Dopamine-D2S receptor inhibition of calcium influx,adenylyl cyclase,and mitogen-activated protein kinase in pituitary cells: distinct Galpha and Gbetagamma requirements

The G protein specificity of multiple signaling pathways of the dopamine-D2S (short form) receptor was investigated in GH4ZR7 lactotroph cells. Activation of the dopamine-D2S receptor inhibited forskolin-induced cAMP production, reduced BayK8644- activated calcium influx, and blocked TRH-mediated p42/p44 MAPK phosphorylation. These actions were blocked by pretreatment with pertussis toxin (PTX), indicating mediation by G(i/o) proteins. D2S stimulation also decreased TRH-induced MAPK/ERK kinase phosphorylation. TRH induced c-Raf but not B-Raf activation, and the D2S receptor inhibited both TRH-induced c-Raf and basal B-Raf kinase activity. After PTX treatment, D2S receptor signaling was rescued in cells stably transfected with individual PTX-insensitive Galpha mutants. Inhibition of adenylyl cyclase was partly rescued by Galpha(i)2 or Galpha(i)3, but Galpha(o) alone completely reconstituted D2S-mediated inhibition of BayK8644-induced L-type calcium channel activation. Galpha(o) and Galpha(i)3 were the main components involved in D2S-mediated p42/44 MAPK inhibition. In cells transfected with the carboxyl-terminal domain of G protein receptor kinase to inhibit Gbetagamma signaling, only D2S-mediated inhibition of calcium influx was blocked, but not inhibition of adenylyl cyclase or MAPK. These results indicate that the dopamine-D2S receptor couples to distinct G(i/o) proteins, depending on the pathway addressed, and suggest a novel Galpha(i)3/Galpha(o)-dependent inhibition of MAPK mediated by c-Raf and B-Raf-dependent inhibition of MAPK/ERK kinase.     

Galphai1 and Galphai3 differentially interact with, and regulate, the G protein-activated K+ channel

G protein-activated K(+) channels (GIRKs; Kir3) are activated by direct binding of Gbetagamma subunits released from heterotrimeric G proteins. In native tissues, only pertussis toxin-sensitive G proteins of the G(i/o) family, preferably Galpha(i3) and Galpha(i2), are donors of Gbetagamma for GIRK. How this specificity is achieved is not known. Here, using a pull-down method, we confirmed the presence of Galpha(i3-GDP) binding site in the N terminus of GIRK1 and identified novel binding sites in the N terminus of GIRK2 and in the C termini of GIRK1 and GIRK2. The non-hydrolyzable GTP analog, guanosine 5'-3-O-(thio)triphosphate, reduced the binding of Galpha(i3) by a factor of 2-4. Galpha(i1-GDP) bound to GIRK1 and GIRK2 much weaker than Galpha(i3-GDP). Titrated expression of components of signaling pathway in Xenopus oocytes and their activation by m2 muscarinic receptors revealed that G(i3) activates GIRK more efficiently than G(i1), as indicated by larger and faster agonist-evoked currents. Activation of GIRK by purified Gbetagamma in excised membrane patches was strongly augmented by coexpression of Galpha(i3) and less by Galpha(i1). Differences in physical interactions of GIRK with GDP-bound Galpha subunits, or Galphabetagamma heterotrimers, may dictate different extents of Galphabetagamma anchoring, influence the efficiency of GIRK activation by Gbetagamma, and play a role in determining signaling specificity.     

Specificity of Gbetagamma signaling to Kir3 channels depends on the helical domain of pertussis toxin-sensitive Galpha subunits

