FIGURE 6.

H2R regulation by GRK2 in COS7-transfected cells. COS7 cells cotransfected with H2R and GRK2K220R, GRK2D110A, or GRK2K220R-D110A constructs are shown. A, concentration-response curves for cAMP production are shown. 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. cAMP production was determined as described under “Experimental Procedures.” Data are the means ± S.E. (n = 3). B, coimmunoprecipitation of GRK2 mutants and H2R is shown. COS 7 cells cotransfected with HA-tagged H2R and GRK2 mutants or Mock were incubated for 10 min with 10 μm amthamine, and cross-linking with 2.5 mm dithiobis(succinimidyl propionate) was done as described under “Experimental Procedures.” Cells were lysed, and the receptor was immunoprecipitated (IP) with anti-HA antibodies. Coprecipitated GRK2 was detected by Western blot (WB).