Mutagenesis of the 'determinant loop' region of human choriogonadotropin beta.
The hormone-specific beta subunits of the four human glycoprotein hormones are homologous, and mapping studies are underway in many laboratories to delineate the amino acid residues responsible for receptor binding and activation. Results on the human choriogonadotropin beta (hCG beta) subunit, obtained using synthetic peptides, chemically modified derivatives, and mutant forms prepared via site-directed mutagenesis, have suggested that amino acid residues enclosed by the purported disulfide loop between Cys-93 and Cys-100 may contribute to receptor binding and perhaps specificity. Indeed, the 93-100 amino acid sequence is referred to as a determinant loop. We have used site-directed mutagenesis to prepare single amino acid residue replacements at positions not previously investigated in full length beta subunits; these include Arg-95, Ser-96, Thr-97, and Thr-98. In addition, Leu-92 was studied in an effort to determine whether changes immediately adjacent to the determinant loop alter receptor binding. The wild-type and mutant cDNAs for hCG beta were subcloned into a Prsv expression vector and transiently transfected into Chinese hamster ovary cells containing a stably integrated gene for bovine alpha. The concentrations of total expressed hCG beta in heterodimer form with the bovine alpha subunit were determined by radioimmunoassays. The mutant gonadotropins were assayed in vitro using a competitive binding assay with [125I]hCG and progesterone production, both in the transformed murine Leydig cell line, MA-10. Mutant beta subunits containing the replacements Lys-92, Ser-95, Asp-96, and Tyr-97 exhibited normal alpha subunit binding.(ABSTRACT TRUNCATED AT 250 WORDS)
Huang
J
J
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, FL 33101.
Ujihara
M
M
Xia
H
H
Chen
F
F
Yoshida
H
H
Puett
D
D
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D001681:Biological Assay; D006063:Chorionic Gonadotropin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018997:Chorionic Gonadotropin, beta Subunit, Human; D006224:Cricetinae; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017354:Point Mutation; D011374:Progesterone / Q000096:biosynthesis; D011487:Protein Conformation; D011967:Receptors, Gonadotropin / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D006063:Chorionic Gonadotropin; D018997:Chorionic Gonadotropin, beta Subunit, Human; D010446:Peptide Fragments; D011967:Receptors, Gonadotropin; D011374:Progesterone