Acetylcholine signaling through muscarinic type 2 receptors activates atrial G protein-gated inwardly rectifying K(+) (Kir3) channels via the betagamma subunits of G proteins (Gbetagamma). Different combinations of recombinant Gbetagamma subunits have been shown to activate Kir3 channels in a similar manner. In native systems, however, only Gbetagamma subunits associated with the pertussis toxin-sensitive Galpha(i/o) subunits signal to K(+) channels. Additionally, in vitro binding experiments supported the notion that the C terminus of Kir3 channels interacts preferentially with Galpha(i) over Galpha(q). In this study we confirmed in two heterologous expression systems a preference of Galpha(i) over Galpha(q) in the activation of K(+) currents. To identify determinants of Gbetagamma signaling specificity, we first exchanged domains of Galpha(i) and Galpha(q) subunits responsible for receptor coupling selectivity and swapped their receptor coupling partners. Our results established that the G proteins, regardless of the receptor type to which they coupled, conferred specificity to Kir3 activation. We next tested signaling through chimeras between the Galpha(i) and Galpha(q) subunits in which the N terminus, the helical, or the GTPase domains of the Galpha subunits were exchanged. Our results revealed that the helical domain of Galpha(i) (residues 63-175) in the background of Galpha(q) could support Kir3 activation, whereas the reverse chimera could not. Moreover, the helical domain of the Galpha(i) subunit conferred "Galpha(i)-like" binding of the Kir3 C terminus to the Galpha(q) subunits that contained it. These results implicate the helical domain of Galpha(i) proteins as a critical determinant of Gbetagamma signaling specificity.     

Functional interaction between Galpha(z) and Rap1GAP suggests a novel form of cellular cross-talk

G(z) is a member of the G(i) family of trimeric G proteins whose primary role in cell physiology is still unknown. In an ongoing effort to elucidate the cellular functions of G(z), the yeast two-hybrid system was employed to identify proteins that specifically interact with a mutationally activated form of Galpha(z). One of the molecules uncovered in this screen was Rap1GAP, a previously identified protein that specifically stimulates GTP hydrolytic activity of the monomeric G protein Rap1 and thus is believed to function as a down-regulator of Rap1 signaling. Like G(z), the precise role of Rap1 in cell physiology is poorly understood. Biochemical analysis using purified recombinant proteins revealed that the physical interaction between Galpha(z) and Rap1GAP blocks the ability of RGSs (regulators of G protein signaling) to stimulate GTP hydrolysis of the alpha subunit, and also attenuates the ability of activated Galpha(z) to inhibit adenylyl cyclase. Structure-function analyses indicate that the first 74 amino-terminal residues of Rap1GAP, a region distinct from the catalytic core domain responsible for the GAP activity toward Rap1, is required for this interaction. Co-precipitation assays revealed that Galpha(z), Rap1GAP, and Rap1 can form a stable complex. These data suggest that Rap1GAP acts as a signal integrator to somehow coordinate and/or integrate G(z) signaling and Rap1 signaling in cells.     

Galpha(16/z) chimeras efficiently link a wide range of G protein-coupled receptors to calcium mobilization

G protein-coupled receptors (GPCRs) represent a class of important therapeutic targets for drug discovery. The integration of GPCRs into contemporary high-throughput functional assays is critically dependent on the presence of appropriate G proteins. Given that different GPCRs can discriminate against distinct G proteins, a universal G protein adapter is extremely desirable. In this report, the authors evaluated two highly promiscuous Galpha(16/z) chimeras, 16z25 and 16z44, for their ability to translate GPCR activation into Ca(2+) mobilization using the fluorescence imaging plate reader (FLIPR) and aequorin. A panel of 24 G(s)- or G(i)-coupled receptors was examined for their functional association with the Galpha(16/z) chimeras. Although most of the GPCRs tested were incapable of inducing Ca(2+) mobilization upon their activation by specific agonists, the introduction of 16z25 or 16z44 allowed all of these GPCRs to mediate agonist-induced Ca(2+) mobilization. In contrast, only 16 of the GPCRs tested were capable of using Galpha(16) to mobilize intracellular Ca(2+). Analysis of dose-response curves obtained with the delta-opioid, dopamine D(1), and Xenopus melatonin Mel1c receptors revealed that the Galpha(16/z) chimeras possess better sensitivity than Galpha(16) in both the FLIPR and aequorin assays. Collectively, these studies help to validate the promiscuity of the Galpha(16/z) chimeras as well as their application in contemporary drug-screening assays that are based on ligand-induced Ca(2+) mobilization.     

