CHARACTERIZATION AND PURIFICATION OF LH/HCG RECEPTORS AND ADENYLATE CYCLASE

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This aspect of the program is concerned with a) characterization of gonadotropin and prolactin receptors of the testis and ovary; b) the physical and functional relationships of the LH receptor site and adenylate cyclase; and c) cAMP-dependent and independent phosphorylation during gonadotropin action. In studies on the properties of testicular lactogern receptors, detergent-solubilized preparations had higher binding capacity than the membrane fraction, due to unmasking of a receptor pool. The crosslinked hormone-receptor complexes were distributed between two subunit sets with Mr of 113 & 103K or 59 & 53K, equivalent to 91 & 81K and 37 & 31K (calculated) for the free receptor. The existence of bands with close Mrs could be attributed to differences in glycosylation or phosphorylation. Parallel crosslinking experiments with other tissues (i.e. ovary, mammary gland, liver and kidney) showed similar resolution. As in the ovary, the 37K species appears to be contained within the 80K species through disulfide linkage. Variants of low ahd high affinity subunits were usually indistinguishable in cross-linking studies with ovarian soluble preparations. In contrast, silver staining profiles of purified ovarian receptors (20.4 nmol/mg protein) showed 88K and 95K variants of the high Mr subunits, while the low Mr subunit was observed as a single band of 41K. The purified receptor subunits were biologically active as demonstrated by hormone binding to free receptors transblotted to nitrocellulose. We have devised a procedure for purification of microgram quantities of active lactogen receptors from rat ovaries. Also, we have shown that extensive internalization and degradation of bound hCG is not required for the early phase of gonadotropic desensitization in the Leydig cell. Studies of the hormone and G-nucleotide effect on membrane phosphorylation have shown that in the presence of submaximal levels of GTP, oLH induces significant increase in phosphorylation of a 44K protein. These findings and our previous observations, suggest that hormone binding to the LH receptor induces a G-nucleotide/Ca2+ dependent phosphorylation of the 44K protein.