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γ-Aminobutyric acid (GABA) release from inhibitory interneurons located within the cerebellar cortex limits the extent of neuronal excitation in part through activation of metabotropic GABA(B) receptors. Stimulation of these receptors triggers a number of downstream signaling events, including activation of GIRK channels by the Gβγ dimer resulting in membrane hyperpolarization and inhibition of neurotransmitter release from presynaptic sites. Here, we identify RGS6, a member of the R7 subfamily of RGS proteins, as a key regulator of GABA(B)R signaling in cerebellum. RGS6 is enriched in the granule cell layer of the cerebellum along with neuronal GIRK channel subunits 1 and 2 where RGS6 forms a complex with known binding partners Gβ(5) and R7BP. Mice lacking RGS6 exhibit abnormal gait and ataxia characterized by impaired rotarod performance improved by treatment with a GABA(B)R antagonist. RGS6(-/-) mice administered baclofen also showed exaggerated motor coordination deficits compared with their wild-type counterparts. Isolated cerebellar neurons natively expressed RGS6, GABA(B)R, and GIRK channel subunits, and cerebellar granule neurons from RGS6(-/-) mice showed a significant delay in the deactivation kinetics of baclofen-induced GIRK channel currents. These results establish RGS6 as a key component of GABA(B)R signaling and represent the first demonstration of an essential role for modulatory actions of RGS proteins in adult cerebellum. Dysregulation of RGS6 expression in human patients could potentially contribute to loss of motor coordination and, thus, pharmacological manipulation of RGS6 levels might represent a viable means to treat patients with ataxias of cerebellar origin.
Voltage-gated Ca(2+) channels translate the electrical inputs of excitable cells into biochemical outputs by controlling influx of the ubiquitous second messenger Ca(2+) . As such the channels play pivotal roles in many cellular functions including the triggering of neurotransmitter and hormone release by CaV2.1 (P/Q-type) and CaV2.2 (N-type) channels. It is well established that G protein coupled receptors (GPCRs) orchestrate precise regulation neurotransmitter and hormone release through inhibition of CaV2 channels. Although the GPCRs recruit a number of different pathways, perhaps the most prominent, and certainly most studied among these is the so-called voltage-dependent inhibition mediated by direct binding of Gβγ to the α1 subunit of CaV2 channels. This article will review the basics of Ca(2+) -channels and G protein signaling, and the functional impact of this now classical inhibitory mechanism on channel function. It will also provide an update on more recent developments in the field, both related to functional effects and crosstalk with other signaling pathways, and advances made toward understanding the molecular interactions that underlie binding of Gβγ to the channel and the voltage-dependence that is a signature characteristic of this mechanism.
Catecholamines and other transmitters released from adrenal chromaffin cells play central roles in the "fight-or-flight" response and exert profound effects on cardiovascular, endocrine, immune, and nervous system function. As such, precise regulation of chromaffin cell exocytosis is key to maintaining normal physiological function and appropriate responsiveness to acute stress. Chromaffin cells express a number of different G protein coupled receptors (GPCRs) that sense the local environment and orchestrate this precise control of transmitter release. The primary trigger for catecholamine release is Ca2+ entry through voltage-gated Ca2+ channels, so it makes sense that these channels are subject to complex regulation by GPCRs. In particular G protein βγ heterodimers (Gbc) bind to and inhibit Ca2+ channels. Here I review the mechanisms by which GPCRs inhibit Ca2+ channels in chromaffin cells and how this might be altered by cellular context. This is related to the potent autocrine inhibition of Ca2+ entry and transmitter release seen in chromaffin cells. Recent data that implicate an additional inhibitory target of Gβγ on the exocytotic machinery and how this might fine tune neuroendocrine secretion are also discussed.
Evidence from Drosophila and cultured cell studies supports a role for heterotrimeric guanosine triphosphate-binding proteins (G proteins) in Wnt signaling. Wnt inhibits the degradation of the transcriptional regulator beta-catenin. We screened the alpha and betagamma subunits of major families of G proteins in a Xenopus egg extract system that reconstitutes beta-catenin degradation. We found that Galpha(o), Galpha(q), Galpha(i2), and Gbetagamma inhibited beta-catenin degradation. Gbeta(1)gamma(2) promoted the phosphorylation and activation of the Wnt co-receptor low-density lipoprotein receptor-related protein 6 (LRP6) by recruiting glycogen synthase kinase 3 (GSK3) to the membrane and enhancing its kinase activity. In both a reporter gene assay and an in vivo assay, c-betaARK (C-terminal domain of beta-adrenergic receptor kinase), an inhibitor of Gbetagamma, blocked LRP6 activity. Several components of the Wnt-beta-catenin pathway formed a complex: Gbeta(1)gamma(2), LRP6, GSK3, axin, and dishevelled. We propose that free Gbetagamma and Galpha subunits, released from activated G proteins, act cooperatively to inhibit beta-catenin degradation and activate beta-catenin-mediated transcription.