GIPC recruits GAIP (RGS19) to attenuate dopamine D2 receptor signaling

Pleiotropic G proteins are essential for the action of hormones and neurotransmitters and are activated by stimulation of G protein-coupled receptors (GPCR), which initiates heterotrimer dissociation of the G protein, exchange of GDP for GTP on its Galpha subunit and activation of effector proteins. Regulator of G protein signaling (RGS) proteins regulate this cascade and can be recruited to the membrane upon GPCR activation. Direct functional interaction between RGS and GPCR has been hypothesized. We show that recruitment of GAIP (RGS19) by the dopamine D2 receptor (D2R), a GPCR, required the scaffold protein GIPC (GAIP-interacting protein, C terminus) and that all three were coexpressed in neurons and neuroendocrine cells. Dynamic translocation of GAIP to the plasma membrane and coassembly in a protein complex in which GIPC was a required component was dictated by D2R activation and physical interactions. In addition, two different D2R-mediated responses were regulated by the GTPase activity of GAIP at the level of the G protein coupling in a GIPC-dependent manner. Since GIPC exclusively interacted with GAIP and selectively with subsets of GPCR, this mechanism may serve to sort GPCR signaling in cells that usually express a large repertoire of GPCRs, G proteins, and RGS.     

Selective regulation of Galpha(q/11) by an RGS domain in the G protein-coupled receptor kinase, GRK2

G protein-coupled receptor kinases (GRKs) are well characterized regulators of G protein-coupled receptors, whereas regulators of G protein signaling (RGS) proteins directly control the activity of G protein alpha subunits. Interestingly, a recent report (Siderovski, D. P., Hessel, A., Chung, S., Mak, T. W., and Tyers, M. (1996) Curr. Biol. 6, 211-212) identified a region within the N terminus of GRKs that contained homology to RGS domains. Given that RGS domains demonstrate AlF(4)(-)-dependent binding to G protein alpha subunits, we tested the ability of G proteins from a crude bovine brain extract to bind to GRK affinity columns in the absence or presence of AlF(4)(-). This revealed the specific ability of bovine brain Galpha(q/11) to bind to both GRK2 and GRK3 in an AlF(4)(-)-dependent manner. In contrast, Galpha(s), Galpha(i), and Galpha(12/13) did not bind to GRK2 or GRK3 despite their presence in the extract. Additional studies revealed that bovine brain Galpha(q/11) could also bind to an N-terminal construct of GRK2, while no binding of Galpha(q/11), Galpha(s), Galpha(i), or Galpha(12/13) to comparable constructs of GRK5 or GRK6 was observed. Experiments using purified Galpha(q) revealed significant binding of both Galpha(q) GDP/AlF(4)(-) and Galpha(q)(GTPgammaS), but not Galpha(q)(GDP), to GRK2. Activation-dependent binding was also observed in both COS-1 and HEK293 cells as GRK2 significantly co-immunoprecipitated constitutively active Galpha(q)(R183C) but not wild type Galpha(q). In vitro analysis revealed that GRK2 possesses weak GAP activity toward Galpha(q) that is dependent on the presence of a G protein-coupled receptor. However, GRK2 effectively inhibited Galpha(q)-mediated activation of phospholipase C-beta both in vitro and in cells, possibly through sequestration of activated Galpha(q). These data suggest that a subfamily of the GRKs may be bifunctional regulators of G protein-coupled receptor signaling operating directly on both receptors and G proteins.     

Different domains in the third intracellular loop of the GLP-1 receptor are responsible for Galpha(s) and Galpha(i)/Galpha(o) activation

It has previously been shown that the GLP-1 receptor is primarily coupled to the adenylate cyclase pathway via activation of Galpha(s) proteins. Recent studies have shown that the third intracellular loop of the receptor is important in the stimulation of cAMP production. We have studied the effect of three synthetic peptide sequences derived from the third intracellular loop of the GLP-1 receptor on signal transduction in Rin m5F cell membranes. The whole third intracellular loop strongly stimulates both pertussis toxin and cholera toxin-sensitive G proteins, while the N-terminal half exclusively stimulates cholera toxin-sensitive G proteins and the C-terminal half only stimulates pertussis toxin-sensitive G-proteins as demonstrated by measurements of GTPase activity. These data confirm that the principal stimulatory G-protein interaction site resides in the third intracellular loop, but also suggest that the GLP-1 receptor is not only coupled to the Galpha(s) but also to the Galpha(i)/Galpha(o) type of G proteins and that distinct domains within the third intracellular loop are responsible for the activation of the different G-protein subfamilies.     

