Structure and function of the luteinizing hormone receptor

The relationship of structure and function of the rat luteinizing hormone receptor (LHR) was studied by site-directed mutagenesis and expression of wild type and mutant receptors in mammalian COS1 cells and overexpression in the baculovirus-insect system. The levels of expression of the holoreceptor (form A) and its truncated splice variant (form B) in baculovirus-infected insect Sf9 cells were more than 120-fold greater than in mammalian COS1 cells. Both forms bound hCG with high affinity. The effect of N-linked glycosylation on hormone binding activity of the LHR was studied by mutating each of the six putative glycosylation sites. Functional glycosyl chains were present at positions 152 and 173. At least one oligosaccharide that is not required for hormone binding activity was present at Asn269, 277 or 291. The putative glycosylation site Asn77 was not glycosylated. The LHR form B expressed in insect cells contains only high mannose carbohydrate chains, which were sufficient for high-affinity hormone binding. Studies on refolding of the denatured and reduced LHR form B preparations demonstrated that only the proximal N-acetylglucosamine residue is required for regeneration of the high affinity hormone binding form of the LHR. Other studies revealed that Cys8, 12, 14 and 22 in exon 1 of the LHR were essential for hormone binding. In addition, mutation of Cys109 or Cys134 in exons 5 and 6 respectively, resulted in 75% reduction in hormone binding activity but did not affect the expression level and stability of mutant receptors. Cys257 and Cys258 (exon 9), Cys321 and Cys331 (exon 11), and Cys417 and Cys492 (exoplasmic loops 1 and 2, respectively) were found to be essential for receptor translocation and/or membrane insertion. Cys282 (exon 10) and Cys314 (Exon 11) were not required for either human chorionic gonadotropin (hCG) binding or membrane insertion. Ligand blotting showed that denatured and reduced LHR form B but not form A could be refolded, indicating that cysteine residues located in the transmembrane and/or extracellular loops of the holoreceptor disrupt the correct formation of disulfide bonds as well as the conformation required for hormone binding.