Characteristics of a soluble gonadotropin receptor from the rat testis.

The gonadal receptor for luteinizing hormone LH and chorionic gonadotropin was extracted in soluble form from a particulate binding fraction of the interstitial cells of the rat testis by treatment with the nonionic detergent Triton X-100. During binding studies with the soluble receptor and 12ZI-labeled human chorionic gonadotropin (hCG), receptor-bound and free forms of the hormone were separated by a double precipitation procedure with polyethylene glycol. The soluble gonadotropin receptors retained hormonal specificity and high affinity for LH and hCG, and showed rapid and reversible gonadotropin binding during incubation with lo-l1 M “jl-hCG. The initial rate of binding of hCG by soluble receptors was higher at 34” than at 24’ or 4”, but degradation of receptors occurred more rapidly at the higher temperature with corresponding loss of binding activity. The equilibrium association constant of the soluble hormone-receptor complex at 24” (0.5 to 1 x 10’” M-I) was detectably lower than that of the particulate receptors for hCG (2.4 X lOlo M-l). The optimum pH for gonadotropin binding was 7.4, and no effects of buffer composition, ionic strength, or calcium concentration upon binding were demonstrable. Exposure of particulate and soluble receptors to trypsin caused loss of gonadotropin binding activity, indicating the protein nature of an essential component of the receptor site. In addition, a significant role of phospholipid in the structural and functional properties of the receptor was suggested by the reduced binding activity observed after treatment of particulate and soluble receptors with phospholipase A, and by the aggregation which occurred after exposure of the soluble receptors to phospholipase C. Gel filtration and density gradient centrifugation of the free receptors, and the receptor-hormone complex formed by equilibration of the soluble receptors with WI-hCG, were performed in solutions containing 0.1% Triton. The soluble receptor and receptor-hormone complex showed adsorption to Sepharose 6B during gel filtration, and were quantitatively bound by blue dextran. For these reasons, blue dextran could not be used as a front marker during gel filtration studies, and 0.01% bovine serum albumin was included in buffers employed for chromatography on Sepharose 6B. The distribution coefficient (Kay) of the receptorhormone complex on Sephadex G-200 was 0.09, and that of free hCG was 0.33. On columns of Sepharose 6B, the K,, of the free receptors and the receptor-hormone complex was 0.32, and that of free hCG was 0.56. By reference to the behavior of standard proteins during filtration on Sepharose 6B, the hydrodynamic radius of the receptor was calculated to be 64 A. Sucrose density gradient centrifugation showed that the sedimentation constant of the free receptor was 6.5 S, and that of the hormone-receptor complex was 7.5 S. Dialysis of the complex to remove Triton X-100 caused conversion to an 8.8 S form, but no aggregation occurred. The density of the 7.5 S hormone-receptor complex in cesium chloride gradients was 1.289. From these values, the molecular weights of the 6.5 S (free) and ‘7.5 S (combined) forms of the receptor were calculated to be 194,000 and 224,000, respectively, and the axial ratios (prolate) of the two forms were 12 and 10.2, respectively. The properties of the gonadotropin receptor extracted by Triton X-100 were consistent with those of a highly asymmetric molecule, predominantly of protein nature, with a minor but functionally important phospholipid component. The retention of high specificity and affinity by the solubilized gonadotropin receptors indicates that the receptor macromolecules possess relatively high conformational stability, and provides an approach to the structural analysis of the hormone binding site.