Mutants of GPR40.

<p>The point mutations of GPR40 tested in this study are represented as a transmembrane snake plot. The blocked area represents plasma membrane with seven putative transmembrane domains.</p

Effect of R104P mutant on GPR40 mediated ERK phosphorylation.

<p>HEK293 cells transiently transfected with wild type (A) or R104P mutant (B) or empty vector (C) were stimulated with different ligands at indicated concentration at 10min or 100 μM LA for various durations (0, 5, 10, 15, 30 and 60 min) at 37°C, and ERK1/2 phosphorylation was detected with Western blot. ERK phosphorylation levels were normalized to the GAPDH level in the same sample. Data are means ± SEM (n = 3). *P < 0.05, **P < 0.01 and ***P < 0.001, versus vehicle control.</p

Linoleic acid induces internalization of GPR40.

<p>HEK293 cell lines stably expressing with myc-tagged wild type or R104P mutant, were incubated with 100 μM Linoleic acid for 0–60 min at 37°C and stained with permeabilized condition. And the cell-surface level of GPR40 was determined by measuring surface myc immunoreactivity with Immunofluorescence microscopy.</p

Effect of R104P mutant on cell surface localization of GPR40.

<p>(A) Translocation of GPR40 was studied with HEK293 cells transiently transfected with wild type or R104P mutant, and Cells were stained with non-permeabilized and permeabilized condition. GPR40 membranes were stained with myc-tagged GPR40 (red, arrows). Cell nuclei were stained with Hoechst 33342 (blue), 2 μg of DNA transfected. (B) Quantitative analysis of wild type and R104P mutant expression with plasmid of different amount as measured by membranes (M) and total protein (T), (shown in panel A). Data are means ± SEM (n = 3). *P < 0.05 and **P < 0.01, WT membranes versus R104P membranes. Scale bar, 10 μm.</p

Calcium response in HEK293 cells expressing various GPR40 mutants in response to LA, DHA and AM8596.

<p>HEK293 cells were transfected with wild type or mutant GPR40 by electroporation. Cells were then loaded with Fluo-4 AM and intracellular calcium changes after stimulation with different compounds were monitored. (A) Intracellular calcium changes of empty vector, wild-type and R104P/Y202C/R211H mutant hGPR40. (B, C and D) Calcium mobilization in empty vector, wild-type, R104P/Y202C/R211H, R104P, Y202C, R211H and Y202C/R211H in response to LA (B), DHA (C) and AM8596 (D). Data are presented as the means ± SEM (n = 3).</p

Effect of expression of GPR40 in HEK 293 cells on calcium response.

<p>(A) HEK293 cells were transiently transfected with different amount of plasmids (wild type or R104P mutant), and GPR40 was detected with Western blot. (B and C) HEK-293 cells were transfected with wild type (B) or R104P mutant (C), then cells were loaded with Fluo-4 AM and intracellular calcium changes after stimulation with linoleic acid were monitored. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.</p