Relative fluorescence models (RFU,MaxMin) are indicated. deficiency in recycling, and/or accelerated degradation. The aberrant trafficking was confirmed with a sortase-A-mediated method for labeling cell surface proteins. These results demonstrate that this conserved proline in TM6 is crucial for intracellular trafficking of PAFR. Keywords:Cell/Trafficking, Chaperones, G Proteins/Coupled Receptors (GPCR), Membrane/Proteins, Methods/Fluorescence, Protein/Cell Homogentisic acid Surface, Protein/Export, Receptors/Recycling == Introduction == Many G-protein coupled receptors (GPCRs)2classified in the rhodopsin-type family have several common residues located in their seven transmembrane (TM) helices (1,2). Studies of Mouse monoclonal to CD22.K22 reacts with CD22, a 140 kDa B-cell specific molecule, expressed in the cytoplasm of all B lymphocytes and on the cell surface of only mature B cells. CD22 antigen is present in the most B-cell leukemias and lymphomas but not T-cell leukemias. In contrast with CD10, CD19 and CD20 antigen, CD22 antigen is still present on lymphoplasmacytoid cells but is dininished on the fully mature plasma cells. CD22 is an adhesion molecule and plays a role in B cell activation as a signaling molecule membrane preparations from cells expressing numerous GPCRs, including the platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) receptor (PAFR) have demonstrated the significance of these conserved residues for ligand acknowledgement and/or GTPS binding (36). The three-dimensional structural analysis of some GPCRs (7) suggests that changes in these residues are likely to result in misfolding of the protein during its biosynthesis in the endoplasmic reticulum (ER). However, little is known about the functions of these residues for passage through the ER quality control. Recent information about pharmacological chaperones, which are able to rescue the intracellular retention of several misfolded proteins by stabilizing their conformation and/or enhancing refolding (810), raises the possibility that the addition of specific ligands to structurally deficient GPCRs retained in the ER might result in their export to the cell surface. Even though binding properties of ligands to mutant GPCRs have been examined using membrane preparations (4,1115), little is known about intracellular trafficking (for example, the internalization and recycling) of these mutants in living cells, mainly because of their ER retention. In this statement, we identified several residues in PAFR that could be Homogentisic acid crucial for correct folding during its biosynthesis in the ER by mutating the residues and determining which caused a deficiency in PAFR expression at the cell surface. We then used a PAFR antagonist or agonist as a pharmacological chaperone to analyze the structurally defective mutants after they were trafficked to the cell surface. Moreover, we used a new technique, site-specific N-terminal labeling of cell surface proteins on living cells by sortase-A (16,17), to evaluate the trafficking of mutant PAFR after activation with agonist. We found that a conserved proline, Pro247, in TM6 of PAFR is usually important not only for ER export but also for trafficking,e.g.internalization, recycling, and/or sorting to lysosomes. == EXPERIMENTAL PROCEDURES == == == == == == Materials == Methylcarbamyl (mc)-PAF C-16 was purchased from Cayman Chemical (Ann Arbor, MI). Chloroquine diphosphate salt was from Sigma. Y-24180 was donated from Yoshitomi Pharmaceutical Industries, Ltd. (Osaka, Japan). == Construction of Mutant GPCRs == N terminally HA-tagged human PAFR (HA-hPAFR), human leukotriene B4type 2 receptor (HA-hBLT2), or human GPR43 (HA-hGPR43) were used as themes to generate mutant receptors using the QuikChange Site-directed Mutagenesis kit (Stratagene, La Jolla, CA) following the manufacturer’s instructions. The mutant receptors were inserted into pcDNA3.1 or pCXN2.1. The primer units utilized are outlined undersupplemental materials. == Cell Culture and Transfection == HeLa cells were cultured in Dulbecco’s altered Eagle’s medium (DMEM, Sigma) supplemented with 10% fetal bovine serum. Chinese hamster ovary-K1 cells were cultured in Ham’s F-12 (Sigma) supplemented with 10% fetal bovine serum. PC12h cells were cultured in DMEM supplemented with 10% horse serum and 5% fetal bovine serum. These cells were transfected Homogentisic acid with a plasmid harboring a wild-type (WT) or mutated receptor using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Stable cell lines with inducible expression of WT or mutant PAFRs were established by transfecting the pTRE plasmid bearing the appropriate PAFRs into a stable HeLa cell collection harboring the Tet repressor (HeLa Tet-On Cell Collection; Clontech, Palo Alto, CA (18)) using Lipofectamine 2000. Cells were produced under Geneticin (1 mg/ml; Invitrogen) and hygromycin (100 g/ml; Wako, Osaka, Japan) selection, isolated, expanded, and then tested for doxycycline (Dox; 1 g/ml; Clontech)-inducible expression of PAFR by Western blotting. The clones utilized for experiments showed very low basal, but highly inducible, receptor expression. For our experiments, after cells were plated and cultured for 16 h, receptor expression was induced by adding 1 g/ml of Dox.