G-protein-coupled receptors (GPCR) play important roles in controlling neurotransmitter and hormone release. Inhibition of voltage-gated Ca(2+) channels (Ca(2+) channels) by G protein betagamma subunits (Gbetagamma) is one prominent mechanism, but there is evidence for additional effects distinct from those on calcium entry. However, relatively few studies have investigated the Ca(2+)-channel-independent effects of Gbetagamma on transmitter release, so the impact of this mechanism remains unclear. We used carbon fiber amperometry to analyze catecholamine release from individual vesicles in bovine adrenal chromaffin cells, a widely used neurosecretory model. To bypass the effects of Gbetagamma on Ca(2+) entry, we stimulated secretion using ionomycin (a Ca(2+) ionophore) or direct intracellular application of Ca(2+) through a patch pipette. Activation of endogenous GPCR or transient transfection with exogenous Gbetagamma significantly reduced the number of amperometric spikes (the number of vesicular fusion events). The charge ("quantal size") and amplitude of the amperometric spikes were also significantly reduced by GPCR/Gbetagamma. We conclude that independent from effects on calcium entry, Gbetagamma can regulate both the number of vesicles that undergo exocytosis and the amount of catecholamine released per fusion event. We discuss possible mechanisms by which Gbetagamma might exert these novel effects including interaction with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex.
Migration of cells up the chemoattractant gradients is mediated by the binding of chemoattractants to G protein-coupled receptors and activation of a network of coordinated excitatory and inhibitory signals. Although the excitatory process has been well studied, the molecular nature of the inhibitory signals remains largely elusive. Here we report that the receptor for activated C kinase 1 (RACK1), a novel binding protein of heterotrimeric G protein betagamma (G betagamma) subunits, acts as a negative regulator of directed cell migration. After chemoattractant-induced polarization of Jurkat and neutrophil-like differentiated HL60 (dHL60) cells, RACK1 interacts with G betagamma and is recruited to the leading edge. Down-regulation of RACK1 dramatically enhances chemotaxis of cells, whereas overexpression of RACK1 or a fragment of RACK1 that retains G betagamma-binding capacity inhibits cell migration. Further studies reveal that RACK1 does not modulate cell migration through binding to other known interacting proteins such as PKC beta and Src. Rather, RACK1 selectively inhibits G betagamma-stimulated phosphatidylinositol 3-kinase gamma (PI3K gamma) and phospholipase C (PLC) beta activity, due to the competitive binding of RACK1, PI3K gamma, and PLC beta to G betagamma. Taken together, these findings provide a novel mechanism of regulating cell migration, i.e., RACK1-mediated interference with G betagamma-dependent activation of key effectors critical for chemotaxis.
Presynaptic inhibitory G protein-coupled receptors (GPCRs) can decrease neurotransmission by inducing interaction of Gbetagamma with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. We have shown that this action of Gbetagamma requires the carboxyl terminus of the 25-kDa synaptosome-associated protein (SNAP25) and is downstream of the well known inhibition of Ca2+ entry through voltage-gated calcium channels. We propose a mechanism in which Gbetagamma and synaptotagmin compete for binding to the SNARE complex. Here, we characterized the Gbetagamma interaction sites on syntaxin1A and SNAP25 and demonstrated an overlap of the Gbetagamma- and synaptotagmin I -binding regions on each member of the SNARE complex. Synaptotagmin competes in a Ca2+-sensitive manner with binding of Gbetagamma to SNAP25, syntaxin1A, and the assembled SNARE complex. We predict, based on these findings, that at high intracellular Ca2+ concentrations, Ca2+-synaptotagmin I can displace Gbetagamma binding and the Gbetagamma-dependent inhibition of exocytosis can be blocked. We tested this hypothesis in giant synapses of the lamprey spinal cord, where 5-HT works via Gbetagamma to inhibit neurotransmission (Blackmer et al., 2001). We showed that increased presynaptic Ca2+ suppresses the 5-HT- and Gbetagamma-dependent inhibition of exocytosis. We suggest that this effect may be due to Ca2+-dependent competition between Gbetagamma and synaptotagmin I for SNARE binding. This type of dynamic regulation may represent a novel mechanism for modifying transmitter release in a graded manner based on the history of action potentials that increase intracellular Ca2+ concentrations and of inhibitory signals through G(i)-coupled GPCRs.
