Abstract. Heterotrimeric G-proteins are intracellular partners of G-protein-coupled receptors (GPCRs). GPCRs act on inactive Ga·GDP/Gbg heterotrimers to promote GDP release and GTP binding, resulting in liberation of Ga from Gbg. Ga·GTP and Gbg target effectors including adenylyl cyclases, phospholipases and ion channels. Signaling is terminated by intrinsic GTPase activity of Ga and heterotrimer reformation -a cycle accelerated by 'regulators of G-protein signaling' (RGS proteins). Recent studies have identified several unconventional G-protein signaling pathways that diverge from this CMLS, Cell. Mol. Life Sci. 62 (2005) 551-577 1420-682X/05/050551-27 DOI 10.1007/s00018-004-4462-3 © Birkhäuser Verlag, Basel, 2005 CMLS Cellular and Molecular Life Sciencesstandard model. Whereas phospholipase C (PLC) b is activated by Ga q and Gbg, novel PLC isoforms are regulated by both heterotrimeric and Ras-superfamily G-proteins. An Arabidopsis protein has been discovered containing both GPCR and RGS domains within the same protein. Most surprisingly, a receptor-independent Ga nucleotide cycle that regulates cell division has been delineated in both Caenorhabditis elegans and Drosophila melanogaster. Here, we revisit classical heterotrimeric G-protein signaling and explore these new, non-canonical G-protein signaling pathways.

Because phospholipase C (PLC-) is activated by G␣ 12/13 and Rho family GTPases, we investigated whether these G proteins contribute to the increased inositol lipid hydrolysis observed in COS-7 cells after activation of certain G protein-coupled receptors. Stimulation of inositol lipid hydrolysis by endogenous lysophosphatidic acid (LPA) or thrombin receptors was markedly enhanced by the expression of PLC-. Expression of the LPA 1 or PAR1 receptor increased inositol phosphate production in response to LPA or SFLLRN, respectively, and these agonist-stimulated responses were markedly enhanced by coexpression of PLC-. Both LPA 1 and PAR1 receptor-mediated activation of PLC-was inhibited by coexpression of the regulator of G protein signaling (RGS) domain of p115RhoGEF, a GTPase-activating protein for G␣ 12/13 but not by expression of the RGS domain of GRK2, which inhibits G␣ q signaling. In contrast, activation of the G q -coupled M1 muscarinic or P2Y 2 purinergic receptor was neither enhanced by coexpression with PLC-nor inhibited by the RGS domain of p115RhoGEF but was blocked by expression of the RGS domain of GRK2. Expression of the Rho inhibitor C3 botulinum toxin did not affect LPA-or SFLLRN-stimulated inositol lipid hydrolysis in the absence of PLC-but completely prevented the PLC--dependent increase in inositol phosphate accumulation. Likewise, C3 toxin blocked the PLC--dependent stimulatory effects of the LPA 1 , LPA 2 , LPA 3 , or PAR1 receptor but had no effect on the agonist-promoted inositol phosphate response of the M1 or P2Y 2 receptor. Moreover, PLC--dependent stimulation of inositol phosphate accumulation by activation of the epidermal growth factor receptor, which involves Ras-but not Rho-mediated activation of the phospholipase, was unaffected by C3 toxin. These studies illustrate that specific LPA and thrombin receptors promote inositol lipid signaling via activation of G␣ 12/13 and Rho.Many extracellular hormones, neurotransmitters, and growth factors exert their physiological effects by mechanisms that in part involve phospholipase C-catalyzed breakdown of phosphatidylinositol (4,5)P 2 into the Ca 2ϩ -mobilizing second-messenger inositol (1,4,5)P 3 and the protein kinase C-activating second-messenger diacylglycerol (Irvine et al., 1987;Rhee, 2001). For example, extracellular stimuli that activate members of the large family of seven transmembrane-spanning heterotrimeric G protein-coupled receptors (GPCRs) activate PLC-␤ isozymes by the release of ␣-subunits of the G q family of G proteins (Smrcka et al., 1991;Taylor et al., 1991;Waldo et al., 1991) or by the release of G␤␥ dimers from activated G i (Blank et al., 1992;Boyer et al., 1992;Camps et al., 1992). In contrast, PLC-␥ enzymes are activated by tyrosine phosphorylation after activation of receptor and nonreceptor tyrosine kinases (Meisenhelder et al., 1989;Wahl et al., 1989).PLC-, which possesses Ras-associating (RA) domains at its carboxyl terminus, was initially identified in Caenorhabditis elegans as a Ras-binding protein (Shib...

GoLoco ('Gα i/o -Loco' interaction) motif proteins have recently been identified as novel GDIs (guanine nucleotide dissociation inhibitors) for heterotrimeric G-protein α subunits. G18 is a member of the mammalian GoLoco-motif gene family and was uncovered by analyses of human and mouse genomes for anonymous open-reading frames. The encoded G18 polypeptide is predicted to contain three 19-amino-acid GoLoco motifs, which have been shown in other proteins to bind Gα subunits and inhibit spontaneous nucleotide release. However, the G18 protein has thus far not been characterized biochemically. Here, we have cloned and expressed the G18 protein and assessed its ability to act as a GDI. G18 is capable of simultaneously binding more than one Gα i1 subunit. In binding assays with the non-hydrolysable GTP analogue guanosine 5 -[γ -thio]triphosphate, G18 exhibits GDI activity, slowing the exchange of GDP for GTP by Gα i1 . Only the first and third GoLoco motifs within G18 are capable of interacting with Gα subunits, and these bind with low micromolar affinity only to Gα i1 in the GDP-bound form, and not to Gα o , Gα q , Gα s or Gα 12 . Mutation of Ala-121 to aspartate in the inactive second GoLoco motif of G18, to restore the signature acidicglutamine-arginine tripeptide that forms critical contacts with Gα and its bound nucleotide [Kimple, Kimple, Betts, Sondek and Siderovski (2002) Nature (London) 416, 878-881], results in gain-of-function with respect to Gα binding and GDI activity.

GPSM2 (G-protein signalling modulator 2; also known as LGN or mammalian Pins) is a protein that regulates mitotic spindle organization and cell division. GPSM2 contains seven tetratricopeptide repeats (TPR) and four Galpha(i/o)-Loco (GoLoco) motifs. GPSM2 has guanine nucleotide dissociation inhibitor (GDI) activity towards both Galpha(o)- and Galpha(i)-subunits; however, a systematic analysis of its individual GoLoco motifs has not been described. We analyzed each of the four individual GoLoco motifs from GPSM2, assessing their relative binding affinities and GDI potencies for Galpha(i1), Galpha(i2), and Galpha(i3) and Galpha(o). Each of the four GPSM2 GoLoco motifs (36-43 amino acids in length) was expressed in bacteria as a GST-fusion protein and purified to homogeneity. The binding of each of the four GST-GoLoco motifs to Galpha(i1)-, Galpha(o)-, and Galpha(s)-subunits was assessed by surface plasmon resonance; all of the motifs bound Galpha(i1), but exhibited low affinity towards Galpha(o). GDI activity was assessed by a fluorescence-based nucleotide-binding assay, revealing that all four GoLoco motifs are functional as GDIs for Galpha(i1), Galpha(i2), and Galpha(i3). Consistent with our binding studies, the GDI activity of GPSM2 GoLoco motifs on Galpha(o) was significantly lower than that toward Galpha(i1), suggesting that the in vivo targets of GPSM2 are most likely to be Galpha(i)-subunits.

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...

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Regulators of G-protein Signaling (RGS proteins)