Abstract
Several residues are conserved in the transmembrane domains (TMs) of G-protein coupled receptors. Here we demonstrate that a conserved proline, Pro(247), in TM6 of platelet-activating factor receptor (PAFR) is required for endoplasmic reticulum (ER) export and trafficking after agonist-induced internalization. Alanine-substituted mutants of the conserved residues of PAFRs, including P247A, were retained in the ER. Because a PAFR antagonist, Y-24180, acted as a pharmacological chaperone to rescue ER retention, this retention is due to misfolding of PAFR. Methylcarbamyl (mc)-PAF, a PAFR agonist, did not increase the cell surface expression of P247A, even though another ER-retained mutant, D63A, was effectively trafficked. Signaling and accumulation of the receptors in the early endosomes were observed in the mc-PAF-treated P247A-expressing cells, suggesting that P247A was trafficked to the cell surface by mc-PAF, and thereafter disappeared from the surface due to aberrant trafficking, e.g. enhanced internalization, 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 the conserved proline in TM6 is crucial for intracellular trafficking of PAFR.
Highlights
Many G-protein coupled receptors (GPCRs)2 classified in the rhodopsin-type family have several common residues located in their seven transmembrane (TM) helices [1, 2]
We identified several residues in platelet-activating factor receptor (PAFR) that could be crucial for correct folding during its biosynthesis in the endoplasmic reticulum (ER) by mutating the residues and determining which caused a deficiency in PAFR expression at the cell surface
Similar to the results obtained with HA-hPAFR, these mutants were impaired in cell surface expression and predominantly accumulated in the ER (Fig. 3, A and B), suggesting that these residues could play a pivotal role in cell surface expression of these GPCRs
Summary
Materials—Methylcarbamyl (mc)-PAF C-16 was purchased from Cayman Chemical (Ann Arbor, MI). Flow Cytometry—For staining, cells were incubated with anti-HA antibody (clone 3F10; Roche Applied Science) in phosphate-buffered saline (PBS) containing 2% goat serum at room temperature for 30 min, followed by staining with phycoerythrin-conjugated anti-rat IgG (Beckman Coulter, Fullerton, CA). After a blocking step using 5% skim milk in TBS-T (20 tured in DMEM supplemented with 10% horse serum and 5% mM Tris-buffered saline (pH 7.4), 0.1% Tween 20), blots were fetal bovine serum These cells were transfected with a plas- probed with the primary antibody for 1 h. The resulting pellets were suspended in sampling buffer and subjected to Western blot analyses using anti-ubiquitin antibody (P4D1) and horseradish peroxidase-conjugated anti-mouse IgG whole antibody (Santa Cruz Biotechnology). After the cells were washed with PBS, they were further incubated in fresh DMEM (0.1% BSA) containing 30 M His6-sortase-A and 10 M Alexa Fluor 488-labeled LPETGG peptide at 37 °C for 15 min. Differences were considered significant at p Ͻ 0.05, 0.01, and 0.001 as indicated
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
