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. 2018 Oct 6;8:250–266. doi: 10.1016/j.isci.2018.10.001

Figure 1.

Figure 1

Metabolic Stress Facilitates the Interaction of GPRC5B and SMS2 and SMS2 Phosphorylation

(A) Interaction between GPRC5B and SMS2 was measured using co-immunoprecipitation (IP). COS7 cells were transfected with expression plasmids for SMS2-Flag and either 5b-WT or mutant 5b-YF. Empty vector (EV) was used as a negative control. SMS2-Flag was immunoprecipitated using Flag beads. GPRC5B in the immunoprecipitates was detected using SDS-PAGE and western blotting.

(B and C) Palmitate exposure enhances GPRC5B-SMS2 interaction. (B) COS7 cells were transfected with the indicated expression plasmids. After 24 hr of transfection, culture medium was replaced with serum-free medium containing palmitate-BSA complex for the indicated times. GPRC5B-Flag was immunoprecipitated using Flag beads. Immunoprecipitates were digested with PNGase F to remove N-linked glycans from GPRC5B and then subjected to SDS-PAGE. Data are means ± SEM (n = 3; ANOVA, **p < 0.01). (C) COS7 cells were co-transfected with expression plasmids for SMS2-AcGFP and GPRC5B-Flag. After 8 hr of palmitate treatment, cells were fixed and labeled with anti-Flag antibody. Alexa 546-labeled secondary antibody was used to visualize GPRC5B. Fluorescence images were obtained with an FV1000 confocal microscope (Olympus). Sensitized emission fluorescence resonance energy transfer (FRET) was measured. FRET values for individual cells are plotted, and means ± SEM superimposed (n = 52; Student's t test). Palm, palmitate; BSA, vehicle control. Scale bar, 10 μm.

(D) SFK inhibitor, Su6656 blocked palmitate-induced SMS2 phosphorylation. Transfected COS7 cells with the indicated expression plasmids were pretreated with 10 μM of Su6656 and then stimulated with 0.5 mM palmitate for 8 hr. After stimulation, SMS2-Flag was immunoprecipitated, and then tyrosine phosphorylation was detected using anti-phosphotyrosine antibody. Data are means ± SEM (n = 3; ANOVA, ***p < 0.001).

(E) GPRC5B-SMS2 interaction enhances SMS2 tyrosine phosphorylation by recruiting SFK under metabolic stress. After 8 hr of palmitate treatment, SMS was immunoprecipitated.

See also Figures S1 and S2.