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. 2011 May 5;286(26):22971–22981. doi: 10.1074/jbc.M110.206870

FIGURE 4.

FIGURE 4.

Spectra FRET analysis of m8-C3V3 R75A mutants. a, ligand binding potency of R75A mutant of mGluR8 is shown. [3H]LY341495 binding to wild-type and R75A mutant-expressing HEK293 cell membranes was measured. Results are presented as the percentage of total specific binding of wild-type. b, shown is the initial rate of the G protein activation ability of the R75A mutant of mGluR8. [35S]GTPγS binding to G protein was measured in the presence of 1 mm l-AP4. Results are presented as the percentage of total specific binding of wild-type. c, shown are fluorescence and difference interference contrast images of HEK293 cells expressing R75A-C3 (i and ii), R75A-V3 (iii and iv), and R75A-C3V3 samples (v and vi). The fluorescence images (i, iii, and v) are shown in gray scale. d, immunoblotting analysis of samples coexpressing WT-C3 (V3) and R75A-V3 (C3). The same amounts of membranes co-expressing WT-C3 (V3) and R75A-V3 (C3) were electrophoresed with (+Me) or without (−Me) β-mercaptoethanol and probed with Anti-GFP antibody. The amount of cDNA in the co-transfection is indicated under the each lane (μg/100-mm dish). e, shown is the amount of expression (pmol/mg of total protein) of Cerulean- or Venus-fused mGluR8 in coexpressing samples estimated from emission spectra of each sample. The amounts of Cerulean, Venus, and total expression are indicated by white, gray, and black bars, respectively. The amount of cDNA in the co-transfection is indicated under each bar (μg/100-mm dish). f, plots of Eapp versus molar ratio between Cerulean and Venus (c/v ratio) in WT+R75A hetero samples with (closed squares) or without (open circles) 10 mm l-glutamate. The c/v ratio was estimated from the values shown in e. The curves were fitted to Equation 8. The inset shows the reciprocal plots fitted to Equation 9. g, comparison of the dose-response plot of FRET efficiency among WT-homo (open circles), WT+R75A hetero (closed triangles), and R75A-homo (open squares) is shown. All the data are expressed as mean ± S.D. of more than three experiments.