Abstract
The human β2-adrenergic receptor (βAR) 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 βAR, followed by β-arrestin binding and receptor internalization. βAR 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 βAR. 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 βAR 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 theV max 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 inV 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 βAR 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 βAR recycling. Our data demonstrate that the cluster of serines within the 355 to 364 βAR domain confer the rapid, GRK-mediated, receptor-level desensitization of the βAR.
Footnotes
- Received January 21, 2000.
- Accepted August 14, 2000.
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Send reprint requests to: Dr. Richard B. Clark, University of Texas-Houston Medical School, Department of Integrative Biology and Pharmacology, P.O. Box 20708, Houston, Texas 77225. E-mail:richard.b.clark{at}uth.tmc.edu
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This study was supported by National Institutes of Health Grants GM31208 (R.B.C.) and HL57445, HL50047 (B.J.K.) and HL03463 (R.H.M.)
- The American Society for Pharmacology and Experimental Therapeutics
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