title: Molecular cloning and pharmacological characterization of giant panda (Ailuropoda melanoleuca) melanocortin-4 receptor.
abstract: The melanocortin-4 receptor (MC4R) is critical in regulating mammalian food intake and energy expenditure. Giant panda (Ailuropoda melanoleuca), famous as the living fossil, is an endangered species endemic to China. We are interested in exploring the functions of the giant panda MC4R (amMC4R) in regulating energy homeostasis and report herein the molecular cloning and pharmacology of the amMC4R. Sequence analysis revealed that amMC4R was highly homologous (>88%) at nucleotide and amino acid sequences to several mammalian MC4Rs. Western blot revealed that the expression construct myc-amMC4R in pcDNA3.1 was successfully constructed and expressed in HEK293T cells. With human MC4R (hMC4R) as a control, pharmacological characteristics of amMC4R were analyzed with binding and signaling assays. Four agonists, including [Nle(4), D-Phe(7)]-α-melanocyte stimulating hormone (NDP-MSH), α- and β-MSH, and a small molecule agonist, THIQ, were used in binding and signaling assays. We showed that amMC4R bound NDP-MSH with the highest affinity followed by THIQ, α-MSH, and β-MSH, with the same ranking order as hMC4R. Treatment of HEK293T cells expressing amMC4R with different concentrations of agonists resulted in dose-dependent increase of intracellular cAMP levels, with similar EC50s for the four agonists. The results suggested that the cloned amMC4R encoded a functional MC4R. The availability of amMC4R and its binding and signaling properties will facilitate the investigation of amMC4R in regulating food intake and energy homeostasis.
authors.affiliation: Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu 225009, People's Republic of China. Electronic address: zqwang@yzu.edu.cn.
authors.forename: Zhi-Qiang
authors.initials: ZQ
authors.lastname: Wang
authors.affiliation: Department of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine, Auburn University, Auburn, AL 36849, United States.
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.affiliation: Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu 225009, People's Republic of China.
authors.forename: Lin
authors.initials: L
authors.lastname: Shi
authors.affiliation: Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu 225009, People's Republic of China.
authors.forename: Ji-Tian
authors.initials: JT
authors.lastname: Chai
authors.affiliation: Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu 225009, People's Republic of China.
authors.forename: Xin-Jun
authors.initials: XJ
authors.lastname: Zhang
authors.affiliation: Department of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine, Auburn University, Auburn, AL 36849, United States. Electronic address: taoyaxi@auburn.edu.
authors.forename: Ya-Xiong
authors.initials: YX
authors.lastname: Tao
mesh_terms: D000818:Animals; D003001:Cloning, Molecular; D057809:HEK293 Cells; D006801:Humans; D044105:Receptor, Melanocortin, Type 4 / Q000235:genetics*; D015398:Signal Transduction; D001503:Ursidae / Q000235:genetics*
chemical_list: D044105:Receptor, Melanocortin, Type 4
keywords: Binding; Cloning; Giant panda; Melanocortin-4 receptor; Signaling