MAPPING OF HCG-RECEPTOR COMPLEXES

Molecular forms of the porcine LH/CG receptor (pLHR) and complexes bet ween 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 o f disulfide bridges. According to previous observations \39\, syntheti c 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-pep tide antibodies (Ab) able to interfere with hormone signalling. Moreov er, the results of peptide mapping indicated that some peptide stretch es may be more involved in signalling rather than in binding. Immunoch emical mapping based on monoclonal antibodies (mAbs) was used to probe the hCG-ectodomain complex. It appeared that mAbs directed to epitope s present on the 'beta-tip' of hCG (assembled from the beta subunit lo ops 3 and 1, and previously designated site IIIb) and on the 'alpha-ti p' (alpha subunit loops I and 3, site IIIa) bound to hCG-receptor comp lexes, whereas a conformational epitope (defined by the alpha-beta int erface between beta seat belt C-terminus and alpha loop 2, site II) wa s masked. Interestingly, we and others previously reported that, in th e hCG-full length receptor complex, site IIIa was shielded to mAb bind ing. A peptide mimicking the second extracellular loop (EL2) of the re ceptor endodomain was found to prevent the binding of a mAb directed t o site IIIa, suggesting that this region of the endodomain may be inte racting with the 'alpha-tip'. In the full-length, membrane anchored pL HR, the EL2 peptide inhibited hCG-induced cAMP production, but not bin ding. The possibility of inhibiting stimulation without inhibition of binding gives support to the 'negative specificity' hypothesis [6]. Th us, the ectodomain of the glycoprotein hormone receptors might be cons idered as a screening device preventing access of any glycoprotein hor mone to the signalling peptide keys of the endodomain, which otherwise would be sensitive to any alpha subunit stimulation. Finally, antibod y binding to site IIIa on the hCG-ectodomain complex was also hindered by an anti-peptide mAb directed against a peptide encoded by the eigh th exon (pE x 8) of the LHR. This suggests that pExs 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. Copyright (C) 1996 Elsevier Science Ireland Ltd.