Proteolytic degradation of regulator of G protein signaling 2 facilitates temporal regulation of Gq/11 signaling and vascular contraction

Abstract

Regulator of G protein signaling 2 (RGS2) controls signaling by receptors coupled to the G class heterotrimeric G proteins. RGS2 deficiency causes several phenotypes in mice and occurs in several diseases, including hypertension in which a proteolytically unstable RGS2 mutant has been reported. However, the mechanisms and functions of RGS2 proteolysis remain poorly understood. Here we addressed these questions by identifying degradation signals in RGS2, and studying dynamic regulation of G-evoked Ca signaling and vascular contraction. We identified a novel bipartite degradation signal in the N-terminal domain of RGS2. Mutations disrupting this signal blunted proteolytic degradation downstream of E3 ubiquitin ligase binding to RGS2. Analysis of RGS2 mutants proteolyzed at various rates and the effects of proteasome inhibition indicated that proteolytic degradation controls agonist efficacy by setting RGS2 protein expression levels, and affecting the rate at which cells regain agonist responsiveness as synthesis of RGS2 stops. Analyzing contraction of mesenteric resistance arteries supported the biological relevance of this mechanism. Because RGS2 mRNA expression often is strikingly and transiently up-regulated and then down-regulated upon cell stimulation, our findings indicate that proteolytic degradation tightly couples RGS2 transcription, protein levels, and function. Together these mechanisms provide tight temporal control of G-coupled receptor signaling in the cardiovascular, immune, and nervous systems.

N-Terminal Targeting of Regulator of G Protein Signaling Protein 2 for F-Box Only Protein 44–Mediated Proteasomal Degradation

“…7. These studies expand on previous observations by us and others (Gu et al, 2008;Kanai et al, 2017;Sjögren et al, 2015) and provide a mechanistic explanation for the increased protein stability that results from alternative translation initiation of RGS2.…”

“…1A), were used to detect effects of proteasome inhibition or RGS2 protein levels. In agreement with previous studies (Kanai et al, 2017), WT, M1, and M5 RGS2 protein levels were significantly increased in transiently transfected HEK-293T cells by the proteasome inhibitor MG-132 (10 µM: Fig. 1B-D).…”

“…In fact, in our previous studies, we demonstrated that siRNAmediated knockdown of ATE-1, while significantly stabilizing RGS4, had no effect on RGS2-Q2L protein expression (Sjögren et al, 2015). In addition, a recent study failed to confirm association between RGS2 and TEB4 (Kanai et al, 2017). Instead, we identified an alternative pathway for This article has not been copyedited and formatted.…”

“…of RGS2 is recognized by FBXO44.RGS2 contains four N-terminal methionine residuesthat can act as alternative translation initiation sites (Met 1 , Met 5 , Met 16 and Met 33 ) (Gu et al, 2008) and the shorter translation variants resulting from initiation at Met 16 or Met 33 yield a protein that is protected from degradation (Kanai et al, 2017). Our hypothesis for the current study was therefore that FBXO44 would target RGS2 through a degron located in the very N-terminus of the protein.…”

Short translational ramp determines efficiency of protein synthesis

“…Modified and wild type mEOS, eGFP and human G protein subunit alpha i1 (Giα; NM_002069) construct DNA were created by PCR using forward primers that code for the certain sequence extracted from our EGFP library expression found in the FACS experiment (KFS, KYY, KIH -high expression, IGK -moderate expression, TVG -low expression). Regulator of G protein signaling 2 (RGS2; NM_002923) constructs were amplified by PCR reaction from previously described constructs 51 . PCR products were cloned in the pBAD or pET16b vector, transformed into Top 10 E.coli cells and sequenced for the correct clones.…”