FIGURE 4.

H2R regulation by RGS in COS7-transfected cells. A, shown is a schematic representation of GRK2 functional domains. Shown are the N-terminal RH domain, the central catalytic domain, and the C-terminal pleckstrin homology domain. The point mutation at residue 220, essential for GRK2 catalytic activity, is identified. Also shown are the HA-tagged RH, KIN-PH, and RH-PH expression constructs. B–D, COS7 cells cotransfected with H2R and RH, KIN-PH, or RH-PH constructs are shown. B, shown is analysis of constructs expression in COS7-transfected cells. Cells were lysed, and equal amounts of proteins were subjected to SDS-PAGE and analyzed by Western blot with HA antibody. Data are representative of at least three independent experiments. C, shown are concentration-response curves for cAMP production. Cells were exposed for 9 min to increasing concentrations of amthamine at 37 °C in the presence of 1 mm IBMX, and cAMP levels were determined. D, desensitization kinetics are shown. Cells were preincubated at different times with 10 μm amthamine, washed, and restimulated with 10 μm amthamine in the presence of 1 mm IBMX. cAMP production was determined as described under “Experimental Procedures.” C and D, data are the means ± S.E. (n = 3).