Expression of a human cDNA encoding the beta 2-adrenergic receptor in Chinese hamster fibroblasts (CHW): functionality and regulation of the expressed receptors.
A human beta-adrenergic receptor cDNA was transfected and expressed in transformed Chinese hamster fibroblasts (CHW). The expressed receptor exhibited a typical beta 2-adrenergic selectivity for agonists and antagonists as assessed by radioligand binding and adenylate cyclase activation. Guanine nucleotide-sensitive high affinity binding of the agonist, isoproterenol, indicated effective coupling of the expressed receptor to a guanine nucleotide-regulatory protein. The level of expression of beta 2-AR in various cell clones varied over 200-fold and was positively correlated with the levels of beta 2-AR mRNA. In cells expressing between 0.04 and 3.0 pmol of beta 2-AR/mg of membrane protein, the efficacy of isoproterenol for stimulating adenylate cyclase increased with increasing numbers of expressed receptors but reached a plateau and started to decrease in clones with higher beta 2-AR density (3.0-8.0 pmol/mg of membrane protein). Preincubation of beta 2-AR-expressing cells with isoproterenol for 15 min led to significant reduction in the level of isoproterenol-sensitive adenylate cyclase activity. This agonist-induced desensitization was also accompanied by phosphorylation of the beta 2-AR. These data indicate that the expressed human beta 2-AR displays typical functional characteristics of adenylate cyclase-coupled receptors including agonist-induced desensitization. Moreover, the availability of this series of cellular clones, which differ markedly in their density of beta 2-AR, provides a unique set of biological reagents for future studies of beta 2-AR function and regulation.
Bouvier
M
M
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.
Hnatowich
M
M
Collins
S
S
Kobilka
B K
BK
Deblasi
A
A
Lefkowitz
R J
RJ
Caron
M G
MG
D000262:Adenylyl Cyclases / Q000032:analysis; D000818:Animals; D002478:Cells, Cultured; D006224:Cricetinae; D004247:DNA / Q000032:analysis*; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D008970:Molecular Weight; D010766:Phosphorylation; D012333:RNA, Messenger / Q000032:analysis; D011943:Receptors, Adrenergic, beta / Q000032:analysis* / Q000235:genetics / Q000502:physiology
D012333:RNA, Messenger; D011943:Receptors, Adrenergic, beta; D004247:DNA; D000262:Adenylyl Cyclases; D007545:Isoproterenol