GRK2 regulates endothelin signalling 427 Figure 1 Kinetics of ETAR desensitization and re-sensitization. (A) Representative trace showing desensitization of ETAR [Ca2þ]i signals fol- lowing ET-1 challenge (50 nM, 3 min) in Fluo4-loaded MSMCs. Data are representative of 16 cells from three or more separate animals. MSMCs were transfected with 0.5 mg eGFP-PH and loaded with Fura-Red and subjected to the standard R1/R2 desensitization protocol: cells were stimulated with ET-1 (50 nM, 30 sec) (R1), followed by a variable period of 10, 20, or 30 min before further ET-1 (50 nM, 30 s) exposure (R2). Representative traces from single cells with either a 10 min (B) or 30 min (C ) wash period are shown. ETAR desensitization was deter- mined as the relative change in R2 response compared with R1 for both eGFP-PH (solid traces) and Fura-Red (broken traces). (D) Cumulative data shown as means + SEM from 14 – 15 cells from three or more different animals. immunoreactivity (Figure 3C, D), whereas GRK5 and 6 expression was unaffected (Figure 3D). To examine the effect of siRNA-mediated GRK2 knockdown on ETAR desensitization, MSMCs were co-transfected with eGFP-PH (0.5 mg) and negative-control (10 nM) or anti-GRK2 (10 nM) siRNAs and subjected to the standard R1/R2 desensitization pro- tocol. In the presence of negative-control siRNA, R2 responses were decreased by 80% for eGFP-PH and by 60% for [Ca2þ]i signals compared with R1, consistent with the degree of receptor desensitization observed in untransfected cells (Figure 4A, B, D). In contrast, in cells transfected with anti-GRK2 siRNA, R2 and R1 responses were similar to those previously seen in D110A,K220RGRK2-transfected MSMCs, with a 50% decrease in eGFP-PH and 30% decrease in Ca2þ signals (Figure 4A, C, D). To assess whether quantitatively similar effects of manipulating cellular GRK2 are seen with respect to the DAG/PKC arm of the ETAR signalling pathway, we have assessed ET-1-stimulated translo- cation of the Ca2þ and DAG-sensitive PKCa and DAG-sensitive PKC1 isoenzymes. Applying the standard desensitization protocol to eGFP-PKCa- (Figure 5A, C) or eGFP-PKC1 (Figure 5B, C)- transfected MSMCs showed that a signiﬁcantly greater recovery with respect to PKC recruitment responses was also observed when cellular GRK2 levels were selectively diminished. 3.4 ET-1-stimulated recruitment of endogenous GRKs To investigate further GRK2-mediated regulation of ETAR signal- ling, we examined the redistribution of this GRK isoenzyme follow- ing ET-1 addition. The MSMCs were treated with ET-1 (50 nM) for 3 min, after which cells were ﬁxed and processed to allow immunocytochemical detection of GRKs. Confocal images show GRK2 recruitment to the plasma membrane following ET-1 exposure (Figure 6A). The predominantly nuclear localization of GRK5 was unaltered following ET-1 challenge (Figure 6B), whereas addition of the Ca2þ-ionophore ionomycin promoted