Regulator of G protein signaling Z1 (RGSZ1) interacts with Galpha i subunits and regulates Galpha i-mediated cell signaling

Regulator of G protein signaling (RGS) proteins constitute a family of over 20 proteins that negatively regulate heterotrimeric G protein-coupled receptor signaling pathways by enhancing endogenous GTPase activities of G protein alpha subunits. RGSZ1, one of the RGS proteins specifically localized to the brain, has been cloned previously and described as a selective GTPase accelerating protein for Galpha(z) subunit. Here, we employed several methods to provide new evidence that RGSZ1 interacts not only with Galpha(z,) but also with Galpha(i), as supported by in vitro binding assays and functional studies. Using glutathione S-transferase fusion protein pull-down assays, glutathione S-transferase-RGSZ1 protein was shown to bind (35)S-labeled Galpha(i1) protein in an AlF(4)(-)dependent manner. The interaction between RGSZ1 and Galpha(i) was confirmed further by co-immunoprecipitation studies and yeast two-hybrid experiments using a quantitative luciferase reporter gene. Extending these observations to functional studies, RGSZ1 accelerated endogenous GTPase activity of Galpha(i1) in single-turnover GTPase assays. Human RGSZ1 functionally regulated GPA1 (a yeast Galpha(i)-like protein)-mediated yeast pheromone response when expressed in a SST2 (yeast RGS protein) knockout strain. In PC12 cells, transfected RGSZ1 blocked mitogen-activated protein kinase activity induced by UK14304, an alpha(2)-adrenergic receptor agonist. Furthermore, RGSZ1 attenuated D2 dopamine receptor agonist-induced serum response element reporter gene activity in Chinese hamster ovary cells. In summary, these data suggest that RGSZ1 serves as a GTPase accelerating protein for Galpha(i) and regulates Galpha(i)-mediated signaling, thus expanding the potential role of RGSZ1 in G protein-mediated cellular activities.     

Receptor-mediated changes at the myristoylated amino terminus of Galpha(il) proteins

G protein-coupled receptors (GPCRs) catalyze nucleotide release in heterotrimeric G proteins, the slow step in G protein activation. G i/o family proteins are permanently, cotranslationally myristoylated at the extreme amino terminus. While myristoylation of the amino terminus has long been known to aid in anchoring G i proteins to the membrane, the role of myristoylation with regard to interaction with activated receptors is not known. Previous studies have characterized activation-dependent changes in the amino terminus of Galpha proteins in solution [Medkova, M. (2002) Biochemistry 41, 9963-9972; Preininger, A. M. (2003) Biochemistry 42, 7931-7941], but changes in the environment of specific residues within the Galpha i1 amino terminus during receptor-mediated G i activation have not been reported. Using site-specific fluorescence labeling of individual residues along a stretch of the Galpha il amino terminus, we found specific changes in the environment of these residues upon interaction with the activated receptor and following GTPgammaS binding. These changes map to a distinct surface of the amino-terminal helix opposite the Gbetagamma binding interface. The receptor-dependent fluorescence changes are consistent with a myristoylated amino terminus in the proximity of the membrane and/or receptor. Myristoylation affects both the rate and intensity of receptor activation-dependent changes detected at several residues along the amino terminus (with no significant effect on the rate of receptor-mediated GTPgammaS binding). This work demonstrates that the myristoylated amino terminus of Galpha il proteins undergoes receptor-mediated changes during the dynamic process of G protein signaling.     

