Differentiation-dependent expression of catecholamine-stimulated adenylate cyclase. Roles of the beta-receptor and G/F protein in differentiating 3T3-L1 adipocytes.

3T3-Ll Preadipocytes possess an adenylate cyclase system that is highly stimulated (-4.5-fold) by 10 PM GTP in the absence of @-adrenergic agonists and other hormones. During the differentiation of preadipocytes into adipocytes, a sharp decline in this activity precedes the manifestation of catecholamine-stimulated (6to 10-fold) adenylate cyclase activity. Preadipocytes exhibit 1900 high affinity @-receptorsJcel1, but the maximal occupation of these receptors by isoproterenol results in only a 20% elevation in the rate of CAMP synthesis. During differentiation, P-receptor number increases by only 60-70% to 3100 sites/cell. /I-Receptors in preadipocyte membranes have a KO of 0.2 n~ for [12613 iodohydroxybenzylpindolol and decrease their affinity for /I-agonists in the presence of GTP. During prolonged treatment with 5 p~ isoproterenol, preadipocytes down regulate more than 80% of their receptors. No significant changes in these properties occurred during adipocyte development. Moreover, p-receptors in preadipocyte and adipocyte membranes exhibited similar affinities and stereoselectivities for a series of b-agonists and &antagonists. In contrast to the unaltered properties of the 8-receptor, the regulation of adenylate cyclase activity by GTP changes markedly during adipocyte development. GTP (10 ELM) stimulates the preadipocyte enzyme equally well in the absence or presence of isoproterenol, while the GTP-mediated enhancement of adipocyte membrane adenylate cyclase activity is highly dependent on the presence of the @adrenergic agonist. These observations suggested that G/F protein exhibited different sets of regulatory (coupling) properties in the presence of similar 8-receptors in undifferentiated and differentiated cells. In complementary experiments, the cholera toxincatalyzed [32P]ADP-ribosylation of 3T3-Ll membranes revealed dramatic differences between the G/F subunits of preadipocytes and adipocytes. In adipocyte membranes, 13-fold more 32P was incorporated into the 42,000-dalton component and 4-fold more labeling was observed in the 49,000to 50,000-dalton doublet than in the corresponding G/F subunits in preadipocyte mem-