title: Effects of TSH receptor mutations on binding and biological activity of monoclonal antibodies and TSH.
abstract: The effects of an extensive series of mutations in the TSH receptor (TSHR) leucine-rich domain (LRD) on the ability of thyroid-stimulating monoclonal antibodies (TSMAbs) and TSH to bind to the receptor and stimulate cyclic AMP production in TSHR-transfected CHO cells has been investigated. In addition, the ability of a mouse monoclonal antibody with blocking (i.e., antagonist) activity (RSR-B2) to interact with mutated receptors has been studied. Several amino acids distributed along an extensive part of the concave surface of the LRD were found to be important for binding and stimulation by the thyroid-stimulating human MAb M22 but did not appear to be important for TSH binding and stimulation. Most of these amino acids important for M22 interactions were also found to be important for the stimulating activity of six different mouse TSMAbs and a hamster TSMAb. Furthermore, most of these same amino acids were important for stimulation by TSHR autoantibodies in a panel of sera from patients with Graves' disease. Amino acid R255 was the only residue found to be unimportant for TSH stimulation but critical for stimulation by all thyroid-stimulating antibodies tested (23 patient serum TSHR autoantibodies, M22, and all seven animal TSMAbs). About half the amino acids (all located in the N-terminal part of the LRD) found to be important for M22 activity were also important for the blocking activity of RSR-B2 and although the epitopes for the two MAbs overlap they are different. As the two MAbs have similar affinities, their epitope differences are probably responsible for their different activities. Overall our results indicate that different TSMAbs and different patient sera thyroid-stimulating autoantibodies interact with the same region of the TSHR, but there are subtle differences in the actual amino acids that make contact with the different stimulators.
authors.affiliation: FIRS Laboratories, RSR Ltd., Parc Ty Glas, Llanishen, Cardiff, CF14 5DU, United Kingdom.
authors.forename: Jane
authors.initials: J
authors.lastname: Sanders
authors.forename: Jane
authors.initials: J
authors.lastname: Bolton
authors.forename: Paul
authors.initials: P
authors.lastname: Sanders
authors.forename: Jennifer
authors.initials: J
authors.lastname: Jeffreys
authors.forename: Nobuhiro
authors.initials: N
authors.lastname: Nakatake
authors.forename: Tonya
authors.initials: T
authors.lastname: Richards
authors.forename: Michele
authors.initials: M
authors.lastname: Evans
authors.forename: Angela
authors.initials: A
authors.lastname: Kiddie
authors.forename: Sara
authors.initials: S
authors.lastname: Summerhayes
authors.forename: Emma
authors.initials: E
authors.lastname: Roberts
authors.forename: Ricardo Núñez
authors.initials: RN
authors.lastname: Miguel
authors.forename: Jadwiga
authors.initials: J
authors.lastname: Furmaniak
authors.forename: Bernard Rees
authors.initials: BR
authors.lastname: Smith
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D000937:Antigen-Antibody Reactions; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000096:biosynthesis; D006111:Graves Disease / Q000276:immunology; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D011989:Receptors, Thyrotropin / Q000235:genetics* / Q000378:metabolism; D013972:Thyrotropin / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000502:physiology; D014162:Transfection
chemical_list: D000911:Antibodies, Monoclonal; D011989:Receptors, Thyrotropin; D013972:Thyrotropin; D000242:Cyclic AMP