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Porcine LH receptor ectodomain was overexpressed in insect cells and lepidopteran larvae using the recombinant baculovirus expression system. A low multiplicity of infection yielded the largest active production, of approximately 107 receptors/cell or 3 μg active receptor/mg total protein in infected cells. The truncated ectodomain solubilized with Triton X-100 bound its ligand with a high affinity which was comparable with that of the native membrane receptor. Increasing the multiplicity of infection resulted in an optimum protein production of 0·6 mg receptor/mg total protein in infected cells. This receptor was largely inactive, probably trapped within aggregation pools. Active receptor could be recovered by dilution of the samples. No secretion of recombinant receptor was ever observed whatever the conditions of infection. Expression of the recombinant receptor in insect larvae was also tested. This low-cost system failed both to increase the amount of active receptor and to induce secretion into the haemolymph. Two methods remain for producing sizeable amounts of active receptor with this baculovirus/insect cell system. One relies on immunoaffinity purification of the active protein and requires large-scale production, and the other is based on the purification of overexpressed inactive receptor followed by renaturation.

International audience; We have previously reported that antipeptide antibodies directed against the second extracellular loop of the cardiac h5-HT4 receptor could block the activation of the L-type Ca channel in human atrial cardiomyocytes. In this paper we investigate the immunological and physiological activity of these antibodies, in a cell system expressing a larger amount of receptors than the atrial cells. The recombinant receptor was expressed at the surface of COS-7 cells under an active form (serotonin, EC50 = 1.81 x 10(-7) M), at a high level (375 +/- 25 fmol receptor/mg total protein) and was able to bind a specific ligand (GR113808) with a high affinity (Kd = 0.28 +/- 0.05 nM). In this system, the same anti-peptide antibodies used for the cardiac cells induced an "agonist-like" effect on the recombinant h5-HT4 receptor. These results are in line with those shown for others G-protein coupled receptors, as adrenoreceptors. In addition, this work showed that the effect of the antibodies is not only dependent on the epitopic region recognised but also on the molecular density and/or the cellular environment of the target receptors. Finally, our results support the hypothesis that the h5-HT4 receptor could be a new target for autoantibodies in patients with atrial arrhythmia.

International audience; The lutropin receptor ectodomain overexpressed under the control of the powerful polyhedrin promoter in baculovirus-infected Sf9 insect cells, is mainly found in an inactive, intracellularly-aggregated form. It is secreted in an active form under the control of the P10 promoter, a somewhat weaker and earlier promoter, at the pride of a lower production. The apparent molecular masses of the two species encoded by the same cDNA are 48 kDa and 60-68 kDa, respectively. The relationship between the extent and type of glycosylation and the extracellular targeting for the recombinant lutropin receptor ectodomains was investigated precisely with endoglycosidases, lectins of various specificities, and a glycosylation inhibitor, and tested with monoclonal and polyclonal antibodies. The results indicate that the strong polyhedrin promoter probably overwhelms the processing capacity of the ER in Sf9 cells, so that only a high- mannose precursor is expressed in large amounts. Only a minute amount of protein is secreted, which has been processed by Sf9 exoglycosidases/glycosyltransferases and bears complex/hybrid oligosaccharides. The weaker P10 promoter allows secretion of a mature and active receptor ectodomain, bearing complex glycosylation. An important O-linked glycosylation is also added post-translationally on this species. In particular, beta-galactose and sialic acid residues were specifically detected in the secreted species, evidence of the induction of the corresponding glycosyltransferases or of their genes.These results suggest that Sf9 cells should eventually be engineered with chaperones and glycosyltransferases in order to improve the production of demanding glycoproteins such as the porcine lutropin ectodomain, so as to open the way to resolution of the three-dimensional structures of these receptors.

International audience; The monoclonal antibody, HT13 recognizes human choriogonadotropin (CG) bound to the extracellular domain of its receptor, but not to the full-length receptor. The HT13 epitope is located in the regions of residues 15-17 and 73-75 of the human CG alpha-subunit. Only one synthetic peptide, lutropin (LH)/CG-receptor-(481-497)-peptide (EL2 peptide), which spans the second putative extracellular loop of the LH/CG-receptor endodomain, prevents recognition of human CG by HT13 mAb. EL2 peptide decreases hormone-induced cAMP production, but not high-affinity binding. An anti-EL2 serum also displays the capacity to inhibit human CG-stimulated cAMP production. These results suggest that the second extracellular loop of the receptor is in contact with the HT13 epitope of human CG alpha-subunit and is involved in signal transduction. A relative orientation of the hormone versus the endodomain is proposed.

