Kinetic analysis of the internalization and recycling of [H-3]TRH and C-terminal truncations of the long isoform of the rat thyrotropin-releasing hormone receptor-1

The C-terminal tail of the long splice variant of the rat thyrotropin-relea sing hormone (TRH) receptor-1 (TRHR-1L) comprises around 93 amino acids. A series of C-terminal truncations was constructed and expressed transiently in HEK-293 cells. The extent of steady-state internalization of these in re sponse to [H-3]TRH was dependent upon the degree of truncation. Little effe ct was produced by deletion of the C-terminal to 50 amino acids, although t here was a substantial decrease in the extent of internalization by deletio n to 45-46 amino acids. The rate of internalization of TRHR-1L in response to ligand was substantially decreased by the acid-wash procedures often use d in the analysis of cellular distribution of receptors with peptide ligand s, and thus an alternative procedure using a Mes-containing buffer was empl oyed in the present study. Apart from a truncation anticipated to eliminate post-translational acylation of the receptor, which altered both the assoc iation and dissociation rates of [H-3]TRH, the kinetics of ligand binding w ere unaffected by C-terminal truncation. Equally, the rate of recycling to the plasma membrane of internalized receptors was unaffected by C-terminal truncation. Although the extent of internalization of the full-length recep tor was impaired by pre-exposure of cells to TRH, this was not true of C-te rminal truncation mutants, which displayed limited steady-state internaliza tion ratios. A mutant with a substantial C-terminal deletion also displayed decreased functional desensitization compared with the full-length recepto r.