Rom cells transfected with wild-type (WT) a2A-AR and are presented as the mean 6 S.E. of four experiments. *, p,0.05 versus WT a2A-AR. (C) Effect of mutation of Lys65 on the subcellular distribution of a2A-AR. a2A-AR and its mutants K65R, K65E and K65Q were tagged with GFP at their C-termini and transiently expressed in HEK293 (upper panel) and HeLa cells (lower panel). Their subcellular distribution was revealed by detecting GFP fluorescence by confocal microscopy. The data shown are representative images of at least three independent experiments. Green, GFP-tagged receptors; blue, DNA staining by DAPI (nuclei). Scale bar, 10 mm. doi:10.1371/journal.pone.0050416.gER (Fig. 5A). To quantify the colocalization of the receptors with the ER marker, Pearson’s coefficient was determined. Pearson’s coefficient of the mutant K65A was significantly higher than those of wild-type a2A-AR and the mutant K65R (Fig. 5B). These datasuggest that the residue Lys65 in the ICL1 likely controls 17460038 the exit of a2A-AR out of the ER.a2-AR Export and Cell-Surface ExpressionFigure 4. Effects of mutating Lys65 to Ala and Arg on a2A-AR-mediated activation of ERK1/2. (A) HEK293 cells were transfected with wildtype (WT) a2A-AR or its mutants K65A and K65R and then stimulated with increasing concentrations of UK14,304 for 5 min. ERK1/2 activation was determined by Western blot analysis using phospho-specific ERK1/2 antibodies. Upper panel, a representative blot of ERK1/2 activation; Lower panel, total ERK1/2 expression. (B) Quantitative data expressed as percentage of ERK1/2 activation obtained in cells transfected with a2A-AR and stimulated with UK14304 at 1 mM and presented as the mean 6 S.E. of three separate experiments. *, p,0.05 versus WT a2A-AR at the same concentration of UK14,304. doi:10.1371/journal.pone.0050416.gDiscussionThe molecular mechanisms underlying export from the ER and subsequent transport to the cell surface of the GPCR superfamily remain poorly elucidated. It has been significant efforts to define the structural determinants for GPCR export and several highly conserved hydrophobic and charged sequences which are required for GPCR export from the ER to the cell surface have been identified [5,15,18,24?4,37,38,49]. We have previously Ornipressin site investigated the role of the ICL1 in the cell-surface transport of GPCRs and identified a Leu residue essential for ER export of a group of GPCRs, including a2B-AR [38]. This Leu residue is extremely conserved amongst the family A GPCRs and indeed, as demonstrated in this manuscript, Leu64 in the ICL1 also plays an obligatory role in a2A-AR export from the ER and transport to the cell surface. Thus, this isolated Leu may provide a common signal directing anterograde transport of multiple nascent GPCRs. The most important finding described in this 113-79-1 manuscript is that different positively charged residues on the ICL1 have differential impacts on the cell-surface transport of distinct GPCRs. During the studies on the function of the ICL1, we have surprisingly found that mutation of Lys65 produced a marked inhibitory effect on the cell-surface transport of a2A-AR. First, intact cell ligand binding and flow cytometry to quantify the cell-surface receptors showed that mutation of Lys65 to Ala reduced a2A-AR cell-surface number by more than 50 . Second, the significant reduction of receptor expression at the cell surface was supported by direct visualization of intracellular localization of the mutated receptors.As revealed b.Rom cells transfected with wild-type (WT) a2A-AR and are presented as the mean 6 S.E. of four experiments. *, p,0.05 versus WT a2A-AR. (C) Effect of mutation of Lys65 on the subcellular distribution of a2A-AR. a2A-AR and its mutants K65R, K65E and K65Q were tagged with GFP at their C-termini and transiently expressed in HEK293 (upper panel) and HeLa cells (lower panel). Their subcellular distribution was revealed by detecting GFP fluorescence by confocal microscopy. The data shown are representative images of at least three independent experiments. Green, GFP-tagged receptors; blue, DNA staining by DAPI (nuclei). Scale bar, 10 mm. doi:10.1371/journal.pone.0050416.gER (Fig. 5A). To quantify the colocalization of the receptors with the ER marker, Pearson’s coefficient was determined. Pearson’s coefficient of the mutant K65A was significantly higher than those of wild-type a2A-AR and the mutant K65R (Fig. 5B). These datasuggest that the residue Lys65 in the ICL1 likely controls 17460038 the exit of a2A-AR out of the ER.a2-AR Export and Cell-Surface ExpressionFigure 4. Effects of mutating Lys65 to Ala and Arg on a2A-AR-mediated activation of ERK1/2. (A) HEK293 cells were transfected with wildtype (WT) a2A-AR or its mutants K65A and K65R and then stimulated with increasing concentrations of UK14,304 for 5 min. ERK1/2 activation was determined by Western blot analysis using phospho-specific ERK1/2 antibodies. Upper panel, a representative blot of ERK1/2 activation; Lower panel, total ERK1/2 expression. (B) Quantitative data expressed as percentage of ERK1/2 activation obtained in cells transfected with a2A-AR and stimulated with UK14304 at 1 mM and presented as the mean 6 S.E. of three separate experiments. *, p,0.05 versus WT a2A-AR at the same concentration of UK14,304. doi:10.1371/journal.pone.0050416.gDiscussionThe molecular mechanisms underlying export from the ER and subsequent transport to the cell surface of the GPCR superfamily remain poorly elucidated. It has been significant efforts to define the structural determinants for GPCR export and several highly conserved hydrophobic and charged sequences which are required for GPCR export from the ER to the cell surface have been identified [5,15,18,24?4,37,38,49]. We have previously investigated the role of the ICL1 in the cell-surface transport of GPCRs and identified a Leu residue essential for ER export of a group of GPCRs, including a2B-AR [38]. This Leu residue is extremely conserved amongst the family A GPCRs and indeed, as demonstrated in this manuscript, Leu64 in the ICL1 also plays an obligatory role in a2A-AR export from the ER and transport to the cell surface. Thus, this isolated Leu may provide a common signal directing anterograde transport of multiple nascent GPCRs. The most important finding described in this manuscript is that different positively charged residues on the ICL1 have differential impacts on the cell-surface transport of distinct GPCRs. During the studies on the function of the ICL1, we have surprisingly found that mutation of Lys65 produced a marked inhibitory effect on the cell-surface transport of a2A-AR. First, intact cell ligand binding and flow cytometry to quantify the cell-surface receptors showed that mutation of Lys65 to Ala reduced a2A-AR cell-surface number by more than 50 . Second, the significant reduction of receptor expression at the cell surface was supported by direct visualization of intracellular localization of the mutated receptors.As revealed b.
