Identification of hormone-binding regions of the luteinizing hormone/human chorionic gonadotropin receptor using synthetic peptides.

A comprehensive series of overlapping synthetic peptides have been used to study the relationship between the primary structure of the ovarian receptor for LH/human CG (hCG) and hormone binding. Twenty-four consecutive, overlap peptides that replicate the entire extracellular domain of the rat luteal receptor have been synthesized by standard solid-phase techniques on an automated synthesizer. Eight additional peptides from the extracellular domain and three peptides replicating the putative extracellular loop regions have also been synthesized. Each peptide was evaluated in RRAs for interaction with hCG by measuring its ability to competitively inhibit binding of 125I-hCG to membrane receptor. Twelve peptides were found to be potent in RRAs and caused a reduction of half-maximal binding of 125I-hCG at concentrations of 10-250 x 10(-6) M. The 12 active peptides (and adjacent inactive peptides) defined at least 4 independent receptor regions that can interact with hormone. One site near the NH2-terminus was localized to receptor residues Arg21-Pro38. Two more sites of hormone interaction were identified by peptides replicating residues Arg102-Thr115 and Tyr253-Phe272. A fourth binding region was identified in the third putative extracellular loop, replicated by rat luteal receptor peptide Lys573-Lys583. The amino acid sequences of the four active rat LH/hCG receptor regions were aligned and compared with published sequences for other glycoprotein hormone receptors. Three regions (Arg102-Thr115, Tyr253-Phe272, and Lys573-Lys583) showed high sequence homology with the human LH/hCG receptor, human TSH receptor, and rat FSH receptor and may represent contact sites for the alpha-subunit of hormone. The other binding region, Arg21-Pro38 had low sequence homology with the other glycoprotein hormone receptors and is postulated to be a binding determinant for beta-hCG/LH. This report demonstrates that synthetic overlap peptides of confirmed sequence can be used to successively identify hormone interaction sites of glycoprotein hormone receptors.