GRK2 regulates endothelin signalling 431 Figure 5 GRK2 depletion decreases ET1-induced desensitiza- tion of ETA receptor-stimulated PKC recruitment. MSMCs were co-transfected with 0.5 mg of either eGFP-PKCa or eGFP-PKC1 and negative-control or anti-GRK2 (10 nM) siRNAs. Representative traces showing eGFP-PKC1 (A) or eGFP-PKCa (B) translocations in MSMCs subjected to the stan- dard R1/R2 desensitization protocol (see Methods) in the pres- ence of negative-control (broken traces) or GRK2 siRNA (black traces). Cumulative data (C ) are shown as means + SEM from 8 – 20 cells from three to four animals. Statistical signiﬁcance is indicated as *P , 0.05; **P , 0.01 (one-way ANOVA, Dun- nett’s post hoc test). Figure 6 ET-1 stimulates recruitment of endogenous GRK2, but not GRK5 to the plasma membrane. MSMCs were challenged with ET-1 (50 nM) for 3 min, before ﬁxation and processing for immunocytochemical detection of GRKs 2 and 5 (see Methods). Representative confocal images show the distribution of GRK2 and GRK5 in MSMCs. Left-hand panels are images of untreated cells, whereas the right-hand panels show: (A) GRK2 recruitment to the plasma membrane after ET-1 challenge (indi- cated by arrows); (B) Lack of movement of GRK5 after ET-1 chal- lenge; (C ) Redistribution of GRK5 from the nucleus to the cytoplasm after ionomycin (2 mM, 15 min) treatment. Data are representative of experiments from cells isolated from three sep- arate animals. phosphorylation of the receptor was enhanced by recombinant over-expression of GRKs 2, 5, or 6.13 In addition, over-expression of recombinant GRK2 and GRK3 increased ETAR phosphorylation in CHO cells.25 Together these and other studies suggest that GRKs are able to cause ETAR phosphorylation, however, such studies are not necessarily predictive of how/if the receptor will be regulated by speciﬁc GRK isoenzymes in native ETAR expression systems. Therefore, a key objective here was to delin- eate for the ﬁrst time the role that endogenous GRK isoenzymes play in ETAR regulation in resistance artery smooth muscle. Owing to the lack of effective and speciﬁc pharmacological GRK inhibitors, we applied previously validated molecular approaches to disrupt or decrease activity of speciﬁc endogenous GRK isoenzymes. Initially, we used dominant-negative (kinase-dead) GRK mutants to disrupt ETAR/GRK isoenzyme-speciﬁc interactions in an attempt to attenuate or prevent the reduction in ETAR responsiveness observed on re-addition of ET-1. The D110A,K220RGRK2 construct,