The carboxy-terminal region of the glycoprotein hormone alpha-subunit: contributions to receptor binding and signaling in human chorionic gonadotropin.
The glycoprotein hormones are heterodimeric and contain a common alpha-subunit, which is noncovalently associated with a hormone-specific beta-subunit. The alpha-subunit has been highly conserved throughout evolution; for example, the five amino acid residues of the carboxy-terminus, Tyr-Tyr-His-Lys-Ser-COOH, are identical in nine of the 10 available amino acid sequences. It has been shown that enzymatic removal of these five amino acid residues, while not affecting holoprotein formation, results in a heterodimer that exhibits very little, if any, binding to the CG/LH receptor. Using site-directed mutagenesis on the human alpha-subunit, we have prepared two deletion mutants, Des-(88-92)alpha and Des-(89-92)alpha, and two point mutants, where each of the two tyrosines, 88 and 89, was replaced with phenylalanine, in order to delineate more specifically the contributions of these aromatic side-chains to receptor binding. The cDNAs for wild-type hCG alpha and mutants were introduced into a pcDNAINEO expression vector, and the cDNA for hCG beta was inserted into a pRSV plasmid; both were transiently cotransfected into DUXB-11 cells. The media were collected, and RIAs showed that all mutants formed heterodimers; moreover, there was no discernable difference in subunit assembly between wild-type hCG alpha and the various mutant alpha-subunits. The gonadotropin mutants were assayed in vitro using a competitive binding assay with [125I]hCG and stimulation of progesterone production in the transformed murine Leydig cell line MA-10.(ABSTRACT TRUNCATED AT 250 WORDS)
Chen
F
F
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Florida 33101.
Wang
Y
Y
Puett
D
D
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D002461:Cell Line, Transformed; D006063:Chorionic Gonadotropin / Q000378:metabolism*; D018997:Chorionic Gonadotropin, beta Subunit, Human; D004247:DNA / Q000235:genetics; D015870:Gene Expression; D015292:Glycoprotein Hormones, alpha Subunit / Q000235:genetics* / Q000502:physiology; D006801:Humans; D007985:Leydig Cells / Q000187:drug effects / Q000378:metabolism; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000378:metabolism; D011374:Progesterone / Q000096:biosynthesis; D011485:Protein Binding; D055503:Protein Multimerization; D011974:Receptors, LH / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction
D006063:Chorionic Gonadotropin; D018997:Chorionic Gonadotropin, beta Subunit, Human; D015292:Glycoprotein Hormones, alpha Subunit; D010446:Peptide Fragments; D011974:Receptors, LH; D011993:Recombinant Fusion Proteins; D011374:Progesterone; D004247:DNA