title: Mapping of HCG-receptor complexes.
abstract: Molecular forms of the porcine LH/CG receptor (pLHR) and complexes between hCG and either the full-length pLHR or its extracellular domain (ectodomain) have been produced in various recombinant systems. In COS cells and in the baculovirus insect cells system, the co-expression of the ecto- and endo-domains reconstituted a functional receptor where the association of the two domains seems to depend upon the presence of disulfide bridges. According to previous observations [39], synthetic peptides mimicking three regions of the ectodomain (21-38, 100-115, 250-272) were found to inhibit hormone binding and stimulation of cAMP production. Antisera raised against these peptides contained anti-peptide antibodies (Ab) able to interfere with hormone signalling. Moreover, the results of peptide mapping indicated that some peptides stretches may be more involved in signalling rather than in binding. Immunochemical mapping based on monoclonal antibodies (mAbs) was used to probe the hCG-ectodomain complex. It appeared that mAbs directed to epitopes present on the 'beta-tip' of hCG (assembled from the beta subunit loops 3 and 1, and previously designated site IIIb) and on the 'alpha-tip' (alpha subunit loops 1 and 3, site IIIa) bound to hCG-receptor complexes, whereas a conformational epitope (defined by the alpha-beta interface between beta seat belt C-terminus and alpha loop 2, site II) was masked. Interestingly, we and others previously reported that, in the hCG-full length receptor complex, site IIIa was shielded to mAb binding. A peptide mimicking the second extracellular loop (EL2) of the receptor endodomain was found to prevent the binding of a mAb directed to site IIIa, suggesting that this region of the endodomain may be interacting with the 'alpha-tip'. In the full-length, membrane anchored pLHR, the EL2 peptide inhibited hCG-induced cAMP production, but not binding. The possibility of inhibiting stimulation without inhibition of binding gives support to the 'negative specificity' hypothesis [6]. Thus, the ectodomain of the glycoprotein hormone receptors might be considered as a screening device preventing access of any glycoprotein hormone to the signalling peptide keys of the endodomain, which otherwise would be sensitive to any alpha subunit stimulation. Finally, antibody binding to site IIIa on the hCG-ectodomain complex was also hindered by an anti-peptide mAb directed against a peptide encoded by the eighth exon (pE x 8) of the LHR. This suggests that pEx8 is vicinal to the alpha-tip of hCG and to EL2 in the hCG-full length receptor complex. Altogether, these observations help to build up a topological model of the hCG-receptor complex.
authors.affiliation: Unité Récepteurs et Communication Cellulaire, Biologie Cellulaire et Moléculaire, INRA-Biotechnologies, Jouy-en-Josas, France.
authors.forename: J J
authors.initials: JJ
authors.lastname: Remy
authors.forename: L
authors.initials: L
authors.lastname: Couture
authors.forename: J
authors.initials: J
authors.lastname: Pantel
authors.forename: T
authors.initials: T
authors.lastname: Haertlé
authors.forename: H
authors.initials: H
authors.lastname: Rabesona
authors.forename: V
authors.initials: V
authors.lastname: Bozon
authors.forename: E
authors.initials: E
authors.lastname: Pajot-Augy
authors.forename: P
authors.initials: P
authors.lastname: Robert
authors.forename: F
authors.initials: F
authors.lastname: Troalen
authors.forename: R
authors.initials: R
authors.lastname: Salesse
authors.forename: J M
authors.initials: JM
authors.lastname: Bidart
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D006063:Chorionic Gonadotropin / Q000737:chemistry / Q000378:metabolism*; D018604:Epitope Mapping; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D011974:Receptors, LH / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins
chemical_list: D006063:Chorionic Gonadotropin; D011974:Receptors, LH; D011994:Recombinant Proteins