Altered patterns of agonist-stimulated cAMP accumulation in cells expressing mutant beta 2-adrenergic receptors lacking phosphorylation sites.
As with many other receptor-effector systems, the responsiveness of the beta-adrenergic receptor (beta AR)/adenylyl cyclase system undergoes desensitization upon agonist exposure. Phosphorylations of the receptor by the cAMP-dependent protein kinase (protein kinase A) and the beta AR kinase appear to play roles in such desensitization phenomena, but the functional significance of the receptor phosphorylation in intact cells has not been previously assessed. In this study, we constructed and expressed in a mammalian fibroblast line the normal (wild type) human beta 2 AR and mutant forms of the receptor that lack the putative phosphorylation sites for these two protein kinases. The two consensus sequences for phosphorylation by protein kinase A were altered by changing serines 261, 262 and 345, 346 to alanines. In another mutant, the 11 serines and threonines at the carboxy terminus of the protein that constitute the putative beta AR kinase phosphorylation sites were changed to alanines or glycines. The mutated receptors did not differ from the wild type in their affinities for agonists or antagonists or in their ability to mediate agonist stimulation of adenylyl cyclase. Moreover, their levels of expression in the cultured cells were the same. When stimulated with the beta AR agonist isoproterenol, cells bearing either the wild type or mutant receptors generated cAMP at essentially identical rates for the first 2 min. Cells bearing wild type receptors then showed a rapid desensitization characterized by a markedly diminished rate of cAMP production after the first few minutes of stimulation. However, cells bearing either of the mutated forms of the receptor showed much less desensitization and continued to generate cAMP at a rate 3-4 times greater than that observed in cells expressing the wild type receptor. In contrast, intact cell cAMP levels stimulated by prostaglandin E1 and forskolin were not different between cells bearing wild type or mutant beta AR. These results suggest an important physiological role for phosphorylation of the beta AR in regulating rapid agonist-induced desensitization in intact cells.
Liggett
S B
SB
Howard Hughes Medical Institute, Department of Medicine (Pulmonary and Cardiology), Duke University Medical Center, Durham, North Carolina 27710.
Bouvier
M
M
Hausdorff
W P
WP
O'Dowd
B
B
Caron
M G
MG
Lefkowitz
R J
RJ
D000595:Amino Acid Sequence; D002460:Cell Line; D000242:Cyclic AMP / Q000502:physiology*; D004252:DNA Mutational Analysis; D015536:Down-Regulation; D006801:Humans; D066298:In Vitro Techniques; D007545:Isoproterenol / Q000494:pharmacology; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011494:Protein Kinases / Q000502:physiology; D011943:Receptors, Adrenergic, beta / Q000502:physiology* / Q000648:ultrastructure; D013329:Structure-Activity Relationship
D011943:Receptors, Adrenergic, beta; D000242:Cyclic AMP; D011494:Protein Kinases; D007545:Isoproterenol