428 G.E. Morris et al. Figure 2 Inhibition of GRK2 reduces the extent of ETAR desensitization. MSMCs were co-transfected with 0.5 mg eGFP-PH and either pcDNA3 (control), D110A,K220RGRK2, D110A,K220RGRK3, K215RGRK5, or K215RGRK6 (0.5 mg). Cells were loaded with Fura-Red and subjected to the standard R1/R2 desensitization protocol (see Methods). Representative images showing both IP3 and Ca2þ changes in MSMCs expressing pcDNA3 (control) or D110A,K220RGRK2 at both the R1 and R2 stimulation points are shown in (A). Representative traces are from cells trans- fected with pcDNA3 (B), D110A,K220RGRK2 (C ), D110A,K220RGRK3 (D), K215RGRK5 (E), or K215RGRK6 (F ). ETAR desensitization was determined as the relative change in R2 response compared with R1 for both eGFP-PH (black traces) and Fura-Red (broken traces). Cumulative data (G) are expressed as means + SEM for the percentage change in R2 relative to R1; n ¼ 7 – 17 cells for each time-point, from at least eight separate experiments from three or more different animals. Statistical signiﬁcance is indicated as **P , 0.01 vs. pcDNA3 (one-way ANOVA and Dun- nett’s post hoc test).