Fig. 2: Cellular colocalization of MC4R and GRP78, and GRP78 downregulation of MC4R trafficking and intracellular signaling. | Experimental & Molecular Medicine

Fig. 2: Cellular colocalization of MC4R and GRP78, and GRP78 downregulation of MC4R trafficking and intracellular signaling.

From: Glucose-regulated protein 78 binds to and regulates the melanocortin-4 receptor

Fig. 2

The intracellular localization of MC4R and GRP78 was analyzed in the absence and presence of the MC4R agonist MTII in HEK 293T cells overexpressing 3xFlag-MC4R and GRP78-Myc plasmids. To observe the effect of GRP78 knockdown, HEK 293T cells were sequentially transfected with siRNA against GRP78 (siGRP78) and the 3xFlag-MC4R plasmid. siGRP78 and 3xFlag-MC4R-expressing HEK 293T cells were treated with 2.5 µg/ml tunicamycin (Tu) to induce ER stress. Surface receptors were measured by ELISA, and intracellular signaling was analyzed by a CRE-mediated luciferase assay. a Time course of MTII treatment of HEK 293T cells expressing 3xFlag-MC4R (labeled with Alexa Fluor 568, red) and GRP78-Myc (labeled with Alexa Fluor 488, green) proteins. After treatment with 1 µM MTII, cells were fixed at 5, 10, 20, and 40 min and immunostained (Scale bar, 20 µm). b Quantification of colocalized MC4R with GRP78. Student’s t test for b: 0 min, n = 31; 5 min, n = 30; 10 min, n = 28; 20 min, n = 24; 40 min, n = 25; ***P < 0.0001 versus 0 min control. c Downregulation of GRP78 in siGRP78-transfected cells compared to that in control-transfected cells. Control plasmids included a mock (empty vector lacking a siRNA sequence) and a scramble (vector containing an siRNA sequence that was designed not to degrade any specific cellular message). d The percentage of sequestered MC4R, shown as a ratio of the total number of receptors to internalized receptors, after GRP78 knockdown. e Expression of ER stress marker proteins after ER stress induction: phosphorylated inositol-requiring enzyme 1α (pIRE1α), phosphorylated eukaryotic initiation factor 2α (peIF2α), and spliced X-box-binding protein 1 (XBP1s). After treatment with 2.5 µg/ml tunicamycin for 3 h in cells transfected with control mock vector or with siGRP78, cell lysates were analyzed for levels of pIRE1α, peIF2α, XBP1s, and β-actin (as a control for normalization) by western blotting. f The ratio of the total receptors to internalized receptors under GRP78 knockdown and ER stress to illustrate the percentage of sequestered MC4Rs. g Relative cAMP-mediated transcriptional activity stimulated with various concentrations of MTII during GRP78 knockdown and ER stress. The results are shown as the mean ± SEM of at least three independent experiments performed in triplicate. One-way ANOVA, Bonferroni post hoc tests for c; two-way ANOVA, Bonferroni post hoc tests for d, f, and g: *P, P < 0.05, **P, ††P < 0.001, and ***P, †††P < 0.001; *, P, versus MC4R only or si-mock; †, P, versus siGRP78 + MC4R + Tu or vehicle; One-tailed Student’s t test for e: P < 0.05, ††P < 0.001, and ***P < 0.001; *, P, versus si-mock; †, P, versus vehicle. The data are presented as the mean ± SEM

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