BETA-3-ADRENOCEPTOR AGONIST-INDUCED DOWN-REGULATION OF G(S)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 [I-125]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 G(s). This effect was specific for G(s) because levels of both G(i)2 alpha and G(q) alpha/G(11)alpha were unaltered by isoprenaline treatment of both beta 2-CHO and beta 3-CHO cells. The effect of isoprenaline on G(s) 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 G(s) 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.