FIGURE 2. Site-directed mutagenesis identifies key residues at the N terminus required for the cell-surface expression of α_2B-AR.

A, the sequence of the α_2B-AR N terminus, in which each amino acid residue (except Gly-3) was mutated to Ala individually or in combination. B, specific [³H]RX821002 binding to intact HEK293T cells transfected with α_2B-AR and its mutants. HEK293T cells were transiently transfected with α_2B-AR and its mutants, and their expression at the cell surface was determined by intact cell ligand binding with [³H]RX821002 as described in the legend of Fig. 1. The data shown are the percentages of the mean value obtained from cells transfected with WT α_2B-AR and are presented as the mean ± S.E. of three separate experiments. *, p < 0.05 versus cells transfected with WT α_2B-AR. C, Western blot analysis of α_2B-AR and its mutants M6A, Y12A, S13A, and Y12A/S13A (YS-AA). HEK293T cells were cultured on 6-well plates and transfected with 0.5 μg of GFP-tagged α_2B-AR or its mutants. The cells were then solubilized in 300 μl of 1× SDS gel loading buffer. Five μl of total cell lysate were separated by 10% SDS-PGAE, receptor expression was visualized by immunoblotting using GFP antibodies (upperpanel), and the blots were stripped and then probed with β-actin antibodies (lower panel). D, ERK1/2 activation by UK14304 in HEK293T cells transiently transfected with α_2B-AR or its mutants M6A, Y12A, S13A, and Y12A/S13A. HEK293T cells were transfected with α_2B-AR (squares) and its mutants (triangles), and the cells were then treated with UK14304 (0.01–10 μm) for 5 min. P-, phosphorylated. Left panel, representative blots of ERK1/2 activation. Right panel, quantitative data expressed as percent of the ERK1/2 activation obtained in the cells transfected with α_2B-AR and stimulated with 10 μM UK14304 (control).There is no significant difference in total ERK1/2 expression between samples (data not shown). Similar results were obtained in at least three separate experiments. E, subcellular localization of α_2B-AR and its mutants. GFP-conjugated WT and mutated α_2B-AR were transiently expressed in HEK293T cells, and their subcellular distribution was revealed by fluorescence microscopy detecting GFP as described under “Experimental Procedures.” The data shown are representative images of three independent experiments. Scale bar, 10 μm.