Mitogenic and binding properties of monoclonal antibodies to the prolactin receptor in Nb2 rat lymphoma cells. Selective enhancement by anti-mouse IgG.
Three monoclonal antibodies (mAbs) (T6, U5, and U6) against prolactin (PRL) receptors in rat liver were studied in the rat lymphoma lactogen-dependent (Nb2-11C) and autonomous (Nb2-SP) cell lines. The mAbs had strong affinity for lactogen receptors (Ka = 12-14 nM-1), similar to that of human growth hormone (hGH) which is a lactogenic hormone. T6 and hGH competed for the same binding site, while U5 and U6 interacted with another epitope. The 125I-hGH-receptor complex could be immunoprecipitated by either U5 or U6, but not by T6. Affinity labeling and immunoblotting revealed that hGH and U6 bind to a protein of 63-65 kDa. T6, U5, and U6 were mitogenic in Nb2-11C cells but their respective potencies were 185-, 70-, and 4700-fold lower than that of hGH. Anti-mouse IgG enhanced the mitogenic effect of all three mAbs and almost completely abolished the differences between them, although their mitogenic activity was still 60-120-fold lower than hGH. Des-13-hGH, a competitive antagonist of hGH which hardly effected the binding of 125I-U5, inhibited the U5-stimulated proliferation of Nb2-11C cells in a noncompetitive manner, indicating that simultaneous binding of both ligands fixed the receptor in a nonactive conformation. A Fab fragment of T6 was not mitogenic, and inhibited the hGH-induced mitogenesis in a competitive manner, but its mitogenicity could be restored by anti-mouse IgG. We suggest that the dimerization or oligomerization of the lactogen receptor in Nb2-11C cells is an obligatory step in the transduction of the mitogenic signal. It may be induced by binding of the mAb to a site, which can be either identical or may even be distinct from that which binds the lactogenic hormone.
Department of Biochemistry and Human Nutrition, Faculty of Agriculture, Hebrew University of Jerusalem, Rehovot, Israel.
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G
Elberg
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PA
Kelly
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J
Djiane
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Binder
A
A
Gertler
D000818:Animals; D000911:Antibodies, Monoclonal; D001667:Binding, Competitive; D002460:Cell Line; D013006:Growth Hormone / Q000378:metabolism; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D007700:Kinetics; D008223:Lymphoma; D051379:Mice; D011485:Protein Binding; D051381:Rats; D011981:Receptors, Prolactin / Q000276:immunology / Q000502:physiology*
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D011981:Receptors, Prolactin; D013006:Growth Hormone