GoLoco Motif Research Articles

Inhibition of GDP/GTP Exchange on Gα Subunits by Proteins Containing G-Protein Regulatory Motifs

A novel Galpha binding consensus sequence, termed G-protein regulatory (GPR) or GoLoco motif, has been identified in a growing number of proteins, which are thought to modulate G-protein signaling. Alternative roles of GPR proteins as nucleotide exchange factors or as GDP dissociation inhibitors for Galpha have been proposed. We investigated the modulation of the GDP/GTP exchange of Gialpha(1), Goalpha, and Gsalpha by three proteins containing GPR motifs (GPR proteins), LGN-585-642, Pcp2, and RapIGAPII-23-131, to elucidate the mechanisms of GPR protein function. The GPR proteins displayed similar patterns of interaction with Gialpha(1) with the following order of affinities: Gialpha(1)GDP >> Gialpha(1)GDPAlF(4)(-) > or = Gialpha(1)GTPgammaS. No detectable binding of the GPR proteins to Gsalpha was observed. LGN-585-642, Pcp2, and RapIGAPII-23-131 inhibited the rates of spontaneous GTPgammaS binding and blocked GDP release from Gialpha(1) and Goalpha. The inhibitory effects of the GPR proteins on Gialpha(1) were significantly more potent, indicating that Gi might be a preferred target for these modulators. Our results suggest that GPR proteins are potent GDP dissociation inhibitors for Gialpha-like Galpha subunits in vitro, and in this capacity they may inhibit GPCR/Gi protein signaling in vivo.

RGS12 and RGS14 GoLoco Motifs Are Gα i Interaction Sites with Guanine Nucleotide Dissociation Inhibitor Activity

RGS12 and RGS14 GoLoco Motifs Are Gα i Interaction Sites with Guanine Nucleotide Dissociation Inhibitor Activity

Activator of G protein signaling 3 is a guanine dissociation inhibitor for Galpha i subunits.

Activator of G protein signaling 3 (AGS3) is a newly identified protein shown to act at the level of the G protein itself. AGS3 belongs to the GoLoco family of proteins, sharing the 19-aa GoLoco motif that is a Galpha(i/o) binding motif. AGS3 interacts only with members of the Galpha(i/o) subfamily. By surface plasmon resonance, we found that AGS3 binds exclusively to the GDP-bound form of Galpha(i3). In GTPgammaS binding assays, AGS3 behaves as a guanine dissociation inhibitor (GDI), inhibiting the rate of exchange of GDP for GTP by Galpha(i3). AGS3 interacts with both Galpha(i3) and Galpha(o) subunits, but has GDI activity only on Galpha(i3), not on Galpha(o). The fourth GoLoco motif of AGS3 is a major contributor to this activity. AGS3 stabilizes Galpha(i3) in its GDP-bound form, as it inhibits the increase in tryptophan fluorescence of the Galpha(i3)-GDP subunit stimulated by AlF(4)(-). AGS3 is widely expressed as it is detected by immunoblotting in brain, testis, liver, kidney, heart, pancreas, and in PC-12 cells. Several different sizes of the protein are detected. By Northern blotting, AGS3 shows 2.3-kb and 3.5-kb mRNAs in heart and brain, respectively, suggesting tissue-specific alternative splicing. Taken together, our results demonstrate that AGS3 is a GDI. To the best of our knowledge, no other GDI has been described for heterotrimeric G proteins. Inhibition of the Galpha subunit and stimulation of heterotrimeric G protein signaling, presumably by stimulating Gbetagamma, extend the possibilities for modulating signal transduction through heterotrimeric G proteins.

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

Studies of the desensitization of G-protein-coupled signal transduction have led to the discovery of a family of GTPase-activating proteins (GAPs) for heterotrimetric G-protein α subunits – the ‘regulator of G-protein signaling’ or RGS proteins 1 Siderovski D.P. et al. Curr. Biol. 1996; 6: 211-212 Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar , 2 De Vries L. Farquhar M.G. Trends Cell Biol. 1999; 9: 138-144 Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar . RGS12 is the largest mammalian RGS protein that is currently known 2 De Vries L. Farquhar M.G. Trends Cell Biol. 1999; 9: 138-144 Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar and has a multi-domain structure reminiscent of both adaptor and scaffold proteins (Fig. 1a). It consists of an N-terminal PDZ (PSD-95/Dlg/ZO-1) domain, which binds the C-termini of both RGS12 itself and the G-protein-coupled interleukin-8 receptor B or CXCR2 ( 3 Snow B.E. et al. J. Biol. Chem. 1998; 273: 17749-17755 Crossref PubMed Scopus (181) Google Scholar ), a phosphotyrosine-binding (PTB) domain (D. P. Siderovski, L. De Vries, and M. A. Diversé-Pierluissi, unpublished), and a central RGS domain, which is a GAP for Gi/o-class heterotrimeric G protein α subunits 3 Snow B.E. et al. J. Biol. Chem. 1998; 273: 17749-17755 Crossref PubMed Scopus (181) Google Scholar . Here, we show that the C-terminus of RGS12 contains a new 19-amino-acid motif, also present in other proteins that bind Gi/o-class α subunits (Fig. 1b). As this motif was initially discovered by comparison of mammalian RGS proteins with Loco, the Drosophila RGS12 homologue, we have named it the Gαi/o-Loco or ‘GoLoco’ motif.