Binding of [³H]prostaglandin E₁ to putative receptors linked to adenylate cyclase of cultured cell clones

A method for assessing the binding of ³H labeled prostaglandin E₁ ([³H]PGE₁) to cell membranes has been developed and used to study the interaction of [³H]PGE₁ with membranes from cultured mammalian cells. Receptor sites were identified by correlation of the potency of a series of compounds to compete for [³H]PGE₁ binding sites and to stimulate adenylate cyclase activity, by correlation of rates of binding and change in enzyme activity, and by the correspondence of [³H]PGE₁ binding activity with the presence or absence of PGE₁ sensitive adenylate cyclase in several clones. In clone B82, a murine L cell, [³H]PGE₁ binds with an activation energy of 14 kcal/mol to a class of sites with an affinity of 0.5 x 10⁸M^-1 and a capacity of 150 fmol/mg of protein. Concentration dependence of adenylate cyclase activation by PGE₁ (K(D)=30 nM) and kinetic analysis of [³H]PGE₁ binding (k₁=4 x 10⁶ liters/mol/min, k-₁ = 0.15/min) verify this affinity. Concentration dependence and specificity of binding and activation of adenylate cyclases in neuroblastoma clones N4TG1 and N18TG2 substantiate the method. In several clones that lack PGE₁ responsive adenylate cyclase, no specific [³H]PGE₁ binding is detectable.