The GoLoco motif: a Gαi/o binding motif and potential guanine-nucleotide exchange factor

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Activation of GABA B receptors in chick dorsal root ganglion (DRG) neurons inhibits the Ca v 2.2 calcium channel in both a voltage-dependent and voltage-independent manner. The voltage-independent inhibition requires activation of a tyrosine kinase that phosphorylates the ␣ 1 subunit of the channel and thereby recruits RGS12, a member of the "regulator of G protein signaling" (RGS) proteins. Here we report that RGS12 binds to the SNARE-binding or "synprint" region (amino acids 726 -985) in loop II-III of the calcium channel ␣1 subunit. A recombinant protein encompassing the Nterminal PTB domain of RGS12 binds to the synprint region in protein overlay and surface plasmon resonance binding assays; this interaction is dependent on tyrosine phosphorylation and yet is within a sequence that differs from the canonical NPXY motif targeted by other PTB domains. In electrophysiological experiments, microinjection of DRG neurons with synprint-derived peptides containing the tyrosine residue Tyr-804 altered the rate of desensitization of neurotransmitter-mediated inhibition of the Ca v 2.2 calcium channel, whereas peptides centered about a second tyrosine residue, Tyr-815, were without effect. RGS12 from a DRG neuron lysate was precipitated using synprint peptides containing phosphorylated Tyr-804. The high degree of conservation of Tyr-804 in the SNAREbinding region of Ca v 2.1 and Ca v 2.2 calcium channels suggests that this region, in addition to the binding of SNARE proteins, is also important for determining the time course of the modulation of calcium current via tyrosine phosphorylation.Multiple G protein-mediated signaling pathways are known to modulate Ca v 2.2 (N-type) calcium channels (1, 2) via direct G protein-ion channel interactions, activation of second messenger cascades, and activation of tyrosine kinases (3, 4). This modulation of voltage-dependent calcium channels is a transient phenomenon. Upon prolonged exposure to a neurotransmitter, neurons become unresponsive or desensitized. Despite the common requirement for the activation of a G proteincoupled receptor kinase (GRK3) for desensitization of the neurotransmitter-mediated inhibition of calcium current (5), G i -and G o -mediated pathways exhibit different rates of desensitization (6) that may result from selective effects of the G␣-directed GTPase-accelerating activity borne by "regulator of G protein signaling" (RGS) 1 proteins (7,8).In dorsal root ganglion (DRG) neurons, the activation of ␥-aminobutyric acid type B (GABA B ) receptors induces both voltage-dependent and voltage-independent inhibition of Ca v 2.2 channels (9). Voltage-independent inhibition requires the activation of a tyrosine kinase that phosphorylates the pore-forming ␣-subunit of the calcium channel (10). The tyrosine-phosphorylated form of the ␣-subunit becomes a target for the phosphotyrosine binding (PTB) domain of RGS12, a member of the RGS protein superfamily that specifically accelerates the rate of desensitization of this response (10).To better understand the molecular b...

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

RGS12 Interacts with the SNARE-binding Region of the Cav2.2 Calcium Channel

Richman

Strock

Hains

et al. 2005

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“…G18.1b was one of six mammalian proteins identified by a motif searching strategy based upon a consensus GPR motif in AGS3 (6,17). 2 Each of these proteins (Rap1GAPII, LGN, RGS12, RGS14, PCP2, and G18.1b) contained at least one GPR motif.…”