Phospholipase D-mediated hydrolysis of phosphatidylcholine is stimulated by protein kinase C and the monomeric G proteins Arf, RhoA, Cdc42, and Rac1, resulting in complex regulation of this enzyme. Using purified proteins, we have identified a novel inhibitor of phospholipase D activity, Gbetagamma subunits of heterotrimeric G proteins. G protein-coupled receptor activation alters affinity between Galpha and Gbetagamma subunits, allowing subsequent interaction with distinct effectors. Gbeta1gamma1 inhibited phospholipase D1 and phospholipase D2 activity, and both Gbeta1gamma1 and Gbeta1gamma2 inhibited stimulated phospholipase D1 activity in a dosedependent manner in reconstitution assays. Reconstitution assays suggest this interaction occurs through the amino terminus of phospholipase D, because Gbeta1gamma1 is unable to inhibit an amino-terminally truncated phospholipase D construct, PLD1.d311, which like full-length phospholipase D isoforms, requires phosphatidylinositol-4,5-bisphosphate for activity. Furthermore, a truncated protein consisting of the amino-terminal region of phospholipase D containing the phox/pleckstrin homology domains was found to interact with Gbeta1gamma1, unlike the PLD1.d311 recombinant protein, which lacks this domain. In vivo, expressed recombinant Gbeta1gamma2 was also found to inhibit phospholipase D activity under basal and stimulated conditions in MDA-MB-231 cells, which natively express both phospholipase D1 and phospholipase D2. These data demonstrate that Gbetagamma directly regulates phospholipase D activity in vitro and suggest a novel mechanism to negatively regulate phospholipase D signaling in vivo.
In a yeast two-hybrid screen designed to identify novel effectors of the G betagamma subunit of heterotrimeric G proteins, we found that G betagamma binds to histone deacetylase 5 (HDAC5), an enzyme involved in a pathway not previously recognized to be directly impacted by G proteins. Formation of the G beta1gamma2-HDAC5 complex in mammalian cells can be blocked by overexpression of G alpha(o), and this inhibition is relieved by activation of alpha2A-adrenergic receptor, suggesting that the interaction occurs in a signal-dependent manner. The C-terminal domain of HDAC5 binds directly to G betagamma through multiple motifs, and overexpression of this domain mimics the C terminus of G protein-coupled receptor kinase 2, a known G betagamma scavenger, in its ability to inhibit the G betagamma/HDAC5 interaction. The C terminus of HDAC4 shares significant similarity with that of HDAC5, and accordingly, HDAC4 is also able to form complexes with G beta1gamma2 in cultured cells, suggesting that the C-terminal domain of class II HDACs is a general G betagamma binding motif. Activation of a G(i/o)-coupled receptor results in a time-dependent activation of MEF2C, an HDAC5-regulated transcription factor, whereas inhibition of the interaction with a G betagamma scavenger inhibits MEF2C activity, suggesting a reduced potency of HDAC5-mediated inhibition. Taken together, these data imply that HDAC5 and possibly other class II HDACs can be added to the growing list of G betagamma effectors.
Receptor for Activated C Kinase 1 (RACK1), a novel G betagamma-interacting protein, selectively inhibits the activation of a subclass of G betagamma effectors such as phospholipase C beta2 (PLCbeta2) and adenylyl cyclase II by direct binding to G betagamma (Chen, S., Dell, E. J., Lin, F., Sai, J., and Hamm, H. E. (2004) J. Biol. Chem. 279, 17861-17868). Here we have mapped the RACK1 binding sites on G betagamma. We found that RACK1 interacts with several different G betagamma isoforms, including G beta1gamma1, Gbeta1gamma2, and Gbeta5gamma2, with similar affinities, suggesting that the conserved residues between G beta1 and G beta5 may be involved in their binding to RACK1. We have confirmed this hypothesis and shown that several synthetic peptides corresponding to the conserved residues can inhibit the RACK1/G betagamma interaction as monitored by fluorescence spectroscopy. Interestingly, these peptides are located at one side of G beta1 and have little overlap with the G alpha subunit binding interface. Additional experiments indicate that the G betagamma contact residues for RACK1, in particular the positively charged amino acids within residues 44-54 of G beta1, are also involved in the interaction with PLCbeta2 and play a critical role in G betagamma-mediated PLCbeta2 activation. These data thus demonstrate that RACK1 can regulate the activity of a G betagamma effector by competing for its binding to the signal transfer region of G betagamma.