1. Amino acid residues of G-protein-coupled receptors critical for endoplasmic reticulum export and trafficking.
- Author
-
Nakamura M, Yasuda D, Hirota N, Yamamoto T, Yamaguchi S, Shimizu T, and Nagamune T
- Subjects
- Amino Acid Sequence, Aminoacyltransferases analysis, Aminoacyltransferases metabolism, Animals, Bacterial Proteins analysis, Bacterial Proteins metabolism, Cysteine Endopeptidases analysis, Cysteine Endopeptidases metabolism, Endoplasmic Reticulum drug effects, Flow Cytometry methods, Fluorescent Antibody Technique methods, Glycosylation, Humans, Microscopy, Confocal methods, Mutation, Platelet Membrane Glycoproteins agonists, Platelet Membrane Glycoproteins genetics, Protein Transport drug effects, Receptors, G-Protein-Coupled agonists, Receptors, G-Protein-Coupled genetics, Receptors, Leukotriene B4 agonists, Receptors, Leukotriene B4 genetics, Staining and Labeling methods, Staphylococcus aureus enzymology, Endoplasmic Reticulum metabolism, Platelet Membrane Glycoproteins analysis, Platelet Membrane Glycoproteins metabolism, Receptors, G-Protein-Coupled analysis, Receptors, G-Protein-Coupled metabolism, Receptors, Leukotriene B4 analysis, Receptors, Leukotriene B4 metabolism
- Abstract
Analysis of the structural features of rhodopsin-type G-protein-coupled receptors (GPCRs) revealed the existence of an additional α-helix, termed helix 8, in the C-terminal tail. Furthermore, these GPCRs were determined to possess several conserved residues in their transmembrane domains. The functional deficiencies of receptors in which these domains or residues have been mutated have not been examined in living cells due to their accumulation in the endoplasmic reticulum (ER), although the ligand affinities of these receptors have been tested in membrane preparations. Recent studies have demonstrated that ER-accumulated receptors are effectively exported from ER using membrane permeable ligands as pharmacological chaperones. Here, we identified several residues of the platelet-activating factor receptor and leukotriene B(4) type-II receptor that are crucial for export from ER. Moreover, we used their specific ligands as pharmacological chaperones to traffic ER-accumulated GPCRs to the cell surface in order to examine the functional deficiencies of each mutant receptor. Here, we introduce the novel technique of site-specific N-terminal labeling of cell surface proteins in living cells with Sortase-A, a transpeptidase isolated from Staphylococcus aureus, to evaluate the trafficking of receptors after agonist stimulation., (Copyright © 2013 Elsevier Inc. All rights reserved.)
- Published
- 2013
- Full Text
- View/download PDF