Molecular mechanism of LH/CG receptor activation.
It is known that the N-terminal half of the LH/CG receptor is responsible for high hCG binding whereas the C-terminal half is capable of receptor activation. Our results suggest that initial hCG binding at the high affinity site in the N-half receptor induces conformational adjustments. This leads to low affinity secondary contacts of the complex of hCG/the N-half receptor with the C-half receptor. This low affinity secondary contact is responsible for activating the receptor. This is based on the following observations. The C-terminal tail of hCG alpha is known to be involved in activation of the LH/CG receptor. In addition to hCG, we examined the C-terminal three residues (His90-Lys91-Ser92) of the common alpha subunit of FSH and TSH. The results show their differential roles in the three hormones. Ser92 is important for binding and cAMP induction of TSH but not for hCG and FSH. Lys91 is important for binding and cAMP induction of hCG, and cAMP induction but not binding of FSH. It is not important for binding or cAMP induction of TSH. His90 is important for all three hormones. When all three residues were truncated, FSH and TSH lose their affinity for binding and cAMP induction, whereas hCG is still capable of binding but not cAMP induction. Therefore, the three amino acids contribute differently in receptor binding and cAMP induction of hCG, FSH and TSH. Our data also indicate that the evolution of the alpha subunit has been constrained in order not to impair any of the hormones. This suggests that each hormone can be independently engineered to improve the potency. To chemically identify the contact site of the alpha C-tail of hCG in the LH/CG receptor, a decamer peptide corresponding to the alpha subunit sequence from His83 to Ser92 (peptide alpha 81-92) was derivatized with UV sensitive reagent, ABG and radio-iodinated. The resulting ABG-125I-peptide alpha 83-92 was capable of binding and activating the LH/CG receptor. Furthermore, it specifically photoaffinity-labeled the LH/CG receptor. In addition, the amino group of alpha Lys91 of peptide alpha 83-92 is crosslinked to a carboxyl group of the receptor, an indication of close association. Reciprocal mutagenesis of alpha Lys91 and Asp397 in exoloop 1 of the LH/CG receptor suggests the complementary of this pair in receptor activation but not the high affinity interaction of hCG and the receptor. In addition, Lys583 of exoloop 3 is also crucial for receptor activation. To test the conformational adjustment, ABG was attached to hCG alpha and reassociated with untreated beta to produce ABG-125I-alpha/beta. The extent of inter-subunit crosslinking of ABG-125I-alpha/beta bound to the receptor was two to three fold less than unbound ABG-125I-alpha/beta. This result indicates structural change at the subunit interface in response to hCG binding to the receptor.
Ryu
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Department of Molecular Biology, University of Wyoming, Laramie 82071-3944, USA.
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Chang
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D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites; D006063:Chorionic Gonadotropin / Q000737:chemistry / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D005640:Follicle Stimulating Hormone / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D010446:Peptide Fragments / Q000378:metabolism; D011974:Receptors, LH / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013972:Thyrotropin / Q000737:chemistry / Q000378:metabolism
D000345:Affinity Labels; D006063:Chorionic Gonadotropin; D010446:Peptide Fragments; D011974:Receptors, LH; D005640:Follicle Stimulating Hormone; D013972:Thyrotropin; D000242:Cyclic AMP