Peptide sequences from the hypervariable regions of two monoclonal anti-idiotypic antibodies against the thyrotropin (TSH) receptor are similar to TSH and inhibit TSH-increased cAMP production in FRTL-5 thyroid cells.
Monoclonal antibodies, D2 and 4G11, selected by the autoantiidiotypic approach following injection of thyrotropin (TSH) into mice, mimic TSH in binding to receptors on thyroid membranes. Based on TSH receptor transfection studies, D2 and 4G11 show unequivocal specificity for the TSH receptor. To see if the complementary determining regions (CDRs) of these antibodies share any primary sequence similarities to regions of TSH critical for receptor binding, we deduced the primary structure of the variable regions of D2 and 4G11 by sequencing the immunoglobulin mRNA. We found that CDR1 of 4G11K and CDR2 of D2 mu show sequence similarity to regions of TSH alpha and TSH beta that had been previously implicated in the interaction of the hormone with its receptor. We tested the inhibitory effects of synthetic peptides from D2 mu-CDR2 and 4G11K-CDR1 on the binding of the corresponding antibodies to rat thyroid FRTL-5 cells and found an EC50 of 0.1 and 1 microM, respectively. TSH-derived peptides with similarity to D2 mu-CDR2 and 4G11K-CDR1 showed a significant but lesser effect on the binding of 4G11 or D2 to thyroid cells. Additionally, we tested the effects of the CDR peptides and TSH-derived peptides on TSH-stimulated cAMP production in FRTL-5 cells and found that D2 mu-CDR2 and 4G11K-CDR1 inhibited this activity, D2 mu-CDR2 most strongly (EC50 10 microM). Thus, linear sequences from the CDRs of these autoantiidiotypic antibodies with similarity to sequences from both subunits of TSH appear to interact with the TSH receptor. These data support previous studies indicating the complexity of the interaction between TSH and its receptor and advance earlier findings that such immunologic approaches are useful in dissecting receptor-ligand interactions.
Taub
R
R
Howard Hughes Medical Institute, University of Pennsylvania School of Medicine, Philadelphia 19104-6145.
Hsu
J C
JC
Garsky
V M
VM
Hill
B L
BL
Erlanger
B F
BF
Kohn
L D
LD
D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000737:chemistry / Q000235:genetics*; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism*; D007147:Immunoglobulin Light Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics*; D007148:Immunoglobulin mu-Chains / Q000235:genetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D012333:RNA, Messenger / Q000235:genetics; D011989:Receptors, Thyrotropin / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013961:Thyroid Gland / Q000187:drug effects / Q000378:metabolism*; D013972:Thyrotropin / Q000235:genetics* / Q000494:pharmacology*; D014162:Transfection
D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D007148:Immunoglobulin mu-Chains; D009838:Oligodeoxyribonucleotides; D010455:Peptides; D012333:RNA, Messenger; D011989:Receptors, Thyrotropin; D013972:Thyrotropin; D000242:Cyclic AMP