Only a portion of the small seatbelt loop in human choriogonadotropin appears capable of contacting the lutropin receptor.
Twenty residues of the human choriogonadotropin (hCG) beta-subunit that are wrapped around alpha-subunit loop 2 like a "seatbelt" stabilize the heterodimer and enable the hormone to distinguish lutropin (LHR), follitropin, and thyrotropin receptors. The N-terminal portion of the seatbelt contains a small disulfide-stabilized loop needed for heterodimer assembly and is thought to mediate hCG-LHR interactions. To test the latter notion, we compared the LHR binding and signal transduction activities of hCG analogs in which the alpha-subunit C terminus (alphaCT) was cross-linked to residues in the small seatbelt loop. Analogs having an intersubunit disulfide between a cysteine in place of alphaCT residue alphaSer-92 and cysteines substituted for loop residues betaArg-94, betaArg-95, or betaSer-96 had high activities in LHR binding and signaling assays despite the fact that both portions of the hormone are thought to be essential for hCG activity. Use of a larger probe blocked hormone activity when the alphaCT was cross-linked to cysteines in place of residues betaArg-95 and betaAsp-99, but not to cysteines in place of residues betaArg-94, betaSer-96, or betaThr-97. This suggested that the side chains of residues betaArg-95 and betaAsp-99, which face in the same outward direction from the heterodimer, are nearer than the others to the LHR interface. The finding that residue 95 can be cross-linked to small alphaCT probes without eliminating hormone activity indicates its side chain does not participate in essential LHR contacts. We suggest that contacts between the small seatbelt loop and the LHR, if any, involve its backbone atoms and possibly the side chain of residue betaAsp-99.
Department of OB-GYN, Robert Wood Johnson (Rutgers) Medical School, Piscataway, New Jersey 08854, USA.
Michael P
MP
Bernard
Win
W
Lin
Donghui
D
Cao
Rebecca V
RV
Myers
Yongna
Y
Xing
William R
WR
Moyle
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D019556:COS Cells; D002244:Carbon / Q000737:chemistry; D006063:Chorionic Gonadotropin / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D005640:Follicle Stimulating Hormone / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D007986:Luteinizing Hormone / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011863:Radioimmunoassay; D011989:Receptors, Thyrotropin / Q000737:chemistry; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D013696:Temperature; D013912:Threonine / Q000737:chemistry
D006063:Chorionic Gonadotropin; D003432:Cross-Linking Reagents; D004220:Disulfides; D008024:Ligands; D011989:Receptors, Thyrotropin; D013912:Threonine; D001224:Aspartic Acid; D012694:Serine; D002244:Carbon; D007986:Luteinizing Hormone; D005640:Follicle Stimulating Hormone; D001120:Arginine; D003545:Cysteine