PRODUCTION OF THE THYROTROPIN RECEPTOR EXTRACELLULAR DOMAIN AS A GLYCOSYLPHOSPHATIDYLINOSITOL-ANCHORED MEMBRANE-PROTEIN AND ITS INTERACTIONWITH THYROTROPIN AND AUTOANTIBODIES

The thyrotrophin (TSH) receptor (TSHR) is synthesized as a single poly peptide with a predicted large extracellular domain (ECD), a seven-tra nsmembrane pass region and a C-terminal intracellular tail. It is a co mmon target for production of autoantibodies. To investigate whether t he ECD is solely responsible for ligand interaction, we directed the e xpression of this domain in isolation on the cell surface by means of a glycosylphosphatidylinositol (GPI) anchor sequence. Immunoblotting d etected TSHR material of M-r 70,000 expressed at high levels. In immun oprecipitation studies, the GPI-anchored ECD was recognized by experim ental and pathological antibodies. The molecule was detected on the ce ll surface by flow cytofluorimetry at up to 10-fold higher amounts tha n the highest expressing full-length receptor alone. Radioligand bindi ng studies confirmed this and showed that the recombinant molecule bou nd TSH with high affinity similar to full-length receptor; however, st udies with human autoimmune sera indicated differences in the degree o f inhibition when compared with full-length receptor. The existence of the GPI anchor was confirmed by cleavage with a GPI-specific phosphol ipase C and biosynthetic labeling with [H-3]ethanolamine. TSHR materia l was also present inside the cell in both soluble and membrane-bound forms. Thus, the recombinant GPI-anchored ECD is the smallest known fr agment of the TSHR that retains high-affinity TSH binding and is expre ssed at high levels on the cell surface as well as internally; this ap proach may well be useful for other membrane proteins.