title: Functional analysis of melanocortin-4-receptor mutants identified in severely obese subjects living in Southern Italy.
abstract: The melanocortin-4 receptor (MC4R) is involved in regulating energy homeostasis; mutations in this gene have been associated with 1-5% of early-onset human obesity. The aim of this study was to functionally characterize MC4R mutations identified in morbidly obese subjects living in Southern Italy. We studied their ligand binding, signaling pathway and subcellular localization. As expected, mutants Q43X and S19fsX51, which produce truncated forms of receptor, were devoid of activity. The activity of mutants W174C and A175T were very different even though the mutations are adjacent and are in the same transmembrane helix (TMH). In fact, the production and expression of mutant A175T on the plasma-membrane (PM) was similar to that of the wild-type (wt) receptor and the mutant retained 70% of wt receptor activity; on the contrary, the production of W174C mutant in the cytoplasm was similar to that of the wt receptor and mutant A175T but was only barely detectable on the PM and was devoid of activity. Confocal microscopy showed that W174C remained entrapped in the endoplasmic reticulum (ER) of the cells. Structural analysis showed that substitution of Trp174, located in the middle of TMH4 and 100% conserved in all known MC4Rs, with Cys could impair the relative orientation of TMH2 and TMH4 thereby affecting the overall protein architecture. Furthermore, co-expression studies showed that mutant A175T but not W174C had a dominant negative effect on the wt receptor activity.
authors.affiliation: Dipartimento di Studi delle Istituzioni e dei Sistemi Territoriali, Facoltà di Scienze Motorie, Università Parthenope, Naples, Italy.
authors.forename: Andreina
authors.initials: A
authors.lastname: Alfieri
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Pasanisi
authors.forename: Salvatore
authors.initials: S
authors.lastname: Salzano
authors.forename: Luciana
authors.initials: L
authors.lastname: Esposito
authors.forename: Domenico
authors.initials: D
authors.lastname: Martone
authors.forename: Domenico
authors.initials: D
authors.lastname: Tafuri
authors.forename: Aurora
authors.initials: A
authors.lastname: Daniele
authors.forename: Franco
authors.initials: F
authors.lastname: Contaldo
authors.forename: Lucia
authors.initials: L
authors.lastname: Sacchetti
authors.forename: Adriana
authors.initials: A
authors.lastname: Zagari
authors.forename: Pasqualina
authors.initials: P
authors.lastname: Buono
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics / Q000502:physiology; D002478:Cells, Cultured; D006801:Humans; D007558:Italy; D008024:Ligands; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D008958:Models, Molecular; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D009767:Obesity, Morbid / Q000235:genetics*; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D044105:Receptor, Melanocortin, Type 4 / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D013329:Structure-Activity Relationship; D014018:Tissue Distribution / Q000187:drug effects; D014162:Transfection; D000521:alpha-MSH / Q000378:metabolism / Q000494:pharmacology
chemical_list: D008024:Ligands; C470072:MC4R protein, human; D050505:Mutant Proteins; D044105:Receptor, Melanocortin, Type 4; D000521:alpha-MSH; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3