Human Missense Mutations in Regulator of G Protein Signaling 2 Affect the Protein Function Through Multiple Mechanisms

Abstract

Regulator of G protein signaling 2 (RGS2) plays a significant role in alleviating vascular contraction and promoting vascular relaxation due to its GTPase accelerating protein activity toward Gq. Mice lacking RGS2 display a hypertensive phenotype, and several RGS2 missense mutations have been found predominantly in hypertensive human subjects. However, the mechanisms whereby these mutations could impact blood pressure is unknown. Here, we selected 16 rare, missense mutations in RGS2 identified in various human exome sequencing projects and evaluated their ability to inhibit intracellular calcium release mediated by angiotensin II receptor type 1 (AT1R). Four of them had reduced function and were further investigated to elucidate underlying mechanisms. Low protein expression, protein mislocalization, and reduced G protein binding were identified as likely mechanisms of the malfunctioning mutants. The Q2L mutant had 50% lower RGS2 than wild-type (WT) protein detected by Western blot. Confocal microscopy demonstrated that R44H and D40Y had impaired plasma membrane targeting; only 46% and 35% of those proteins translocated to the plasma membrane when coexpressed with G Q209L compared with 67% for WT RGS2. The R188H mutant had a significant reduction in G binding affinity (10-fold increase in compared with WT RGS2 in a flow cytometry competition binding assay). This study provides functional data for 16 human RGS2 missense variants on their effects on AT1R-mediated calcium mobilization and provides molecular understanding of those variants with functional loss in vitro. These molecular behaviors can provide insight to inform antihypertensive therapeutics in individuals with variants having reduced function.

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

“…Consequently, RGS2 -/mice are hypertensive and show prolonged responses to these vasoconstrictors (Heximer et al, 2003). In addition, mutations associated with hypertension in humans (Yang et al, 2005) display reduced protein expression (Bodenstein et al, 2007;Phan et al, 2017). We demonstrated that pharmacological enhancement of RGS2 protein levels correlates with increased function.…”

“…were created from the pCMV-RGS2-ProLabel-C3 construct cloned previously (Sjogren et al, 2012) using QuikChange II site-directed mutagenesis (Agilent Technologies, Santa Clara, CA) according to the manufacturer's instructions. RGS2-V5 (in pcDNA3.2-DEST) was created by Gateway® cloning previously in the lab (Phan et al, 2017). K/R mutations were created using QuikChange II site-directed mutagenesis according to the manufacturer's instructions.…”

Adverse Effects of Methylglyoxal on Transcriptome and Metabolic Changes in Visceral Adipose Tissue in a Prediabetic Rat Model

“…In a study of non-obese diabetic rats with hyperglycaemia, glycated processes in adipose tissue after MG exposure not only impaired microcirculation in epididymal adipose tissue but also led to hypoxia and insulin resistance [ 5 ]. Based on our transcriptome profiling results, downregulation of the RGS2 gene, which is involved in the regulation of angiotensin-activated signaling [ 40 ], indirectly contributed to vascular complications in adipose tissue after MG exposure. Direct glycation processes altered and impaired the structure and function of intracellular and extracellular matrix proteins and led to microvascular changes in adipose tissue.…”

Genetic Analysis of Rare Human Variants of Regulators of G Protein Signaling Proteins and Their Role in Human Physiology and Disease

“…By contrast, if a LoF in RGS4 or other RGS protein is found to contribute to disease progression, such a RGS inhibitor may exacerbate the disease and should thus be avoided. A prime example of the power of this analysis is a recent report on RGS2 human variants (Phan et al, 2017) that are linked to hypertension (Yang et al, 2005). The authors found that these RGS2 human variants, located in the N terminus, reduced RGS2-mediated inhibition of Ca 2+ signaling via protein degradation and mislocalization and provided a mechanism for their association with hypertension.…”

“…In a subsequent study, it was found that the Q2L mutation destabilized RGS2 protein (but was reversed by proteasomal inhibition), whereas Q2R (another SNV found in both cases and controls) did not (Bodenstein et al, 2007). A more recent study demonstrated that four human variants, including Q2L, R188H, R44H, and D40Y, showed reduced capacity to inhibit Ca 2+ release by the angiotensin II receptor (Phan et al, 2017). For further review of RGS2 function in physiology, see Bansal et al (2007).…”