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1.
Horm Metab Res ; 47(10): 735-52, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-26361260

RESUMEN

The availability of human monoclonal antibodies (MAbs) to the TSHR has enabled major advances in our understanding of how TSHR autoantibodies interact with the receptor. These advances include determination of the crystal structures of the TSHR LRD in complex with a stimulating autoantibody (M22) and with a blocking type autoantibody (K1-70). The high affinity of MAbs for the TSHR makes them particularly suitable for use as ligands in assays for patient serum TSHR autoantibodies. Also, M22 and K1-70 are effective at low concentrations in vivo as TSHR agonists and antagonists respectively. K1-70 has important potential in the treatment of the hyperthyroidism of Graves' disease and Graves' ophthalmopathy. Small molecule TSHR antagonists described to date do not appear to have the potency and/or specificity shown by K1-70. New models of the TSHR ECD in complex with various ligands have been built. These models suggest that initial binding of TSH to the TSHR causes a conformational change in the hormone. This opens a positively charged pocket in receptor-bound TSH which attracts the negatively charged sulphated tyrosine 385 on the hinge region of the receptor. The ensuing movement of the receptor's hinge region may then cause activation. Similar activation mechanisms seem to take place in the case of FSH and the FSHR and LH and the LHR. However, stimulating TSHR autoantibodies do not appear to activate the TSHR in the same way as TSH.


Asunto(s)
Autoanticuerpos/inmunología , Receptores de Tirotropina/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Glicosilación , Humanos , Modelos Moleculares , Receptores de Tirotropina/agonistas , Receptores de Tirotropina/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología
2.
J Mol Endocrinol ; 42(5): 381-95, 2009 May.
Artículo en Inglés | MEDLINE | ID: mdl-19221175

RESUMEN

The TSH receptor (TSHR) ligands M22 (a thyroid stimulating human monoclonal antibody) and TSH, bind to the concave surface of the leucine rich repeats domain (LRD) of the TSHR and here, we show that M22 mimics closely the binding of TSH. We compared interactions produced by M22 with the TSHR in the M22-TSHR crystal structure (2.55 A resolution) and produced by TSH with the TSHR in a TSH-TSHR comparative model. The crystal structure of the TSHR and a comparative model of TSH based on the crystal structure of FSH were used as components to build the TSH-TSHR model. This model was built based on the FSH-FSH receptor structure (2.9 A) and then the structure of the TSHR in the model was replaced by the TSHR crystal structure. The analysis shows that M22 light chain mimics the TSHbeta chain in its interaction with TSHR LRD, while M22 heavy chain mimics the interactions of the TSHalpha chain. The M22-TSHR complex contains a greater number of hydrogen bonds and salt bridges and fewer hydrophobic interactions than the TSH-TSHR complex, consistent with a higher M22 binding affinity. Furthermore, the surface area formed by TSHR residues N208, Q235, R255, and N256 has been identified as a candidate target region for small molecules which might selectively block binding of autoantibodies to the TSHR.


Asunto(s)
Autoanticuerpos/química , Autoanticuerpos/metabolismo , Inmunoglobulinas Estimulantes de la Tiroides/química , Inmunoglobulinas Estimulantes de la Tiroides/metabolismo , Proteínas/química , Proteínas/metabolismo , Receptores de Tirotropina/química , Receptores de Tirotropina/metabolismo , Tirotropina/metabolismo , Aminoácidos , Humanos , Proteínas Repetidas Ricas en Leucina , Ligandos , Modelos Moleculares , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Propiedades de Superficie , Tirotropina/química
3.
J Mol Endocrinol ; 41(3): 145-64, 2008 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-18606720

RESUMEN

The crystal structures of the leucine-rich repeat domain (LRD) of the FSH receptor (FSHR) in complex with FSH and the TSH receptor (TSHR) LRD in complex with the thyroid-stimulating autoantibody (M22) provide opportunities to assess the molecular basis of the specificity of glycoprotein hormone-receptor binding. A comparative model of the TSH-TSHR complex was built using the two solved crystal structures and verified using studies on receptor affinity and activation. Analysis of the FSH-FSHR and TSH-TSHR complexes allowed identification of receptor residues that may be important in hormone-binding specificity. These residues are in leucine-rich repeats at the two ends of the FSHR and the TSHR LRD structures but not in their central repeats. Interactions in the interfaces are consistent with a higher FSH-binding affinity for the FSHR compared with the binding affinity of TSH for the TSHR. The higher binding affinity of porcine (p)TSH and bovine (b)TSH for human (h)TSHR compared with hTSH appears not to be dependent on interactions with the TSHR LRD as none of the residues that differ among hTSH, pTSH or bTSH interact with the LRD. This suggests that TSHs are likely to interact with other parts of the receptors in addition to the LRD with these non-LRD interactions being responsible for affinity differences. Analysis of interactions in the FSH-FSHR and TSH-TSHR complexes suggests that the alpha-chains of both hormones tend to be involved in the receptor activation process while the beta-chains are more involved in defining binding specificity.


Asunto(s)
Hormona Folículo Estimulante/metabolismo , Inmunoglobulinas Estimulantes de la Tiroides/metabolismo , Receptores de HFE/metabolismo , Receptores de Tirotropina/metabolismo , Aminoácidos , Animales , Bovinos , Hormona Folículo Estimulante/química , Humanos , Inmunoglobulinas Estimulantes de la Tiroides/química , Modelos Moleculares , Estructura Secundaria de Proteína , Receptores de HFE/química , Receptores de Tirotropina/química , Porcinos
4.
Thyroid ; 14(8): 560-70, 2004 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-15320966

RESUMEN

The properties of a human monoclonal antibody to the thyrotropin receptor (TSHR) (M22) with the characteristics of patient sera thyroid stimulating autoantibodies is described. Similar concentrations (pmol/L) of M22 Fab and porcine TSH had similar stimulating effects on cyclic adenosine monophosphate (cAMP) production in TSHR-transfected Chinese hamster ovary cells whereas higher doses of intact M22 immunoglobulin G (IgG) were required to cause the same level of stimulation. Patient sera containing TSHR autoantibodies with TSH antagonist (blocking) activity inhibited M22 Fab and IgG stimulation in a similar way to their ability to block TSH stimulation. Thyroid-stimulating monoclonal antibodies (TSmAbs) produced in mice inhibited 125I-TSH binding and 125I-M22 Fab binding to the TSHR but the mouse TSmAbs were less effective inhibitors than M22. These competition studies emphasized the close relationship between the binding sites on the TSHR for TSH, TSHR autoantibodies with TSH agonist activity, and TSHR autoantibodies with TSH antagonist activity. Recombinant M22 Fab could be produced in Escherichia coli and the recombinant and hybridoma produced Fabs were similarly active in terms of inhibition of TSH binding and cAMP stimulation. The crystal structure of M22 Fab was determined to 1.65 A resolution and is that of a standard Fab although the hypervariable region of the heavy chain protrudes further from the framework than the hypervariable region of the light chain. The M22 antigen binding site is rich in aromatic residues and its surface is dominated by acidic patches on one side and basic patches on the other in agreement with an important role for charge-charge interactions in the TSHR-autoantibody interaction.


Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Autoanticuerpos/química , Autoanticuerpos/genética , Receptores de Tirotropina/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Autoanticuerpos/inmunología , Células CHO , Clonación Molecular , Cricetinae , Cristalografía por Rayos X , Escherichia coli , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/inmunología , Radioisótopos de Yodo , Pruebas de Neutralización , Estructura Terciaria de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Relación Estructura-Actividad , Glándula Tiroides/inmunología , Tirotropina/farmacocinética
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