Anti-human FSH receptor monoclonal antibodies: immunochemical and immunocytochemical characterization of the receptor.
The extracellular domain of the human FSH receptor was expressed in Escherichia coli as a fusion protein with ubiquitin. It was tagged with a poly-His tract which was used for its purification. Immunization of mice allowed the preparation of high affinity antireceptor monoclonal antibodies. The latter fell into two categories: some of them were inhibited hormone binding and adenylate cyclase activation whereas others were devoid of these properties. None of the antibodies had agonistic activity (i.e., stimulated adenylate cyclase). Immunoaffinity chromatography allowed us to purify the native receptor in 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 ovaries showed the presence of a major band of 87 kDa and of a minor band of 81 kDa. Endoglycosidase digestion and pulse-chase experiments showed the former to be the mature receptor and the latter the precursor containing mannose-rich carbohydrates. Thus, as in the case for the LH receptor, there was an accumulation (albeit to a lower degree) of the precursor in target cells. We did not detect variant forms of the protein corresponding to the alternative mRNA transcripts previously described. Additive binding to the receptor of several antibodies, but not of the same antibody, allowed us to establish a sandwich-type ELISA for the receptor (sensitivity approximately 1 fmol) and to obtain evidence against the existence of previously described oligomeric forms of the protein. All monoclonal antibodies were able to label the receptor immunocytochemically in transfected cells, and two of them were also able to detect it at the markedly lower physiological concentrations, i.e., in human Sertoli and granulosa cells.
Vannier
B
B
Unité de Recherches Hormones et Reproduction, Institut National de la Santé et de la Recherche Médicale, Hôpital de Bicêtre, Le Kremlin-Bicêtre, France.
Loosfelt
H
H
Meduri
G
G
Pichon
C
C
Milgrom
E
E
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal; D000242:Cyclic AMP / Q000096:biosynthesis; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D005640:Follicle Stimulating Hormone / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D007739:L Cells; D051379:Mice; D010053:Ovary / Q000737:chemistry / Q000166:cytology; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D011233:Precipitin Tests; D011962:Receptors, FSH / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification; D014162:Transfection
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D011962:Receptors, FSH; D011994:Recombinant Proteins; D005640:Follicle Stimulating Hormone; D000242:Cyclic AMP; D000262:Adenylyl Cyclases