Photoaffinity labeling of the lutropin receptor with synthetic peptide for carboxyl terminus of the human choriogonadotropin alpha subunit.
Human choriogonadotropin (hCG) consists of an alpha subunit and a beta subunit. The existing evidence from various studies using truncation, substitution, synthetic hormone peptides, and hCG crystals suggests that the C-terminal region of the alpha subunit contacts the luteinizing hormone/choriogonoadotropin (LH/CG) receptor and is involved in receptor activation. Despite a deluge of the speculation and the important role of the alpha C-terminal region, direct evidence for its interaction with the receptor has been elusive. Because of the significant biological activity, it is imperative to prove the interaction of the alpha C-terminal region. For this purpose, decamer peptides corresponding to the alpha subunit sequence from His83 to Ser92 (alpha 83-92) were derivatized with the N-hydroxysuccinimide ester of 4-azidobenzoylglycine (ABG) and radioiodinated. The resulting ABG-125I-alpha 83-92 was capable of binding and activating the LH/CG receptor. Furthermore, UV-sensitive ABG-125I-alpha 83-92 exclusively photoaffinity-labeled an approximately of 86-kDa molecule. This labeled molecule was shown to be the LH/CG receptor by various methods including immunoprecipitation by anti-LH/CG receptor antiserum. In addition, evidence is presented that the amino group of alpha Lys91 of alpha 83-92 is in such close proximity to a carboxyl group of the receptor that this pair is cross-linked to form an amide, a zero length cross-link. This low affinity contact of alpha 83-92 and the receptor is sufficient for receptor activation and is crucial for the full understanding of the mechanistics of the receptor activation steps.
Kundu
G C
GC
Department of Molecular Biology, University of Wyoming, Laramie 82071-3944, USA.
Ji
I
I
McCormick
D J
DJ
Ji
T H
TH
D000345:Affinity Labels; D000595:Amino Acid Sequence; D001386:Azides; D002460:Cell Line; D006063:Chorionic Gonadotropin / Q000737:chemistry / Q000378:metabolism*; D003432:Cross-Linking Reagents; D015292:Glycoprotein Hormones, alpha Subunit / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007457:Iodine Radioisotopes; D007668:Kidney; D007700:Kinetics; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism*; D011974:Receptors, LH / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013388:Succinimides; D014162:Transfection
D000345:Affinity Labels; D001386:Azides; D006063:Chorionic Gonadotropin; D003432:Cross-Linking Reagents; D015292:Glycoprotein Hormones, alpha Subunit; D007457:Iodine Radioisotopes; D010446:Peptide Fragments; D011974:Receptors, LH; D011994:Recombinant Proteins; D013388:Succinimides; C046991:N-hydroxysuccinimidyl-4-azidobenzoyl glycine; C001426:N-hydroxysuccinimide