Fig. 1: Identification of GRP78 as a binding protein for the third intracellular loop of MC4R in the hypothalamus.
From: Glucose-regulated protein 78 binds to and regulates the melanocortin-4 receptor

Bacterially expressed GST alone and GST-MC4Ri3 proteins were incubated with mouse hypothalamic extracts. Pulldown complexes were separated by SDS-PAGE and visualized by silver staining (a, upper gel). Coomassie blue staining shows immobilized GST alone or GST-MC4Ri3 proteins (a, lower gel). An ~78-kDa band specific to the GST-MC4Ri3 pulldown sample was isolated and sequenced by MALDI-TOF MS/MS analysis. b Amino acid (a.a.) sequence of GRP78. Peptide sequences obtained by MALDI-TOF MS/MS are indicated in red. c Schematic diagram of globular domains within Chinese hamster GRP78 (haGRP78); signal sequence (S: 1–18), nucleotide-binding domain (NBD: 19–407), substrate-binding domains (SBDβ, 419–528 and SBDα, 529–654) with an ER retention signal KDEL motif (aa 651–654) and the five globular domain fragments (a.a. 1–252, 253–502, 1–385, 386–502, 503–654). Domain fragments are represented as D1, D23, D12, D3, and D4, respectively. d His-tag pulldown of bacterially expressed and purified GRP78 domain fragments with 3xFlag-MC4R expressed in HEK 293T cells. MC4R was incubated with His-tagged proteins coupled to Ni-NTA agarose beads. e GST pulldown assay showing the interaction between GST-MC4Ri3 and the third globular domain of GRP78. To confirm the MC4R–GRP78 interaction in vitro, coimmunoprecipitation was performed using membrane proteins obtained from HEK 293T cells transfected with 3xFlag-MC4R or GRP78-Myc plasmids. f Membrane proteins were immunoprecipitated with anti-Flag and then immunoblotted with anti-Myc. g Membrane proteins were immunoprecipitated with anti-Myc and then immunoblotted with anti-Flag