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Abstract
Many G-protein coupled receptors (GPCRs) of the rhodopsin-type family have several common amino acid residues in their transmembrane domains. Furthermore, the crystal structure analyses revealed that their membrane-proximal, C-terminal tails form an additional a-helix with amphipathic properties, termed helix 8. Although the binding properties of ligands to the receptors deficient in these domains were tested using membrane preparations, the functional analyses of these mutants have not been performed, mainly due to their endoplasmic reticulum (ER)-retention. Recent information on pharmacological chaperones raises the possibility that the addition of membrane permeable ligands to structurally deficient GPCRs retained in the ER might facilitate their export to the cell surface. Here, we identified several domains in platelet-activating factor receptor and leukotriene B4 type-II receptor that could be crucial for correct folding during its biosynthesis in the ER by mutating the residues and determining which caused a deficiency in the expression at the cell surface. We then used several ligands as pharmacological chaperones to analyze the structurally defective mutants after they were trafficked to the cell surface. Moreover, we describe a new technique, site-specific N-terminal labeling of cell surface proteins on living cells by sortase-A, a transpeptidase from Staphylococcus aureus, to evaluate the trafficking of the mutant receptor after stimulation with agonist.
Research papers (academic journals)
