Beta 2-adrenergic receptor regulation after transfection into a cell line deficient in the cAMP-dependent protein kinase.
A mouse beta 2-adrenergic receptor (beta 2AR) DNA clone was transfected and expressed in a mouse adrenocortical tumor cell line (Kin8) that lacks both beta 2AR and cAMP-dependent protein kinase (PKA). The receptor displayed a characteristic beta 2AR agonist binding profile that was similar to that observed in beta 2AR-transfected PKA+ mouse adrenocortical tumor cells (Y1). Isoproterenol treatment of beta 2AR-transfected Kin8 and Y1 cells resulted in a rapid loss of surface beta 2AR, as determined by the binding of the hydrophilic beta 2AR radioligand [3H]CGP 12177 [( 3H]CGP), followed by a decrease in adenylate cyclase activity. Sequestration of beta 2AR in Kin8 cells was beta 2AR agonist specific, temperature dependent, and rapidly reversible. Repeated treatment and recovery from isoproterenol incubation resulted in a cycling of surface [3H]CGP binding. The reappearance of [3H]CGP binding following short isoproterenol treatment was not affected by cycloheximide treatment of the cells. Prolonged incubation of beta 2AR-transfected Kin8 cells with isoproterenol resulted in the down-regulation of beta 2AR protein without a change in beta 2AR mRNA levels. Polysome profiles of control and down-regulated cells revealed that translation of beta 2AR mRNA is inefficient and does not change upon prolonged agonist treatment. Protein synthesis was required to reverse the down-regulation of beta 2AR. These results indicate that neither sequestration nor down-regulation of beta 2AR depends on PKA.
Allen
J M
JM
Howard Hughes Medical Institute, Department of Biochemistry, University of Washington, Seattle 98195.
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D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D002451:Cell Compartmentation / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D004705:Endocytosis; D005786:Gene Expression Regulation; D007545:Isoproterenol / Q000494:pharmacology; D051379:Mice; D011132:Polyribosomes / Q000378:metabolism; D011412:Propanolamines / Q000378:metabolism; D014176:Protein Biosynthesis; D011494:Protein Kinases / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D011943:Receptors, Adrenergic, beta / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D011412:Propanolamines; D012333:RNA, Messenger; D011943:Receptors, Adrenergic, beta; D011494:Protein Kinases; D000262:Adenylyl Cyclases; D007545:Isoproterenol; C033394:CGP 12177