Gbetagamma-activated inwardly rectifying K(+) (GIRK) channel activation kinetics via Galphai and Galphao-coupled receptors are determined by Galpha-specific interdomain interactions that affect GDP release rates

Gbetagamma-activated inwardly rectifying K(+) (GIRK) channels have distinct gating properties when activated by receptors coupled specifically to Galpha(o) versus Galpha(i) subunit isoforms, with Galpha(o)-coupled currents having approximately 3-fold faster agonist-evoked activation kinetics. To identify the molecular determinants in Galpha subunits mediating these kinetic differences, chimeras were constructed using pertussis toxin (PTX)-insensitive Galpha(oA) and Galpha(i2) mutant subunits (Galpha(oA(C351G)) and Galpha(i2(C352G))) and examined in PTX-treated Xenopus oocytes expressing muscarinic m2 receptors and Kir3.1/3.2a channels. These experiments revealed that the alpha-helical N-terminal region (amino acids 1-161) and the switch regions of Galpha(i2) (amino acids 162-262) both partially contribute to slowing the GIRK activation time course when compared with the Galpha(oA(C351G))-coupled response. When present together, they fully reproduce Galpha(i2(C352G))-coupled GIRK kinetics. The Galpha(i2) C-terminal region (amino acids 263-355) had no significant effect on GIRK kinetics. Complementary responses were observed with chimeras substituting the Galpha(o) switch regions into the Galpha(i2(C352G)) subunit, which partially accelerated the GIRK activation rate. The Galpha(oA)/Galpha(i2) chimera results led us to examine an interaction between the alpha-helical domain and the Ras-like domain previously implicated in mediating a 4-fold slower in vitro basal GDP release rate in Galpha(i1) compared with Galpha(o). Mutations disrupting the interdomain contact in Galpha(i2(C352G)) at either the alphaD-alphaE loop (R145A) or the switch III loop (L233Q/A236H/E240T/M241T), significantly accelerated the GIRK activation kinetics consistent with the Galpha(i2) interdomain interface regulating receptor-catalyzed GDP release rates in vivo. We propose that differences in Galpha(i) versus Galpha(o)-coupled GIRK activation kinetics are due to intrinsic differences in receptor-catalyzed GDP release that rate-limit Gbetagamma production and is attributed to heterogeneity in Galpha(i) and Galpha(o) interdomain contacts.     

Inhibition of a Gi-activated potassium channel (GIRK1/4) by the Gq-coupled m1 muscarinic acetylcholine receptor

The G protein-coupled inwardly rectifying K+ channel, GIRK1/GIRK4, can be activated by receptors coupled to the Galpha(i) subunit. An opposing role for Galpha(q) receptor signaling in GIRK regulation has only recently begun to be established. We have studied the effects of m1 muscarinic acetylcholine receptor (mAChR) stimulation, which is known to mobilize calcium and activate protein kinase C (PKC) by a Galpha(q)-dependent mechanism, on whole cell GIRK1/4 currents in Xenopus oocytes. We found that stimulation of the m1 mAChR suppresses both basal and dopamine 2 receptor-activated GIRK 1/4 currents. Overexpression of Gbetagamma subunits attenuates this effect, suggesting that increased binding of Gbetagamma to the GIRK channel can effectively compete with the G(q)-mediated inhibitory signal. This G(q) signal requires the use of second messenger molecules; pharmacology implicates a role for PKC and Ca2+ responses as m1 mAChR-mediated inhibition of GIRK channels is mimicked by PMA and Ca2+ ionophore. We have analyzed a series of mutant and chimeric channels suggesting that the GIRK4 subunit is capable of responding to G(q) signals and that the resulting current inhibition does not occur via phosphorylation of a canonical PKC site on the channel itself.     

