Effects of the mutation of Leu residues and their neighboring positively charged residues in the ICL1 on the cell-surface and total expression of α_2A-AR and α_2B-AR.

<p>(A) The sequence of the ICL1 of α_2A-AR, α_2B-AR and α_2C-AR. (B) Ligand dose-dependent binding of α_2A-AR in intact HEK293 cells. HEK293 cells cultured on 6-well plates were transfected with α_2A-AR and then split onto 24-well plates. The cells were incubated with increasing concentrations of [³H]-RX821002 (0.3125, 0.625, 1.25, 2.5, 5, 10, and 20 nM) and the ligand bound to the receptor was measured by liquid scintillation spectrometry as described in the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050416#s2" target="_blank">Materials and Methods</a>”. The nonspecific binding was determined in the presence of rauwolscine (10 µM). Similar results were obtained in at least three different experiments. (C) Quantification of the cell-surface and total expression of α_2A-AR and its mutants in which Leu64 and Lys65 were mutated to Ala individually or in combination. (D) Quantification of the cell-surface and total expression of α_2B-AR and its mutants in which Leu48 and Arg49 were mutated to Ala. In (C) and (D), HEK293 cells were transfected with α₂-AR and the cell-surface expression of the receptors was measured by intact cell binding assays using [³H]-RX821002 at 20 nM. The mean values of specific [³H]-RX821002 binding were 27255±415 and 16785±452 cpm from cells transfected with wild-type (WT) α_2A-AR and α_2B-AR, respectively. Wild-type α_2A-AR and α_2B-AR and their mutants were tagged with GFP and transiently expressed in HEK293 cells. Total receptor expression was determined by flow cytometry measuring the GFP signal as described in the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050416#s2" target="_blank">Materials and Methods</a>”. (E) Quantification of the cell-surface expression of α_2A-AR and its mutants by flow cytometry. HEK293 cells were transfected with HA-tagged α_2A-AR or its individual mutant and the cell-surface expression of the receptors was measured by flow cytometry following staining with anti-HA antibodies in nonpermeabilized cells as described in the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050416#s2" target="_blank">Materials and Methods</a>”. The data shown in (C), (D), and (E) are percentages of the mean value obtained from cells transfected with wild-type α₂-AR and are presented as the mean ± S.E. of at least three different experiments. *, <i>p</i><0.05 <i>versus</i> respective WT α₂-AR.