THYROTROPIN INTERNALIZATION IS DIRECTED BY A HIGHLY CONSERVED MOTIF IN THE 7TH TRANSMEMBRANE REGION OF ITS RECEPTOR

The thyrotropin (TSH) receptor is a member of G protein-coupled seven- transmembrane-segment receptors. It is characterized by a large extrac ellular domain linked to the seven transmembrane segments and ending w ith a cytoplasmic tail. Sequence alignment shows that a highly conserv ed motif, NPXXY where X is any amino acid, exists at the boundary betw een the seventh transmembrane domain and proximal part of the cytoplas mic tail of virtually all G protein-coupled receptors. This motif has been implicated as an internalization signal for several cell surface receptors, such as the low density lipoprotein (LDL), insulin and insu lin-like growth factor-1 (IGF-1) receptors. The potential effects of t his motif on the TSH receptor signal transduction and receptor-mediate d TSH internalization was analysed by replacement of the tyrosine(678) residue with an alanine residue. This mutation does not impair high a ffinity TSH binding, but completely abolishes the ability of cAMP resp onse upon TSH stimulation. It also significantly reduces TSH internali zation. The role of the cytoplasmic tail of the TSH receptor in recept or-mediated internalization was also assessed. Deletion of up to 56 am ino acids from the C-terminus of the cytoplasmic tail enhances TSH int ernalization as compared to the wild-type receptor. We conclude that t yrosine(678) in the NPXXY motif is required for efficient receptor-med iated TSH internalization and G protein coupling. The cytoplasmic tail of the TSH receptor may contain sequence domains which could modulate the effects of the NPXXY internalization signal.