Molecular interactions between the TSH receptor and a Thyroid-stimulating monoclonal autoantibody.
OBJECTIVE
To study the molecular interactions between the thyroid-stimulating hormone (TSH) receptor (TSHR) and a human thyroid-stimulating monoclonal autoantibody (M22).


METHODS
Amino acid mutations were introduced in the variable region gene sequences of M22 and the wild-type (WT) or mutated M22 Fab expressed in Escherichia coli. The ability of WT or mutated M22 Fab to inhibit binding of (125)I-TSH or (125)I-M22 to the TSHR and to stimulate cyclic adenosine monophosphate (AMP) production in Chinese hamster ovary cells expressing WT TSHRs was studied. Mutated TSHRs were also used in these studies in combination with WT or mutated M22 Fab to further identify interacting residues in the TSHR-M22 complex.


RESULTS
Out of 11 amino acid changes in the heavy chain (HC) of M22, 7 had an effect on M22 Fab biological activity, while in the case of 1 mutation the Fab was not expressed. In particular, stimulating activity of M22 Fab mutated at HC residues, D52, D54, and Y56 was markedly reduced. Mutation of M22 light chain (LC) D52 also reduced M22 Fab stimulating activity, while mutations at two further residues (LC D51 and LC D93) showed no effect. Reverse charge mutations at M22 HC D52 and TSHR R80 provided experimental evidence that these two residues interacted strongly with each other.


CONCLUSIONS
Mutation of both the TSHR and M22 Fab has allowed identification of some residues critical for the receptor-autoantibody interaction. This approach should lead to detailed mapping of the amino acids important for M22 biological activity.
Sanders
Jane
J
FIRS Laboratories, RSR Ltd., Parc Ty Glas, Llanishen, Cardiff, United Kingdom.
Miguel
Ricardo Núñez
RN
Bolton
Jane
J
Bhardwaja
Anshu
A
Sanders
Paul
P
Nakatake
Nobuhiro
N
Evans
Michele
M
Furmaniak
Jadwiga
J
Smith
Bernard Rees
BR
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D000937:Antigen-Antibody Reactions / Q000235:genetics / Q000276:immunology; D001323:Autoantibodies / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000276:immunology; D018828:Immunoglobulins, Thyroid-Stimulating; D007457:Iodine Radioisotopes; D016297:Mutagenesis, Site-Directed; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011989:Receptors, Thyrotropin / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D013972:Thyrotropin / Q000378:metabolism
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D018828:Immunoglobulins, Thyroid-Stimulating; D007457:Iodine Radioisotopes; D011989:Receptors, Thyrotropin; C094372:thyrotropin-binding inhibitory immunoglobulin; D013972:Thyrotropin