Bradykinin recognizes different molecular forms of the B2 kinin receptor in the presence and absence of guanine nucleotides.

We have previously reported that [3H]bradykinin [( 3H]BK) identifies high- and low-affinity B2 kinin receptor sites in bovine myometrial membranes which are sensitive and insensitive respectively to guanine nucleotides. Here we show that these receptor-binding sites are solubilized by the detergent CHAPS. Equilibrium binding in soluble preparations revealed that [3H]BK identified a maximal number of binding sites (Bmax) of 1119 +/- 160 fmol/mg of protein, with an equilibrium dissociation constant (KD) of 314 +/- 70 pM and with a typical B2 kinin receptor specificity. Dissociation of equilibrium binding was biphasic. In the presence of the GTP analogue guanosine 5'[beta gamma-imido]triphosphate (Gpp[NH]p, [3H]BK bound to the soluble receptors with a KD of 929 +/- 129 pM and a Bmax. of 706 +/- 38 fmol/mg of protein. The Gpp(NH)p-promoted decrease in the apparent affinity and Bmax., which was half-maximal at 0.5 microM, was due at least in part to an increase in the dissociation rate of the slowly dissociating component of the equilibrium binding. Recoveries of guanine-nucleotide-sensitivity and of rapidly and slowly dissociating binding components were essentially identical, whether or not the receptor had been occupied by an agonist before solubilization. Sucrose-density-gradient sedimentation profiles revealed that [3H]BK recognized two different molecular forms of the receptor in the absence or presence of guanine nucleotides. These results provide for the first time direct evidence that guanine nucleotides promote a change in the structure of the B2 kinin-receptor complex. We propose that this structural change is due to dissociation of a guanine-nucleotide-regulatory (G-)protein.