FUNCTIONAL-PROPERTIES OF POLYCLONAL ANTIBODIES RAISED AGAINST THE N-TERMINUS REGION (RESIDUES 9-30) OF THE FOLLICLE-STIMULATING-HORMONE (FSH) RECEPTOR - SIGNIFICANCE OF THIS RECEPTOR REGION IN FSH RECOGNITION AND SIGNAL-TRANSDUCTION

In this study, we raised polyclonal antibodies in rabbits against a sy nthetic peptide corresponding to a unique region of the FSH receptor, residues 9-30, with no sequence homology to receptors for LH and TSH, and examined their characteristics relevant to receptor function. Bind ing of [I-125]human (h) FSH to membrane-bound receptors was inhibited in a concentration-dependent manner by the anti-FSH receptor-(9-30) pe ptide antibody. Preimmune serum had no effect. Line-weaver-Burke plot analysis of [I-125]hFSH binding to membrane receptors in the presence or absence of antireceptor peptide antibody indicated that the antibod y effectively competed with FSH at a hormone-binding site on the recep tor. Also, antireceptor peptide antibody, but not preimmune serum, inh ibited the ability of FSH to stimulate the conversion of androstenedio ne to estradiol in cultured immature rat Sertoli cells. Stimulation of estradiol synthesis by Sertoli cells caused by cholera toxin or forsk olin (which are known to act through the G(s)-protein and catalytic un it of adenylate cyclase, respectively) was not inhibited by antirecept or peptide antibody. Indirect immunofluorescence staining of cultured rat Sertoli cells showed binding of antibody to plasma membrane recept or. No fluorescent staining of receptor was observed when cells were i ncubated with preimmune serum or antireceptor peptide antibody in the presence of excess receptor-(9-30) peptide or hFSH. These results were consistent with specific labeling of membrane-bound FSH receptors by anti-receptor-(9-30) peptide antibody. When detergent-solubilized memb rane preparations from rat Sertoli cells were fractionated by sodium d odecyl sulfate-polyacrylamide gel electrophoresis under nonreducing co nditions and then subjected to Western blot analysis, antireceptor pep tide antibody, but not preimmune rabbit serum, specifically recognized intact FSH receptor. Although the antireceptor peptide antibody occup ied the N-terminus 9-30 epitope region in the FSH receptor, it did not induce postbinding events, such as receptor patching (aggregation), a s shown by indirect immunofluorescence staining of rat Sertoli cells a nd the estradiol response. In contrast, a polyclonal antibody against the FSH holoreceptor capable of interacting with multiple epitopes on the receptor could induce FSH-like effects, such as receptor patching and estradiol response in Sertoli cells. In conclusion, antibody raise d against the N-terminus region (9-30) of the FSH receptor recognized intact FSH receptor, inhibited FSH binding, and behaved as an antagoni st, suggesting that this N-terminus epitope region of the receptor is involved in hormone binding. Moreover, the differential effects of thi s antibody and antibody raised against the FSH holo-receptor on postbi nding events suggest that the N-terminus (9-30) region of the FSH rece ptor alone may not be directly involved in signal transduction. The si te-directed antibodies reported herein should offer a useful probe for further study of FSH receptor structure, biosynthesis, and mode of ac tion.