Monoclonal antibodies as probes to study the human growth hormone-binding domain to lactogenic rat liver receptors.
A set of monoclonal antibodies (MAb) to human GH (hGH) was used to study the hormone binding orientation to its receptors (R) from female rat liver. The hGH antigenic region left exposed after its binding to liver microsomes was detected by measuring the ability of various [125I]MAb to bind to the preformed hGH-R complexes. Results indicated that a cluster of epitopes defined by the MAb, termed AE5, AC8, and AE12, remains accessible in the hGH-R complex whereas overlapping epitopes 3C11 and HG3 would define a hGH region involved in the binding site. Supporting these findings, solubilization and HPLC gel filtration of [125I]MAb-hGH-R complexes showed a radioactive peak of about 450,000 mol wt for MAb AE5 or AC8, but not for MAb 3C11 or HG3. [125I]MAb AE12 behaved differently, suggesting that epitope AE12 may be masked or altered in hGH-R-solubilized complexes. MAb directed to the putative hGH-binding site (MAb 3C11, HG3, and the closely related MAb 10C1 and NA71) failed to inhibit binding of the preformed [125I]MAb AE5-hGH complex to the receptors, suggesting a hormone modification after MAb AE5 binding. Accordingly competition experiments indicated an increase in the affinity of hGH for its receptors induced by this MAb. A higher hGH concentration was required to obtain 50% [125I]hGH binding to liver microsomes in the presence of MAb AE5 than in its absence. As the MAb used define epitopes that were previously correlated with the hGH structure, we concluded that a high flexible region (sequences 134-150) is exposed in the hGH-R complex. Furthermore, some MAb directed to this region enhance the hormone affinity for its rat liver receptors, probably through an induced conformational change.
Instituto de Química y Fisicoquímica Biológicas (UBA-CONICET), Facultad de Farmacia y Bioquímica, Buenos Aires, Argentina.
L P
LP
Roguin
C E
CE
Cohen
L A
LA
Retegui
D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D002851:Chromatography, High Pressure Liquid; D000939:Epitopes / Q000276:immunology; D005260:Female; D013006:Growth Hormone / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D008862:Microsomes, Liver / Q000378:metabolism*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000276:immunology; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018000:Receptors, Peptide; D012995:Solubility
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000939:Epitopes; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D018000:Receptors, Peptide; C028202:placental lactogen receptor; D013006:Growth Hormone