Receptor density and cAMP accumulation: analysis in CHO cells exhibiting stable expression of a cDNA that encodes the beta 2-adrenergic receptor.
The relationship between hormone receptor number and hormone-stimulated cAMP accumulation was probed in CHO cells that were transfected with the cDNA encoding the beta-adrenergic receptor under the control of the SV40 early promoter (expression vector pSV2BAR). CHO cells were cotransfected with pSV2BAR and expression vector pHOMER that directs the expression of a neomycin-resistance gene, and stable transfectants were selected. Clones expressing receptor at levels from 30 (wild-type) to 6000 fmol/mg membrane protein were isolated and further characterized for receptor mRNA content (measured by solution hybridization with a single-stranded cDNA probe), steady-state expression of receptor (measured by immunoblotting and indirect immunofluorescence), and their ability to accumulate intracellular cAMP in response to a beta-adrenergic agonist. Receptor mRNA content and the steady-state level of receptor protein and its expression at the cell surface were found to increase with receptor density as measured by radioligand binding. Over a 200-fold range of receptor expression, CHO transfectants displayed increasing efficacy of agonist-stimulated cAMP accumulation and increasing maximal cAMP accumulation in response to agonist. These data provide for the first time an analysis of the relationship between the density of a G-protein-linked receptor and a receptor-mediated response under conditions where the levels of G-proteins and adenylate cyclase are unaltered.
George
S T
ST
Diabetes & Metabolic Diseases Research Program, State University of New York, Stony Brook 11794-8651.
Berrios
M
M
Hadcock
J R
JR
Wang
H Y
HY
Malbon
C C
CC
D000818:Animals; D002460:Cell Line; D002999:Clone Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000378:metabolism*; D004247:DNA / Q000235:genetics*; D005260:Female; D005796:Genes; D007545:Isoproterenol / Q000494:pharmacology*; D010053:Ovary; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D011943:Receptors, Adrenergic, beta / Q000235:genetics / Q000378:metabolism*
D012333:RNA, Messenger; D011943:Receptors, Adrenergic, beta; D004247:DNA; D000242:Cyclic AMP; D007545:Isoproterenol