Mechanisms of Nonhormonal Activation of Adenylate Cyclase Based on Target Analysis

Radiation inactivation was used to examine the mechanism of activation of adenylate cyclase in the cultured renal epithelial cell line LLC-PK₁ with hormonal (vasopressin) and nonhormonal (GTP, forskolin, fluoride, and chloride) activating ligands. Intact cells were frozen, irradiated at -70 °C (0–14 Mrad), thawed, and assayed for adenylate cyclase activity in the presence of activating ligands. The In (adenylate cyclase activity) vs. radiation dose relation was linear (target size 162 kDa) for vasopressin-(2 μm)stimulated activity and concave downward for unstimulated (10 mM Mn²⁺), NaF- (10 mM) stimulated, and NaCl- (100 mM) stimulated activities. Addition of 2 μM vasopressin did not alter the In activity vs. dose relation for NaF- (10 mM) stimulated activity. The dose-response relations for adenylate cyclase activation and for transition in the In activity vs. dose curve shape were measured for vasopressin and NaF. On the basis of our model for adenylate cyclase subunit interactions reported previously [Verkman, A. S., Skorecki, K. L., & Ausiello, D. A. (1986) Am. J. Physiol. 260, C103—C123] and of new mathematical analyses, activation mechanisms for each ligand are proposed. In the unstimulated state, equilibrium between αβ and α + β favors αβ; dissociated a binds to GTP (rate-limiting step), which then combines with the catalytic (C) subunit to form active enzyme. Vasopressin binding to receptor provides a rapid pathway for GTP binding to α, GTP and its analogues accelerate the rate of α_GTP formation. Forskolin inhibits the spontaneous deactivation of activated C. Activation by fluoride may occur without αβ dissociation or GTP addition through activation of C by an αβ-F complex.