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ABSTRACT

The mechanism by which the TSH receptor is activated is unknown. Current knowledge leads us to consider that G protein-coupled receptors are activated by positioning of their ligand in the pocket formed by the hydrophobic transmembrane segments. Furthermore, activation of an N-terminally truncated LH receptor lacking most of the extracellular domain has been described, suggesting the existence of a mechanism involving a direct interaction between LH and the transmembrane segments. The high conservation of the transmembrane segments among G protein-coupled receptors is a strong indication for a common mechanism of receptor activation.

To test this hypothesis for the TSH receptor we have constructed four N-terminally truncated TSH receptor mutants with 5 or 69 amino acids of the extracellular domain joined to signal peptide regions consisting of the first 23 or 33 amino acids. The four fragments were amplified by PCR and subcloned into pBSK+. Sequences were confirmed after subcloning in M13. After joining the four fragments in pBSK+, the four TSH receptor constructs were subcloned in pSVL and transiently or stably expressed in COS and Chinese hamster ovary (CHO) cells respectively.

In contrast to results obtained for the LH receptor, stimulation of the transfectants with 10 μm human chorionic gonadotrophin or 350 mU TSH/ml did not increase cyclic AMP (cAMP) concentrations in cultures of transiently transfected COS cells or stably transfected CHO cells. However, mRNA for the TSH receptor could be detected by RNase protection assay in all stable transfectants used for stimulation of cAMP.

These results suggest that activation of the receptor does not implicate direct interaction of TSH with the transmembrane domains. However, our experiments could not investigate whether binding of TSH to the extracellular part of the TSH receptor can induce conformational changes of the transmembrane part, which might trigger activation of the receptor or any other role of the extracellular receptor domain as a cofactor for TSH receptor activation.

ABSTRACT

The availability of high affinity antibodies to the human TSH receptor (TSHR) would help in defining its functional domains, but this requires the production of pure receptor as immunogen. We have expressed the extracellular domain (ECD) of the TSHR (residues 21–414) as a fusion protein with maltose-binding protein (MBP) in Escherichia coli, using the pMAL-cRl vector. The major protein in an electrophoretically separated, crude bacterial lysate had a molecular mass of 89 kDa, in agreement with the size predicted for the MBP-ECD fusion product. Its identity was confirmed by Western blotting in which it was recognized by two polyclonal antibodies to synthetic peptides of the TSHR and an anti-MBP. Following purification on an amylose column, 15 mg pure MBP-ECD per litre of culture were produced, which was 5% of the total bacterial protein. Following extensive dialysis in a buffer which produces slight denaturation, MBP-ECD was cleaved with factor Xa. The identity of each protein was confirmed by Western blotting.

To investigate the possibility of using the fusion protein as an immunogen we produced rabbit polyclonal antibodies to the ECD which were able to produce immunofluorescent staining of Chinese hamster ovary cells that expressed the TSHR, and revealed a protein of 95 kDa in Western blots of the same cells, in addition to a protein of 55 kDa. Only the protein of 55 kDa was detected in Western blots of human thyroid membranes. Subsequently, immunoglobulins from mice immunized with MBP-ECD were shown to contain TSH-binding inhibiting activity and to inhibit TSH-mediated cyclic AMP production; these mice had a lower serum thyroxine level when compared with mice immunized with the MBP—β galactosidase fusion protein MBP-GAL.

The study shows the feasibility of using recombinant TSHR expressed in E. coli (i) to produce antibodies which recognize the native receptor and thus could be applied to studies of TSHR expression (e.g. in thyroid tumours), (ii) to establish animal models of autoimmune hypothyroidism and (iii) as the starting material in denaturation and refolding experiments which may help in defining structure—function relationships.