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Vol. 58, Issue 5, 1162-1173, November 2000
2-Adrenergic Receptor by the GRK Pathway
Department of Integrative Biology and Pharmacology (A.S., B.W.,
Z.-F.H., J.F., R.B.C.), University of Texas-Houston Medical School,
Houston, Texas; and Departments of Pediatrics (R.H.M.) and Molecular
Physiology and Biophysics (B.J.K.), Baylor College of Medicine,
Houston, Texas
The human 
2-adrenergic receptor (AR) 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 AR, followed by -arrestin
binding and receptor internalization. AR 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 AR. 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
AR 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 µM epinephrine for 5 min caused a 3.5-fold increase in
the EC50 value and a 42% decrease in the
Vmax value for epinephrine stimulation of
adenylyl cyclase. Substitution of all three serines completely
inhibited the epinephrine-induced shift in the EC50. Both
double mutants, S355,356A and S356,364A, showed a nearly complete loss
of the EC50 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
Vmax, 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 AR 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 AR recycling. Our data
demonstrate that the cluster of serines within the 355 to 364 AR
domain confer the rapid, GRK-mediated, receptor-level desensitization
of the AR.
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