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

TLDR

RGS proteins accelerate GTP hydrolysis in vitro, yet the specificity with which they down‑regulate distinct G‑protein‑coupled receptor systems remains unknown. The study aimed to delineate the GTPase specificity of RGS12 and to catalogue four alternatively spliced human RGS12 mRNA variants. Four RGS12 isoforms (6.3, 5.7, 3.1, and 3.7 kb) differing in PDZ domain presence and tissue expression were identified, and in vitro biochemical assays together with interaction‑trap experiments were employed to assess GTPase activity and PDZ binding. RGS12 functions as a G_iα‑activating protein, its N‑terminal PDZ domain selectively binds C‑terminal (A/S)-T.

Abstract

Regulator of G-protein signaling (RGS) proteins increase the intrinsic guanosine triphosphatase (GTPase) activity of G-protein α subunits <i>in vitro</i>, 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 <i>RGS12</i> mRNA. Two <i>RGS12</i> 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. <i>In vitro</i> biochemical assays indicate that RGS12 is a GTPase-activating protein for G_i class α 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-<i>X</i>-(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.