Beta 3-adrenoceptor agonist-induced down-regulation of Gs alpha and functional desensitization in a Chinese hamster ovary cell line expressing a beta 3-adrenoceptor refractory to down-regulation.
Chinese hamster ovary (CHO) cells transfected to express human beta 2- or beta 3-adrenoceptors (beta 2-CHO and beta 3-CHO cells) were exposed to the beta-adrenoceptor agonist isoprenaline at various concentrations and for differing times. Sustained exposure of the beta 2-CHO but not beta 3-CHO cells to isoprenaline resulted in a time- and concentration-dependent down-regulation of the receptor as measured by a reduction in specific binding of [125I]cyanopindolol. Such maintained exposure of cells expressing either receptor to the agonist produced a marked down-regulation of immunologically detectable levels of the alpha subunit of the stimulatory guanine-nucleotide-binding protein Gs. This effect was specific for Gs because levels of both G12 alpha and Gq alpha/G11 alpha were unaltered by isoprenaline treatment of both beta 2-CHO and beta 3-CHO cells. The effect of isoprenaline on Gs alpha down-regulation was some 30-fold more potent in the beta 2-CHO than in the beta 3-CHO cells. Time courses of isoprenaline-induced down-regulation of Gs alpha were not different, however, in the two cell lines. Isoprenaline treatment of the beta 3-CHO cells produced a desensitization of agonist-mediated regulation of adenylyl cyclase, manifested by a 4-fold reduction in the potency and a 30% reduction in maximal effect of the agonist, whereas desensitization of the beta 2-CHO cells was considerably greater (25-fold reduction in potency and 70% reduction in maximal effect). These results demonstrate that agonist-induced down-regulation of the G-protein which interacts with a receptor can be produced by both beta 2- and beta 3-adrenoceptors. Despite apparent concurrence of down-regulation of receptors and G-proteins in other systems [e.g. Adie, Mullaney, McKenzie and Milligan (1992) Biochem. J. 285, 529-536], agonist-induced receptor down-regulation does not appear to be a prerequisite for down-regulation of the G-protein. Furthermore, the results suggest that agonist-induced down-regulation of a G-protein may be sufficient, in the absence of receptor regulation, to induce some agonist desensitization of effector function.
SB Pharmaceuticals, Pinnacles, Harlow, U.K.
J
J
Chambers
J
J
Park
D
D
Cronk
C
C
Chapman
F R
FR
Kennedy
S
S
Wilson
G
G
Milligan
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D015536:Down-Regulation / Q000187:drug effects*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology*; D008969:Molecular Sequence Data; D011943:Receptors, Adrenergic, beta / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D022702:Receptors, Adrenergic, beta-3
D011943:Receptors, Adrenergic, beta; D022702:Receptors, Adrenergic, beta-3; D019204:GTP-Binding Proteins; D007545:Isoproterenol