A label selection approach to assess the role of individual amino groups in human choriogonadotropin receptor binding.
Human choriogonadotropin (hCG) was trace-labeled with [3H]acetic anhydride and then incubated with transformed murine Leydig cells (MA-10). The bound hormone was recovered, subunits (alpha and beta) were separated and then cleaved, and the peptides were purified by high performance liquid chromatography. Analysis of the labeling patterns of peptides from the initial preparation and the bound hCG fraction enabled us to determine the protection factor of several amino groups, which is a measure of the effects of acetylation on receptor binding. The largest protection factors, indicating lower labeling in the bound fraction, were found on beta and involved the alpha-amino group/Lys2 (analyzed together) and Lys104, which exhibited 6-fold and 5-fold selections against binding, respectively. Thus, acetylation at either of these amino groups does not prevent binding but results in selection against hormone association with receptor. Other amino groups were analyzed (e.g. Lys122 on beta; the alpha-amino group and lysines 44/45 (analyzed as a pair), 51, and 75 on alpha), and the selection factors indicated either no change or only modest changes (2-fold) in selection for or against binding. These results indicate that the alpha-amino group/Lys2 and Lys104 of the hormone-specific beta subunit participate, either directly or indirectly, in receptor binding.
Yadav
S P
SP
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Florida 33101.
Brew
K
K
Majercik
M H
MH
Puett
D
D
D000107:Acetylation; D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D006063:Chorionic Gonadotropin / Q000737:chemistry / Q000378:metabolism*; D066298:In Vitro Techniques; D007985:Leydig Cells / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D011974:Receptors, LH / Q000378:metabolism*; D013329:Structure-Activity Relationship
D006063:Chorionic Gonadotropin; D011974:Receptors, LH; D008239:Lysine