FIGURE 2.

GluR6 surface expression in HeLa cells was reduced when either Ser⁸⁴⁶ or Ser⁸⁶⁸ was mutated to mimic phosphorylation. A, GluR6 surface expression was evaluated in HeLa cells expressing FLAG-GluR6 (WT, S846A, S846D, S868A, S868D, or S846D,S868D) by immunofluorescence using confocal microscopy as described under “Experimental Procedures.” B, quantitative FACS analysis shows significantly decreased surface expression of GluR6 S846D and GluR6 S868D compared with GluR6 WT. HeLa cells were transfected with FLAG-GluR6 IRES-EGFP (GluR6 WT, S846A, S846D, S868A, S868D, or S846D,S868D). GluR6 surface expression was analyzed by FACS as described under “Experimental Procedures.” GluR6 S846D surface expression was decreased by 45%, GluR6 S868D by 31%, and GluR6 S846D,S868D by 69% compared with GluR6 WT. Data represent mean ± S.E.M. of the fold-increase in surface expression compared with GluR6 based on the fluorescence intensity of surface-expressed GluR6 in transfected cells. *, p < 0.01, Student's t test. C, quantitative FACS analysis shows that phosphomimetic mutations of GluR6 do not affect GluR6 total expression level. Data represent mean ± S.E.M. of the fold-increase in total expression compared with GluR6 WT based on the fluorescence intensity of total GluR6 in transfected cells.