytoplasmic loop, a region of the receptor believed to be important for G protein coupling. This observation has led to the hypothesis that the two isoforms of the D2 receptor may utilize different signal transduction pathways when present in the same cell. The D2 and D3 receptors, although mostly different, show some common amino acid sequences within the third cytoplasmic loop. Thus, it is possible that the D2 and D3 receptors may employ similar signal transduction pathways. To test these hypotheses directly, NG108-15 neuroblastoma-glioma hybrid cells were stably transfected to express either the D2S, D2L, or D3 DA receptors. All transfected but not untransfected NG108-15 cells demonstrated a dose-dependent reduction in the peak whole-cell potassium (K+) current in response to receptor activation by DA or the DA receptor agonists quinpirole (QUIN) and apomorphine (APO). The modulation of K+ current by D2S receptor stimulation was prevented by pretreatment of the cells with cholera toxin (20 micrograms/ml for 18 h), whereas pertussis toxin pretreatment (500 ng/ml for 4 h) completely blocked the effects of D2L and D3 receptor activation. These observations suggest that the signal transduction mechanisms involved in coupling the two isoforms of the D2 receptor to the K+ current are different, whereas the D2L and D3 receptor coupling mechanisms may be similar. In direct support of this hypothesis, it was observed that the intracellular application of a polyclonal antibody that is specific for the GO alpha subunit completely blocked the ability of D2L and D3 receptors to modulate outward K+ currents. In contrast, the D2S-mediated modulation of K+ currents was blocked by intracellular application of an antibody recognizing GS alpha but not GO alpha. These findings demonstrate that D2S and D2L receptors are able to couple to a common effector in a cell via two G protein pathways.