Dual signaling of human Mel1a melatonin receptors via G(i2), G(i3), and G(q/11) proteins

Mel 1a melatonin receptors belong to the super-family of guanine nucleotide-binding regulatory protein (G protein)-coupled receptors. So far, interest in Mel 1a receptor signaling has focused mainly on the modulation of the adenylyl cyclase pathway via pertussis toxin (PTX)-sensitive G proteins. To further investigate signaling of the human Mel 1a receptor, we have developed an antibody directed against the C terminus of this receptor. This antibody detected the Mel 1a receptor as a protein with an apparent molecular mass of approximately 60 kDa in immunoblots after separation by SDS-PAGE. It also specifically precipitated the 2-[125I]iodomelatonin (125I-Mel)-labeled receptor from Mel 1a-transfected HEK 293 cells. Coprecipitation experiments showed that G(i2), G(i3), and G(q/11) proteins couple to the Mel 1a receptor in an agonist-dependent and guanine nucleotide-sensitive manner. Coupling was selective since other G proteins present in HEK 293 cells, (G(i1), G(o), G(s), G(z), and G12) were not detected in receptor complexes. Coupling of the Mel 1a receptor to G(i) and G(q) was confirmed by inhibition of high-affinity 125I-Mel binding to receptors with subtype-selective G protein alpha-subunit antibodies. G(i2) and/or G(i3) mediated adenylyl cyclase inhibition while G(q/11) induced a transient elevation in cytosolic calcium concentrations in HEK 293 cells stably expressing Mel 1a receptors. Melatonin-induced cytosolic calcium mobilization via PTX-insensitive G proteins was confirmed in primary cultures of ovine pars tuberalis cells endogenously expressing Mel 1a receptors. In conclusion, we report the development of the first antibody recognizing the cloned human Mel 1a melatonin receptor protein. We show that Mel 1a receptors functionally couple to both PTX-sensitive and PTX-insensitive G proteins. The previously unknown signaling of Mel 1a receptors through G(q/11) widens the spectrum of potential targets for melatonin.     

Functional coupling with Galpha(q) and Galpha(i1) protein subunits promotes high-affinity agonist binding to the neurotensin receptor NTS-1 expressed in Escherichia coli

To analyze the coupling of Galpha subunits to the rat neurotensin receptor NTS-1 (NTR), fusion proteins were expressed in Escherichia coli with various Galpha subunits covalently linked to the receptor C-terminus. The presence of Galpha(q) or Galpha(i/q), in which the six C-terminal residues of Galpha(i1) were replaced with those from Galpha(q), increased the percentage of receptors in the agonist high-affinity state. This effect was less pronounced for wild-type Galpha(i1) and not observed for Galpha(i/s). Functional coupling of neurotensin receptor to Galpha was demonstrated by neurotensin-induced [(35)S]GTPgammaS binding for the Galpha(q), Galpha(i/q) and Galpha(i1) subunits, but not for Galpha(i/s). Our results extend previous findings of the dual coupling of NTR to pertussis toxin-sensitive and -insensitive G-proteins in Chinese hamster ovary cells with preference for the latter.     

Heterologous facilitation of G protein-activated K(+) channels by beta-adrenergic stimulation via cAMP-dependent protein kinase

