Fluorescent localization of the beta-adrenergic receptor on DDT-1 cells. Down-regulation by adrenergic agonists.
Continuous incubation of cultured cells with beta-adrenergic agonists results in the desensitization of adrenergic responsiveness accompanied by the down-regulation of cell surface beta-adrenergic receptors (beta AR). Previous studies have relied on measurements of ligand binding activity for the detection of the beta AR in the cell. In the present study, we have raised a monoclonal antibody to a synthetic peptide corresponding to amino acid numbers 226-239 of the hamster beta 2AR. This antibody was used to localize the beta AR in hamster smooth-muscle DDT-1 cells by immunofluorescence, without regard for the ability of the receptor to bind ligands. The beta AR was found to be localized primarily at the plasma membrane of these cells, with a nonhomogeneous pattern of distribution. A rapid loss of beta AR-specific immunofluorescence, which paralleled receptor down-regulation as measured by ligand-binding activity, was seen with beta-adrenergic agonists, but not with antagonists. In addition, a transient increase in fluorescence was observed after short times of exposure of the cells to agonists. This fluorescence increase may reflect a ligand-induced conformational change in the receptor.
Zemcik
B A
BA
Department of Biochemistry and Molecular Biology, Merck Sharp & Dohme Research Laboratories, Rahway, NJ 07065.
Strader
C D
CD
D000818:Animals; D000911:Antibodies, Monoclonal; D002478:Cells, Cultured; D011232:Chemical Precipitation; D006224:Cricetinae; D005455:Fluorescent Antibody Technique; D007545:Isoproterenol / Q000494:pharmacology*; D008024:Ligands; D011943:Receptors, Adrenergic, beta / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D008024:Ligands; D011943:Receptors, Adrenergic, beta; D007545:Isoproterenol