Kinetic analysis of the internalization and recycling of [3H]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-releasing 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 response to [(3)H]TRH was dependent upon the degree of truncation. Little effect was produced by deletion of the C-terminal to 50 amino acids, although there was a substantial decrease in the extent of internalization by deletion 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 used in the analysis of cellular distribution of receptors with peptide ligands, and thus an alternative procedure using a Mes-containing buffer was employed in the present study. Apart from a truncation anticipated to eliminate post-translational acylation of the re-ceptor, which altered both the association and dissociation rates of [(3)H]TRH, the kinetics of ligand binding were 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 receptor was impaired by pre-exposure of cells to TRH, this was not true of C-terminal truncation mutants, which displayed limited steady-state internalization ratios. A mutant with a substantial C-terminal deletion also displayed decreased functional desensitization compared with the full-length receptor.
Drmota
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Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, University of Glasgow, Glasgow G12 8QQ, Scotland, U.K.
Milligan
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D004705:Endocytosis; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D018025:Receptors, Thyrotropin-Releasing Hormone / Q000737:chemistry / Q000378:metabolism*; D013973:Thyrotropin-Releasing Hormone / Q000378:metabolism*; D014316:Tritium
D017931:DNA Primers; D008024:Ligands; D020033:Protein Isoforms; D018025:Receptors, Thyrotropin-Releasing Hormone; D014316:Tritium; D013973:Thyrotropin-Releasing Hormone