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ELIZABETH SHANE, MARY YEH, ANDREW S. FEIGIN, JOHN M. OWENS, JOHN P. BILEZIKIAN, Cytosol Activator Protein from Rat Reticulocytes Requires the Stimulatory Guanine Nucleotide-Binding Protein for Its Actions on Adeiiylate Cyclase, Endocrinology, Volume 117, Issue 1, 1 July 1985, Pages 255–263, https://doi.org/10.1210/endo-117-1-255
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Abstract
Rat reticulocytes contain a cytosol activator protein (RCAP) that augments catecholamine-sensitive adenylate cyclase activity in reticulocyte membranes. A highly purified preparation of RCAP, obtained by Sephacryl S-200 chromatography, was used to elucidate further its mechanism of action. The specific activity of the S-200 fraction to augment isoproterenol responsiveness was increased approximately 1,100-fold over the starting material, from 1.2 to 1,300 nmol cAMP formed per mg RCAP. The mol wt of RCAP is approximately 20,000. The effect of RCAP to enhance isoproterenol responsiveness was apparent within 20 sec, virtually abolishing the normal lag time of hormone-activated adenylate cyclase. In addition to its effects on catecholamine-responsive adenylate cyclase, RCAP significantly increased basal [21 ± 3 (±SEM) to 41 ± 4 pmol/mg protein-30 min; P < 0.02], guanyl-5′-yl-imidodiphosphate-associated (3173 ± 213 to 4339 ± 365 pmol/mg-30 min; P < 0.03), and fluoride-associated (5152 ± 64 to 5807 ± 58 pmol/mg-30 min; P < 0.05) adenylate cyclase activities. RCAP also altered the characteristics of agonist binding to the β-adrenergic receptor of reticulocyte membranes, causing an increase in the apparent ICK) for isoproterenol from 0.7 ± 0.2 to 7.9 ± 1.6 nM (P < 0.001). Similar to its effects on reticulocytes, RCAP enhanced isoproterenol- and prostaglandin E2-sensitive adenylate cyclase activity in the wild-type S49 lymphoma cell and shifted the binding isotherm for isoproterenol rightward. In cyc–, the mutant that lacks the stimulatory guanine nucleotide-binding protein (N8) and in UNC, the mutant in which receptors are uncoupled from N, RCAP was ineffective. Moreover, RCAP decreased agonist affinity for the β-adrenergic receptor in wildtype S49 cells, but not in cye– or UNC cells. These observations suggest that RCAP requires a functional N8 unit for its effects on hormone-sensitive adenylate cyclase activity. (Endocrinology117: 255–263, 1985)