The LH/hCG receptor is a G protein-coupled receptor with an N-terminal extracellular domain involved in hormone—receptor interaction. The recombinant porcine receptor, stably expressed in Chinese hamster ovary (CHO) cells, has the same characteristics (Kd and cAMP production) as in Leydig cells. Six synthetic peptides derived from the receptor ectodomain and two polyclonal anti-peptide sera were tested in the homologous system porcine LH and porcine LH receptor. Their ability to inhibit hormone binding and signal transduction on CHO cells expressing the recombinant receptor was evaluated. Peptides 25–40 and 107–121 exhibited a high transduction inhibition as compared with hormone binding, peptides 21–36, 102–111, and 102–121 inhibited hormone binding more efficiently than signal transduction, and peptide 7–24 exhibited inhibition of both hormone binding and hormone-induced cAMP production. Immuno-globulins against peptides 21–36 and 102–111 inhibited both hormone binding and receptor activation suggesting that these sequences are located on the receptor surface. The data suggest that multiple, discontinuous regions of the extracellular domain of porcine LH receptor are involved in hormone binding and signal transduction. Two minimum critical sequences, 21–24 and 102–107, are involved in hormone binding and vicinal segments may be implicated in signal transduction.

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.

Overexpression of the porcine LH receptor (pLHR) ectodomain has been achieved using the baculovirusinsect cell system but mostly in an aggregated form with no secretion. In order to carry this out, new baculoviruses were selected to produce the pLHR ectodomain in insect Sf9 cells and caterpillars. In pLHR-P10–297 and pLHR-mel-319 baculoviruses, pLHR cDNA was under the control of the P10 promoter and the polyhedrin gene promoter respectively. The constructs contained either the porcine signal peptide (pLHR-P10–297) or the insect signal peptide of melittin (pLHR-mol-319). Infected cells produced 1 × 105−3 × 105 receptors/cell 3 days after infection. The recombinant LH receptor ectodomains produced were secreted in a biologically active form and bound the hormone with high affinity. Infected caterpillars produced a larger amount of active pLHR ectodomain than insect cells. The products were not secreted into the haemolymph however. Promoter and/or signal peptide modifications therefore enabled pLHR recombinant ectodomain secretion in a biologically active form, using the baculovirus—lepidopteran cell system. Moreover, moderate levels of expression seem to allow the production of biologically active ectodomain.

International audience; The porcine follitropin receptor-encoding cDNA (pFSHR) was cloned using reverse transcription-polymerase chain reaction (RT-PCR). Total RNA from porcine granulosa cells was used as template. Two overlapping cDNA fragments encoding, respectively, aa 1 to 290 and aa 191 to 694 of the pFSHR were obtained. Taken together, the two fragments represented the whole coding sequence, assuming a comparable length for the FSHR from the porcine, rat and human species. Functionality of the cloned receptor was assessed by expression experiments: COS cells transfected with the pl;SHR cDNA exhibited high-affinity specific binding for [I-125]hFSH and FSH-dependent cAMP production, The primary sequence of the porcine FSHR N-terminal hormone-binding domain showed high percentages of identity with the sequences from ovine, human, and rat origins. A truncated form of the pFSHR cDNA, lacking aa 75 to 124 in the N-terminal domain, was also cloned and sequenced. A PCR-derived cDNA fragment of 1.45 kb was used as gene-specific hybridisation probe to map the pFSHR-encoding gene by radioactive in situ hybridization. This gene was found co-localized (as in human) with the porcine lutropin hormone receptor (pLHR)-encoding gene on the q2.2-q2.3 region of pig chromosome 3.

The glycoprotein hormone receptors differ from other G protein-coupled receptors by their large extracellular domain which mediates ligand binding. Cooperation between the G-protein coupled membrane domain, the extracellular domain and the hormone in establishing high-affinity binding and efficient transduction is likely to exist. Expression plasmids encoding the full-length porcine LH-hCG receptor (1-696), its extracellular (1-297) and membrane domain (298-696), as well as the α and β subunits of hCG were constructed. We report that coexpression in COS cells of the two LH-hCG receptor domains restores cell surface high-affinity hormone binding and hormone dependent adenylyl cyclase activation, suggesting sufficient interactions between the two receptor domains to reconstitute a complete functional molecule. Moreover, the two hormone subunits and the two receptor domains are able to associate within coexpressing COS cells into an active complex

International audience; We have investigated the potential contraceptive effects of immunization against the luteinizing hormone (LH) receptor in male mice at the prepubertal stage. Two N-terminal fragments of the porcine LH receptor encoding amino acids 1-297 and 1-370 were produced in large quantities through the Baculovirus insect cell system. We have immunized three-week-old mice from two Balb/c stocks of differing fecundity with Sf9 insect cells producing the short (1-297) or long (1-370) recombinant LH receptor. A booster injection was performed at six weeks using purified antigens. Ten days later, the immunized male mice were mated over a period of two weeks with adult untreated females. After weaning of the first litters, the same partners were mated once again under the same conditions. There was no decrease in the antiserum titers against the antigens over a two-month period. The circulating testosterone decreased as the anti-LH receptor antibodies increased. The fertility of the treated male mice was reduced up to 75%, depending on the mouse stock, the antigen used and the time separating immunization and mating. The impaired fertility was mostly due to male sterilization (up to 60% of sterile mates). The delay between mating and birth was enhanced by the treatment, reflecting delayed fertility and/or delayed male behaviour acquisition.