THE HUMAN FOLLITROPIN ALPHA-SUBUNIT C-TERMINUS COLLABORATES WITH A BETA-SUBUNIT CYSTINE NOOSE AND AN ALPHA-SUBUNIT LOOP TO ASSEMBLE A RECEPTOR-BINDING DOMAIN COMPETENT FOR SIGNAL-TRANSDUCTION

FSH is a member of the pituitary/placental glycoprotein hormone family including luteinizing hormone, thyroid-stimulating hormone,and chorio nic gronadotropin. These heterodimeric hormones share a common alpha-s ubunit and a highly homologous but distinct beta-subunit. The determin ant loop of the FSH beta-subunit acts both as a specificity discrimina tor and as an essential receptor-binding site, The three-dimensional s tructure of hCG illustrates the proximity of the determinant loop to t ile carboxyl-terminal residues of the common alpha-subunit. Thus, site -directed mutagenesis was used to make high-resolution substitutions a t this carboxyl-terminal locus, The effects of those substitutions wer e studied. Twelve single mutations and one composite mutation were mad e of the region of hFSH alpha 74-92, each residue substituted by alani ne, Side chain replacement in this region of FSH proved to be detrimen tal to binding, hFSH with mutations of either alpha S85A, alpha T86A, alpha K91A, or alpha S92A only retained 10% or less of the hFSH recept or-binding activity, while compared to these, mutants alpha H79A, alph a Y88A, and alpha Y89A retained slightly more binding activity, The si ngle mutant alpha F74A and composite mutant alpha V76A/E77A binding ac tivity was reduced to half of that elf wild-type (WT) hFSH. In contras t, mutations of either alpha K75A, alpha T80A, alpha H83A, or alpha H9 0A did not adversely affect receptor binding, demonstrating the specif icity of observed effects. The hFSH and mutant hormones were tested in an in vitro bioassay for stimulation of progesterone production. Thos e mutations that did not affect receptor binding (alpha K75A, alpha T8 0A alpha H83A, and alpha H90A) did not affect signal transduction, mea sured by progesterone responses. After comparison of wild-type and mut ant hFSH activities determined in radioreceptor assays (ID50) and in v itro bioassays (ED50), it became evident that signal transduction corr elated with receptor binding.