| Abstract
| - The extracellular domain of the human FSH receptor was expressedin Escherichia coli as afusion protein with ubiquitin. It was tagged with a poly-His tractwhich was used for its purification.Immunization of mice allowed the preparation of high affinityantireceptor monoclonal antibodies. Thelatter fell into two categories: some of them were inhibited hormonebinding and adenylate cyclaseactivation whereas others were devoid of these properties. None ofthe antibodies had agonistic activity(i.e., stimulated adenylate cyclase). Immunoaffinitychromatography allowed us to purify the native receptorin a single step either from a permanently transfected L cell line(75% recovery) or from human ovaries(33% recovery). Immunoblotting of the receptor in human ovariesshowed the presence of a major bandof 87 kDa and of a minor band of 81 kDa. Endoglycosidase digestionand pulse−chase experimentsshowed the former to be the mature receptor and the latter theprecursor containing mannose-richcarbohydrates. Thus, as in the case for the LH receptor, there wasan accumulation (albeit to a lowerdegree) of the precursor in target cells. We did not detectvariant forms of the protein corresponding tothe alternative mRNA transcripts previously described. Additivebinding to the receptor of severalantibodies, but not of the same antibody, allowed us to establish asandwich-type ELISA for the receptor(sensitivity ∼1 fmol) and to obtain evidence against the existence ofpreviously described oligomericforms of the protein. All monoclonal antibodies were able to labelthe receptor immunocytochemicallyin transfected cells, and two of them were also able to detect it atthe markedly lower physiologicalconcentrations, i.e., in human Sertoli and granulosacells.
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