Section: Figmentioning

confidence: 99%

Identification and Characterization of AGS4

Cao

Cismowski

Sato

et al. 2004

Activators of G-protein signaling 1-3 (AGS1-3) were identified in a functional screen of mammalian cDNAs that activated G-protein signaling in the absence of a receptor. We report the isolation and characterization of an additional AGS protein (AGS4) from a human prostate leiomyosarcoma cDNA library. AGS4 is identical to G18.1b, which is encoded by a gene within the major histocompatibility class III region of chromosome 6. The activity of AGS4 in the yeast-based functional screen was selective for G i2 /G i3 and independent of guaninenucleotide exchange by G i ␣. RNA blots indicated enrichment of AGS4/G18.1b mRNA in heart, placenta, lung, and liver. Immunocytochemistry with AGS4/G18.1b-specific antisera indicated a predominant nonhomogeneous, extranuclear distribution within the cell following expression in COS7 or Chinese hamster ovary cells. AGS4/ G18.1b contains three G-protein regulatory motifs downstream of an amino terminus domain with multiple prolines. Glutathione S-transferase (GST)-AGS4/G18.1b fusion proteins interacted with purified G i ␣, and peptides derived from each of the G-protein regulatory motifs inhibited guanosine 5 -3-O-(thio)triphosphate (GTP␥S) binding to purified G i ␣ 1 . AGS4/G18.1b was also complexed with G i ␣ 3 in COS7 cell lysates following cell transfection. However, AGS4/G18.1b did not alter the generation of inositol phosphates in COS7 cells cotransfected with the G␤␥-regulated effector phospholipase C-␤2. These data suggest either that an additional signal is required to position AGS4/G18.1b in the proper cellular location where it can access heterotrimer and promote subunit dissociation or that AGS4 serves as an alternative binding partner for G i ␣ independent of G␤␥ participating in G-protein signaling events that are independent of classical G-protein-coupled receptors at the cell surface.

“…1 AGS3 was identified as a receptor-independent activator of G-protein signaling in a yeastbased functional screen of mammalian cDNAs (2,3). AGS3-LONG is a 650-amino acid protein containing seven tetratricopeptide repeats and four G-protein regulatory (GPR) motifs that preferentially bind G␣ i (2,(7)(8)(9)(10)(11). The GPR motif is also found in Pcp2, G18.1b, LGN, and the GTPase-activating proteins RGS12, RGS14 (regulator of G-protein signaling), RAP1GAPI (partial GPR motif), and Rap1GAPII (3).…”

mentioning

confidence: 99%

AGS3 and Signal Integration by Gαs- and Gαi-coupled Receptors

Sato

Gettys

Lanier

2004

AGS3-LONG and AGS3-SHORT contain G-protein regulatory motifs that interact with and stabilize the GDPbound conformation of G␣ i > G␣ o . AGS3 and related proteins may influence signal strength or duration as well as the adaptation of the signaling system associated with sustained stimulation. To address these issues, we determined the effect of AGS3 on the integration of stimulatory (G␣ s -mediated vasoactive intestinal peptide receptor) and inhibitory (G␣ i -mediated ␣ 2 -adrenergic receptor (␣ 2 -AR)) signals to adenylyl cyclase in Chinese hamster ovary cells. AGS3-SHORT and AGS3-LONG did not alter the VIP-induced increase in cAMP or the inhibitory effect of ␣ 2 -AR activation. System adaptation was addressed by determining the influence of AGS3 on the sensitization of adenylyl cyclase that occurs following prolonged activation of a G␣ i -coupled receptor. Incubation of cells with the ␣ 2 -AR agonist UK14304 (1 M) for 18 h resulted in a ϳ1.8-fold increase in the vasoactive intestinal peptide-induced activation of adenylyl cyclase, and this was associated with a decrease in membrane-associated G␣ i3 . Both effects were blocked by AGS3-SHORT. AGS3-SHORT also decreased the rate of G␣ i3 decay. A mutant AGS3-SHORT incapable of binding G-protein was inactive. These data suggest that AGS3 and perhaps other G-protein regulatory motif-containing proteins increase the stability of G␣ i in the membrane, which influences the adaptation of the cell to prolonged activation of G␣ i -coupled receptors.The activation/deactivation cycle for heterotrimeric G-protein involves the exchange of GDP for GTP and the subsequent hydrolysis of GTP by G␣ subunits. These events are generally initiated by a G-protein-coupled receptor at the cell surface. However, increasing evidence indicates the existence of unexpected regulatory mechanisms for the G-protein activation/ deactivation cycle and a role for G-protein subunits in signaling events within the cell that do not involve a cell-surface receptor (1). These unexpected regulatory mechanisms include accessory proteins that accelerate GTPase activity, serve as novel binding partners for G␣ and G␤␥ subunits, and/or regulate guanine nucleotide exchange independent of a G-protein coupled receptor (1-6).