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.
Elberg
G
G
Department of Biochemistry and Human Nutrition, Faculty of Agriculture, Hebrew University of Jerusalem, Rehovot, Israel.
Kelly
P A
PA
Djiane
J
J
Binder
L
L
Gertler
A
A
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