Zero-length cross-linking reveals that tight interactions between the extracellular and transmembrane domains of the luteinizing hormone receptor persist during receptor activation.
Several molecular models of glycoprotein hormone receptor activation have been proposed. It has been suggested that ligand binding to the ectodomain (ECD) leads to major changes in intramolecular interactions between the ECD and the transmembrane domain. We studied these intramolecular modifications by generating a recombinant LH/CG receptor (LHR) bearing an intramolecular cleavage site. We did this by inserting a furin site at position 316 in the hinge region of the ECD (LHR_Fur316). Affinity for human chorionic gonadotropin (hCG) and cAMP production upon hCG stimulation was identical to those of wild-type LHR. Western blot analysis showed that the LHR_Fur316 receptor was cleaved into two subunits linked by disulfide bridges. Chemical shedding of the ECD from the transmembrane domain did not increase basal adenylate cyclase activity, indicating that the first 294 residues did not act as an inverse agonist. The truncated LHR_316 was still activated by hCG but with an EC50 higher than that for the wild-type receptor. Zero length cross-linking was used to study intramolecular interactions between the two domains of LHR_Fur316. Cross-linking efficiency was similar for the basal and activated states, which indicated that the two domains interacted closely in the basal state, and this tight interaction persisted during activation. Our data suggest that activation of the LHR results from subtle modifications of intramolecular interactions between the two domains and low-affinity binding of hCG to the extracellular loops or residues preceding the first transmembrane segment.
Karges
Beate
B
Faculté de Médecine Paris Sud, 94270 Le Kremlin Bicêtre, France.
Gidenne
Stephane
S
Aumas
Chantal
C
Haddad
François
F
Kelly
Paul A
PA
Milgrom
Edwin
E
de Roux
Nicolas
N
D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D002469:Cell Separation; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D000242:Cyclic AMP / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D045683:Furin / Q000737:chemistry; D018698:Glutamic Acid / Q000737:chemistry; D006801:Humans; D015151:Immunoblotting; D008239:Lysine / Q000737:chemistry; D008954:Models, Biological; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011974:Receptors, LH / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D013997:Time Factors; D014162:Transfection
D003432:Cross-Linking Reagents; D004220:Disulfides; D011974:Receptors, LH; D011994:Recombinant Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D000242:Cyclic AMP; D045683:Furin; D008239:Lysine