GTPase Activating Specificity of RGS12 and Binding Specificity of an Alternatively Spliced PDZ (PSD-95/Dlg/ZO-1) Domain

Abstract

Regulator of G-protein signaling (RGS) proteins increase the intrinsic guanosine triphosphatase (GTPase) activity of G-protein ␣ subunits in vitro, but how specific G-protein-coupled receptor systems are targeted for down-regulation by RGS proteins remains uncharacterized. Here, we describe the GTPase specificity of RGS12 and identify four alternatively spliced forms of human RGS12 mRNA. Two RGS12 isoforms of 6.3 and 5.7 kilobases (kb), encoding both an N-terminal PDZ (PSD-95/ Dlg/ZO-1) domain and the RGS domain, are expressed in most tissues, with highest levels observed in testis, ovary, spleen, cerebellum, and caudate nucleus. The 5.7-kb isoform has an alternative 3 end encoding a putative C-terminal PDZ domain docking site. Two smaller isoforms, of 3.1 and 3.7 kb, which lack the PDZ domain and encode the RGS domain with and without the alternative 3 end, respectively, are most abundantly expressed in brain, kidney, thymus, and prostate. In vitro biochemical assays indicate that RGS12 is a GTPaseactivating protein for G i class ␣ subunits. Biochemical and interaction trap experiments suggest that the RGS12 N terminus acts as a classical PDZ domain, binding selectively to C-terminal (A/S)-T-X-(L/V) motifs as found within both the interleukin-8 receptor B (CXCR2) and the alternative 3 exon form of RGS12. The presence of an alternatively spliced PDZ domain within RGS12 suggests a mechanism by which RGS proteins may target specific G-protein-coupled receptor systems for desensitization.The mammalian "regulators of G-protein signaling" (RGS) 1 gene family was first identified by sequence and functional similarity to fungal and nematode genes captured in genetic screens for negative regulators of specific G-protein-coupled receptor (GPCR) signals (1-3). In vitro biochemical analyses soon established that this gene family encodes potent accelerators ("GAPs") of the intrinsic GTP hydrolysis activity of Gprotein ␣ subunits, revealing a molecular mechanism by which RGS proteins drive G-proteins into their inactive GDP-bound form and hence down-regulate GPCR signal transduction in vivo (reviewed in Refs. 4 and 5). However, the mechanisms by which individual RGS proteins desensitize pathways activated by particular GPCRs remain to be elucidated. Tightly regulated transcription has been described for RGS1 (3), RGS2 (6), and RGS3-RGS11 (7), and palmitoylation of the cysteine-rich N terminus of G␣-interacting protein (GAIP) has also been observed (8); however, transcriptional regulation and post-translational modifications of particular RGS family members can each only be expected to afford a gross level of intracellular control over the temporal and spatial expression of G␣-directed GAP activity.We and others have hypothesized that regions outside the RGS fold contribute to regulation of G␣ GAP activity and/or targeting of individual RGS proteins to particular receptor signaling pathways (4, 5, 9, 10). Here, we report the GAP activity of RGS12 and identify a PDZ-like N-terminal sequence within two splice forms. PDZ domains...

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

“…RGS12 has the potential to serve as a signal transduction "nexus" that modulates multiple signaling pathway components by virtue of its multi-domain architecture including Nterminal PDZ and PTB domains, a central RGS-box with G␣ i/ o-specific GTPase-accelerating activity, C-terminal Rasbinding domains, and a GoLoco motif (13,18,20,21). Our previous studies of the involvement of RGS12 in modulating presynaptic GABA B -receptor signaling in chick DRG neurons suggest that RGS12, in binding to the calcium channel, modulates channel activity directly in addition to its ability to accelerate G␣-subunit GTPase activity (10).…”

“…Fusion of the RGS12 N terminus with the 26-kDa GST moiety allows for a higher signal-to-noise ratio in SPR measurements, because the biosensor response is directly proportional to the molecular mass of the analyte binding to the biosensor (18). However, as mass transport and GST/GST dimerization artifacts can complicate kinetic determinations (19), independent SPR experiments were performed using low density peptide surfaces and varying concentrations of the We also tested the ability of Tyr-804-and Tyr-815-containing peptides to interact with full-length, endogenous RGS12 by micro-affinity chromatography of chick DRG neuron lysates using these biotinylated peptides bound to streptavidin resin.…”

“…4A). As a positive control, a 20-amino acid biotinylated peptide comprising the C terminus of the interleukin-8 receptor CXCR2, which we have previously shown interacts with the N-terminal RGS12 PDZ domain (18), was used. The recombinant RGS12 N terminus was observed to bind both the control CXCR2 peptide and the Tyr(P)-804/Tyr-815 channel-loop peptide surfaces (Fig.…”

GIPC Recruits GAIP (RGS19) To Attenuate Dopamine D2Receptor Signaling

“…□ D The online version of this article contains supplementary material accessible at http://www.molbiolcell.org. complex RGS may assemble by itself to the signaling machinery (Snow et al, 1998), whereas dynamic recruitment of the simple RGS may rely on accessory proteins.…”

“…© 2004 by The American Society for Cell Biology complex RGS may assemble by itself to the signaling machinery (Snow et al, 1998), whereas dynamic recruitment of the simple RGS may rely on accessory proteins. The PDZ-domain-containing protein GIPC was identified by virtue of its interaction with GAIP, a member of the RZ RGS subfamily (De Vries et al, 1998b).…”