Molecular cloning and pharmacological characterization of porcine melanocortin-3 receptor

Abstract

Mouse gene targeting studies revealed that the melanocortin-3 receptor (MC3R) affected feeding efficiency and fat storage in mice. The functions of the MC3R in other mammalian species remain to be investigated. We are interested in exploring the functions of the porcine MC3R (pMC3R) in regulating fat storage because of the economical importance of swine industry. Although nucleotide sequences of MC3Rs from several species have been reported, pMC3R had not been cloned and sequenced. We reported herein the molecular cloning and pharmacological analysis of the pMC3R. Sequence analysis revealed that pMC3R was highly homologous (O80%) at nucleotide and amino acid sequences to human, rat, and mouse MC3Rs. With human MC3R (hMC3R) as a control, the binding and signaling properties of pMC3R were investigated using several agonists The results showed that pMC3R bound NDP-MSH with the highest affinity followed by D-Trp 8 -gMSH, g-, and a-MSH. The same ranking was also found for hMC3R, although pMC3R had two-to ninefold higher affinities for these ligands. Both pMC3R and hMC3R bound AgRP with high affinity. D-Trp 8 -g-MSH was the most potent agonist to stimulate cAMP generation followed by NDP-, a-, and g-MSH. This ranking was the same as that of hMC3R. The availability of pMC3R and its pharmacological characteristics will facilitate the investigation of pMC3R in regulating food intake and fat storage.

TRIENNIAL GROWTH SYMPOSIUM: Neural regulation of feed intake: Modification by hormones, fasting, and disease1,2

“…Short-term nutrient restriction as well as body condition do not correlate with changes in POMC gene expression (Henry et al, 2000;Adam et al, 2002;Archer et al, 2002), unless altered photoperiod was added to the experimental design Archer et al, 2004). The role of a-MSH is suggested as that of appetite inhibition and is mediated through actions on the melanocortin-3 receptor (MC3R) and MC4R, both cloned for sheep and pigs (Iqbal et al, 2001a: Fan et al. 2008a.…”

Functional characterization of nine novel naturally occurring human melanocortin-3 receptor mutations

“…After 1h incubation at 37 C, cells were then placed on ice to terminate the reaction, media were aspirated and intracellular cAMP were extracted with 0.5N percholoric acid containing 180µg/ml theophylline. The cAMP concentrations were measured by radioimmunoassay [13, 32]. All determinations were performed in triplicate and the experiment was performed at least three times.…”

Rescue of defective MC4R cell-surface expression and signaling by a novel pharmacoperone Ipsen 17

“…Mutations of human MC4R used in this study were created by site-directed mutagenesis methodology as described in our previous report (Fan et al 2008).…”

“…Human embryonic kidney (HEK) 293 cells were grown in an atmosphere of 5% CO 2 in DMEM containing 10 mM HEPES, 10% newborn calf serum, 100 units/ml penicillin, and 100 mg/ml streptomycin, as described previously (Fan et al 2008). For establishment of stably transfected cells, HEK 293 cells were seeded at a density of 5!10 7 cells/100-mm dish (Corning, Inc., Beijing, China) and transfected after 48 h using a calcium phosphate precipitation protocol as described previously (Chen & Okayama 1987).…”