The human beta(2)-adrenergic receptor (betaAR) is rapidly desensitized in response to saturating concentrations of agonist by G protein-coupled receptor kinases (GRKs) and cAMP-dependent protein kinase A (PKA) phosphorylation of the betaAR, followed by beta-arrestin binding and receptor internalization. betaAR sites phosphorylated by GRK in vivo have not yet been identified. In this study, we examined the role of the carboxyl terminal serines, 355, 356, and 364, in the GRK-mediated desensitization of the betaAR. Substitution mutants of these serine residues were constructed in which either all three (S355,356,364A), two (S355,356A and S356, 364A), or one of the serines (S356A and S364A) were modified. These mutants were constructed in a betaAR in which the serines of the PKA consensus site were substituted with alanines (designated PKA(-)) to eliminate any PKA contribution to desensitization, and they were stably transfected into human embryonic kidney 293 cells. Treatment of the PKA(-) mutant with 10 microM epinephrine for 5 min caused a 3. 5-fold increase in the EC(50) value and a 42% decrease in the V(max) value for epinephrine stimulation of adenylyl cyclase. Substitution of all three serines completely inhibited the epinephrine-induced shift in the EC(50). Both double mutants, S355,356A and S356,364A, showed a nearly complete loss of the EC(50) shift, whereas the single substitutions, S356A and S364A, caused only a slight decrease in desensitization. None of the mutations altered the epinephrine-induced decrease in V(max,) which seems to be downstream of the receptor. The triple mutation caused a 45% decrease in epinephrine-induced internalization and a 90 to 95% reduction in phosphorylation of the betaAR relative to the PKA(-) (1.9+/- 0.2- and 16.6+/-3.8-fold phosphorylation over basal, respectively). The double mutants caused an intermediate reduction in internalization (20-21%) and phosphorylation (43-52%). None of the serine mutations altered the rate of betaAR recycling. Our data demonstrate that the cluster of serines within the 355 to 364 betaAR domain confer the rapid, GRK-mediated, receptor-level desensitization of the betaAR.