N-Terminal Residues Control Proteasomal Degradation of RGS2, RGS4, and RGS5 in Human Embryonic Kidney 293 Cells

Abstract

Regulator of G protein signaling (RGS) proteins modulate G protein-coupled receptor (GPCR) signaling. The N termini of some RGS4-family proteins provide receptor specificity and also contain an N-end rule determinant that results in ubiquitylation and decreased protein expression. The relevance of these mechanisms to other RGS proteins is not fully understood. Thus we examined function, receptor specificity, and expression of R4 subfamily RGS proteins (RGS2, -3, -4, -5, and -8). Although the N terminus plays a key role in protein stability in human embryonic kidney (HEK) 293 cells, we were unable to demonstrate specificity of RGS2, -3, -4, -5, or -8 for muscarinic receptors (M 1 , M 3 , and M 5 ). However, cellular RGS activity (8 ϭ 3 Ͼ 2) was strongly correlated with expression; RGS4 and -5 had minimal expression and activity. Stabilizing mutations of RGS4 and -5 (C2S) enhanced expression and function with a greater influence on RGS4 than on RGS5. We were surprised to find that a predicted destabilizing mutation in RGS8 (A2C) did not markedly affect expression and had no effect on function. In contrast, a destabilizing mutation in RGS2 (RGS2-Q2L) recently identified as a rare N-terminal genetic variant in a Japanese hypertensive cohort (J Hypertens 23:1497-1505, 2005) showed significantly reduced expression and inhibition of angiotensin II (AT 1 ) receptor-stimulated accumulation of inositol phosphates. We were surprised to find that RGS2-Q2R, also predicted to be destabilizing, showed nearly normal expression and function. Thus, proteasomal regulation of RGS expression in HEK293 cells strongly controls RGS function and a novel RGS2 mutation with decreased protein expression could be relevant to the pathophysiology of hypertension in humans.

Pristimerin Inhibits Breast Cancer Cell Migration by Up-regulating Regulator of G Protein Signaling 4 Expression

“…RGS4 is predominantly degraded by proteasome and the N-end rule pathway (Lee et al, 2005;Bodenstein et al, 2007) and pristimerin inhibits proteasome activity (Tiedemann et al, 2009). Accordingly, RGS4 may play an important role in the inhibition of pristimerin on breast cancer cell migration.…”

Regulation of Protease-activated Receptor 1 Signaling by the Adaptor Protein Complex 2 and R4 Subfamily of Regulator of G Protein Signaling Proteins

“…An HA epitope-tagged G␣ q placed within an internal loop was provided by Dr. Philip Wedegaertner (Thomas Jefferson University, Philadelphia, PA). HA-tagged RGS2, RGS4-C2S, and RGS5-C2S have been described previously (21). HA-tagged RGS3 was purchased from the Missouri S&T cDNA Resource Center (Rolla, MO).…”

The evolution of regulators of G protein signalling proteins as drug targets – 20 years in the making: IUPHAR Review 21

“…Furthermore, re-expression of RGS4 in invasive cancer cell lines in which RGS4 protein is down-regulated suppresses cancer cell invasion and migration (Xie et al, 2009). Apart from RGS4, several other members of the R4 family, including RGS2 and RGS5 are substrates for the ubiquitinproteasomal pathway and are rapidly and constitutively degraded (Davydov and Varshavsky, 2000;Lee et al, 2005;Bodenstein et al, 2007;Lee et al, 2011;Sjögren et al, 2015). This mechanism may be a way for physiological systems to very rapidly adapt to new environments.…”