title: Obesity-linked variants of melanocortin-4 receptor are misfolded in the endoplasmic reticulum and can be rescued to the cell surface by a chemical chaperone.
abstract: Melanocortin-4 receptor (MC4R) is a G protein-coupled receptor expressed in the brain where it controls food intake. Many obesity-linked MC4R variants are poorly expressed at the plasma membrane and are retained intracellularly. We have studied the intracellular localization of four obesity-linked MC4R variants, P78L, R165W, I316S, and I317T, in immortalized neurons. We find that these variants are all retained in the endoplasmic reticulum (ER), are ubiquitinated to a greater extent than the wild-type (wt) receptor, and induce ER stress with increased levels of ER chaperones as compared with wt-MC4R and appearance of CCAAT/enhancer-binding protein homologous protein (CHOP). Expression of the X-box-binding-protein-1 (XBP-1) with selective activation of a protective branch of the unfolded protein response did not have any effect on the cell surface expression of MC4R-I316S. Conversely, the pharmacological chaperone 4-phenyl butyric acid (PBA) increased the cell surface expression of wt-MC4R, MC4R-I316S, and I317T by more than 40%. PBA decreased ubiquitination of MC4R-I316S and prevented ER stress induced by expression of the mutant, suggesting that the drug functions to promote MC4R folding. MC4R-I316S rescued to the cell surface is functional, with a 52% increase in agonist-induced cAMP production, as compared with untreated cells. Also direct inhibition of wt-MC4R and MC4R-I316S ubiquitination by a specific inhibitor of the ubiquitin-activating enzyme 1 increased by approximately 40% the expression of the receptors at the cell surface, and the effects of PBA and ubiquitin-activating enzyme 1 were additive. These data offer a cell-based rationale that drugs that improve MC4R folding or decrease ER-associated degradation of the receptor may function to treat some forms of hereditary obesity.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Slot 516, 4301 West Markham, Little Rock, AR 72205, USA.
authors.forename: Susana
authors.initials: S
authors.lastname: Granell
authors.forename: Sameer
authors.initials: S
authors.lastname: Mohammad
authors.forename: Ramanagouda
authors.initials: R
authors.lastname: Ramanagoudr-Bhojappa
authors.forename: Giulia
authors.initials: G
authors.lastname: Baldini
mesh_terms: D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D000242:Cyclic AMP / Q000096:biosynthesis; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D009765:Obesity / Q000235:genetics* / Q000473:pathology; D010654:Phenylbutyrates / Q000494:pharmacology*; D017510:Protein Folding / Q000187:drug effects; D044105:Receptor, Melanocortin, Type 4 / Q000819:agonists / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D054875:Ubiquitination / Q000187:drug effects; D056811:Unfolded Protein Response / Q000187:drug effects
chemical_list: D050505:Mutant Proteins; D010654:Phenylbutyrates; D044105:Receptor, Melanocortin, Type 4; D011993:Recombinant Fusion Proteins; D000242:Cyclic AMP