To investigate possible effects of adrenergic stimulation on G protein-activated inwardly rectifying K(+) channels (GIRK), acetylcholine (ACh)-evoked K(+) current, I(KACh), was recorded from adult rat atrial cardiomyocytes using the whole cell patch clamp method and a fast perfusion system. The rise time of I(KACh ) was 0. 4 +/- 0.1 s. When isoproterenol (Iso) was applied simultaneously with ACh, an additional slow component (11.4 +/- 3.0 s) appeared, and the amplitude of the elicited I(KACh) was increased by 22.9 +/- 5.4%. Both the slow component of activation and the current increase caused by Iso were abolished by preincubation in 50 microM H89 (N-[2-((p -bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, a potent inhibitor of PKA). This heterologous facilitation of GIRK current by beta-adrenergic stimulation was further studied in Xenopus laevis oocytes coexpressing beta(2)-adrenergic receptors, m(2 )-receptors, and GIRK1/GIRK4 subunits. Both Iso and ACh elicited GIRK currents in these oocytes. Furthermore, Iso facilitated ACh currents in a way, similar to atrial cells. Cytosolic injection of 30-60 pmol cAMP, but not of Rp-cAMPS (a cAMP analogue that is inhibitory to PKA) mimicked the beta(2)-adrenergic effect. The possibility that the potentiation of GIRK currents was a result of the phosphorylation of the beta-adrenergic receptor (beta(2)AR) by PKA was excluded by using a mutant beta(2)AR in which the residues for PKA-mediated modulation were mutated. Overexpression of the alpha subunit of G proteins (Galpha(s)) led to an increase in basal as well as agonist-induced GIRK1/GIRK4 currents (inhibited by H89). At higher levels of expressed Galpha(s), GIRK currents were inhibited, presumably due to sequestration of the beta/gamma subunit dimer of G protein. GIRK1/GIRK5, GIRK1/GIRK2, and homomeric GIRK2 channels were also regulated by cAMP injections. Mutant GIRK1/GIRK4 channels in which the 40 COOH-terminal amino acids (which contain a strong PKA phosphorylation consensus site) were deleted were also modulated by cAMP injections. Hence, the structural determinant responsible is not located within this region. We conclude that, both in atrial myocytes and in Xenopus oocytes, beta-adrenergic stimulation potentiates the ACh-evoked GIRK channels via a pathway that involves PKA-catalyzed phosphorylation downstream from beta(2)AR.     

Na+ promotes the dissociation between Galpha GDP and Gbeta gamma,activating G protein-gated K+ channels

G protein-gated K(+) channels (GIRK, or Kir3) are activated by the direct binding of Gbetagamma or of cytosolic Na(+). Na(+) activation is fast, Gbetagamma-independent, and probably via a direct, low affinity (EC(50), 30-40 mm) binding of Na(+) to the channel. Here we demonstrate that an increase in intracellular Na(+) concentration, [Na(+)](in), within the physiological range (5-20 mm), activates GIRK within minutes via an additional, slow mechanism. The slow activation is observed in GIRK mutants lacking the direct Na(+) effect. It is inhibited by a Gbetagamma scavenger, hence it is Gbetagamma-dependent; but it does not require GTP. We hypothesized that Na(+) elevates the cellular concentration of free Gbetagamma by promoting the dissociation of the Galphabetagamma heterotrimer into free Galpha(GDP) and Gbetagamma. Direct biochemical measurements showed that Na(+) causes a moderate decrease (approximately 2-fold) in the affinity of interaction between Galpha(GDP) and Gbetagamma. Furthermore, in accord with the predictions of our model, slow Na(+) activation was enhanced by mild coexpression of Galpha(i3). Our findings reveal a previously unknown mechanism of regulation of G proteins and demonstrate a novel Gbetagamma-dependent regulation of GIRK by Na(+). We propose that Na(+) may act as a regulatory factor, or even a second messenger, that regulates effectors via Gbetagamma.     

Evidence for transduction of mu but not kappa opioid modulation of extracellular signal-regulated kinase activity by G(z) and G(12) proteins

Chronic treatment with micro or kappa opioid agonists (>/=2 h) inhibits EGF-induced ERK activation in opioid receptor overexpressing COS-7 cells. Although acute mu and kappa opioids activate ERK via a pertussis toxin-sensitive G protein, pertussis toxin insensitivity of the chronic mu (but not kappa) action was observed. Here, we tested several pertussis toxin-insensitive G proteins as candidates to transduce acute and/or chronic opioid modulation of ERK. Overexpressed Galpha(z) (but not Galpha(12)) transduced acute mu (but not kappa) ERK activation in pertussis toxin-treated COS-7 cells. Chronic mu (but not kappa) inhibited EGF stimulation of ERK in pertussis toxin-treated cells overexpressing Galpha(z) or Galpha(12). Transfection of Galpha(13) or Galpha(q) blocked inhibition under the same conditions. Overexpressed interfering and non-interfering Galpha(z) mutants differentially affected mu inhibition of ERK consistent with G(z) transduction. In this and prior studies, Galpha(z) and Galpha(12) immunoreactivity were detected in untransfected COS-7 cells, suggesting that these G proteins may be endogenous mediators of chronic mu inhibitory actions on ERK.