Figure 1
From: RGS7 is recurrently mutated in melanoma and promotes migration and invasion of human cancer cells
Effects of RGS7 mutation on RGS7 stability and activity. (a) The Human RGS7 protein, with conserved domains indicated as blocks, including the Dishevelled domain (DEP); G Protein Gamma-like domain (GGL); RGS domain (RGS). Somatic mutations indicated with arrows. Red triangles indicate deleterious mutations. (b) Three dimensional structure of RGS7 N-Terminus, predicting that R44 is involved in an H-bond network with S58 and D61. (c) Cells expressing wild-type or mutant RGS7 were treated with cycloheximide (CHX), collected at different time points and then immunoblotted with anti-FLAG antibody. Anti-Cyclin D1 was used as a control and anti-GAPDH was used for normalization. (d) A Schematic representation of the BRET-based assay to monitor G protein signaling cycle. Activation of the D2R causes the G protein heterotrimer to dissociate into Gα and Gβγ subunits. Released Gβγ subunits tagged with Venus fluorescent protein interacts with Nluc–tagged reporter G protein receptor kinase (GRK) to produce the BRET signal. Upon termination of D2R activation by antagonist haloperidol, Gαo subunit hydrolyses GTP and reassociates with Gβγ subunits, quenching the BRET signal. (e) Time course of normalized BRET responses recorded in a representative experiment. Left. The deactivation phase after antagonist application is shown. Wild-type RGS7 or mutant were transfected at equal amount of cDNA (210 ng) together with dopamine D2 receptor, Gαo, and BRET sensor pair. Right. Quantification of the exponential decay kinetics of the response. BRET values were averaged from four or six replicates. *P < 0.0001. (f) Correlation analysis between expression levels of RGS7 and activity. Expression levels of RGS7 (x axis) were determined by Western blotting (Supplementary Fig. 9c) and plotted against kGAP (y axis). Mean ± SEM were shown.
