Domestic Animal Endocrinology 26 (2004) 75–86 Functional and phylogenetic analyses of a melanocortin-4 receptor mutation in domestic pigs  K.-S. Kim a,1 , J.M. Reecy a , W.H. Hsu b , L.L. Anderson a,b , M.F. Rothschild a,∗ a Department of Animal Science and Center for Integrated Animal Genomics, Iowa State University, Ames, IA 50011, USA b Department of Biomedical Sciences, Iowa State University, Ames, IA 50011, USA Abstract The melanocortin-4 receptor (MC4R), a G protein-coupled seven-transmembrane receptor, which is expressed in the brain, plays an important role in the control of mammalian energy homeostasis. A missense mutation (Asp298Asn) was identified in the porcine MC4R gene, which is associated with growth and food intake traits. The Asn298 mutation occurs within a highly conserved motif, NPLIY, of all members of G protein-coupled receptors; whereas, Asp298 is conserved in all five melanocortin receptor subtypes. Functional analysis of the porcine MC4R variant was performed with an in vitro gene expression system in 293 cells. Ligand binding (NDP-MSH) did not differ between Asp298 and Asn298 MC4R proteins. However, the Asn298 MC4R variant was unable to stimulate cAMP production in response to NDP-MSH stimulation; whereas, the Asp298 variant could stimulate cAMP accumulation. These results demonstrate that the Asp298 is required for normal MC4R signaling to the adenylyl cyclase. Sequencing of the MC4R gene of seven diverse genera within the Suiformes that include Hippopotamidae (hippos), Tayassuidae (peccaries) and Suidae (pigs), revealed 62 nucleotide variations in MC4R. Phylogenetic relationships of MC4R variations are consistent with those previously described from morphological and physiological data among the subfamilies of the Suiformes. These findings revealed that a single missense mutation (Asp298Asn) of aspartic acid (Asp) to asparagine (Asn) in MC4R gene decreased cAMP content and MC4R signaling, but with no difference in the ligand binding was associated with growth and feed intake traits in domestic pigs. © 2003 Elsevier Inc. All rights reserved. Keywords: Melanocortin-4 receptor; Mutation; Domestic pigs  This research was presented in part at the 84th annual meeting of The Endocrine Society, San Francisco, CA, 2002, abstract P2-338. ∗ Corresponding author. Tel.: +1-515-294-6202; fax: +1-515-294-2401. E-mail address: mfrothsc@iastate.edu (M.F. Rothschild). 1 Present address: Department of Animal Science, Michigan State University, East Lansing, MI 48824, USA. 0739-7240/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.domaniend.2003.12.001