Amino acid residues of G-protein-coupled receptors critical for endoplasmic reticulum export and trafficking.
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.
Department of Biochemistry and Molecular Biology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan. moto-nakamura@umin.net
Motonao
M
Nakamura
Daisuke
D
Yasuda
Nobuaki
N
Hirota
Teruyasu
T
Yamamoto
Satoshi
S
Yamaguchi
Takao
T
Shimizu
Teruyuki
T
Nagamune
D000595:Amino Acid Sequence; D019881:Aminoacyltransferases / Q000032:analysis / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000032:analysis / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods; D005455:Fluorescent Antibody Technique / Q000379:methods; D006031:Glycosylation; D006801:Humans; D018613:Microscopy, Confocal / Q000379:methods; D009154:Mutation; D010980:Platelet Membrane Glycoproteins / Q000819:agonists / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects; D043562:Receptors, G-Protein-Coupled / Q000819:agonists / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D018102:Receptors, Leukotriene B4 / Q000819:agonists / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods; D013211:Staphylococcus aureus / Q000201:enzymology
D001426:Bacterial Proteins; D010980:Platelet Membrane Glycoproteins; D043562:Receptors, G-Protein-Coupled; D018102:Receptors, Leukotriene B4; C039351:platelet activating factor receptor; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases