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

• Published in: Molecular pharmacology 2017-10, Vol.92 (4), p.451-458
• Main Authors: Phan, Hoa T.N., Sjögren, Benita, Neubig, Richard R.
• Format: Article
• Language: English
• Subjects: Angiotensin II - pharmacology; Animals; CHO Cells; Cricetinae; Cricetulus; Dose-Response Relationship, Drug; HEK293 Cells; Humans; Mutation, Missense - physiology; Protein Structure, Secondary; RGS Proteins - agonists; RGS Proteins - chemistry; RGS Proteins - physiology
• ISSN: 0026-895X; 1521-0111
• DOI: 10.1124/mol.117.109215

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 Gαq. 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 Gαq Q209L compared with 67% for WT RGS2. The R188H mutant had a significant reduction in Gαq binding affinity (10-fold increase in Ki 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.