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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 price 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.

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.