The melanocortins are a family of peptide hormones derived from proopiomelanocortin, which is synthesized primarily in the pituitary, but also in the central nervous system, skin, placenta, and other tissues. Through binding to five melanocortin receptors (MCR), these hormones play diverse roles in physiological regulation. All the MCRs are members of the superfamily of G protein-coupled receptors (GPCRs) and are coupled to Gs. Thus, their activation stimulates the cAMP second messenger signal transduction pathway. Melanocortin-4 receptor (MC4R) has important roles in weight regulation, sexual function, and inflammation.^1,2 Mice deficient in MC4R have increased lipid deposition associated with elevated adiposity,³ while mutations in MC4R in humans are associated with early onset or severe obesity.^4,5 Mice treated with a selective MC4R agonist have increased sexual function that is not observed in MC4R-null mice⁶ and central injection of an MC4R antagonist impairs male sexual behavior.⁷ Selective MC4R activation has been linked with the suppression of fever and reduced production of inflammatory mediators.² These results and others point to the need to identify novel agonists and antagonists for MC4R, both to further elucidate the function of this receptor and to selectively regulate different aspects of its function in vivo. STEP™ (Surface Transfection and Expression Protocol) is a novel, patented, solid phase transient transfection technology that overcomes many of the disadvantages of other transfection approaches. STEP™ is exclusively licensed to Originus, Inc. from the University of Michigan (US patents 6897067, 6902933 and 7056741). In this method, a proprietary STEP™ transfection complex containing DNA and an optimized mixture of native and recombinant proteins is evenly applied and processed on the culture surface of multi-well plates. Adherent cells, supplied by the user, are applied directly to the wells of the plate and allowed to grow over the STEP™ coating. Using this method, the probability of receptor-mediated endocytosis of the STEP™ DNA complex increases dramatically compared to solution-phase transfection, enhancing both the transfection efficiency and the co-transfection efficiency for multiple plasmids. Cayman’s Melanocortin 4 Receptor STEP™ Reporter Assay Kit (Luminescence) consists of a 96-well plate coated with both MC4R and Secretory Alkaline Phosphatase (SEAP) reporter constructs (MC4R STEP™ Plate). Cells grown on the STEP™ complex will express MC4R at the cell surface. Binding of agonists to MC4R initiates a signal transduction cascade resulting in expression of SEAP which is secreted into the cell culture media. Aliquots of media are removed at timed intervals, beginning at approximately six hours, and SEAP activity is measured following addition of a luminescence-based alkaline phosphatases substrate provided in the kit. The kit is simple to use and can be easily adapted to high throughput screening for therapeutic compounds regulating the activation of MC4R. A MC4R agonist, α-MSH (α-melanocyte-stimulating hormone), is included in the kit for use as a positive control. The kit provides sufficient reagent to measure SEAP activity at three time points, using the black plates included.

1 Wikberg, J.E.S., Mutulis, F. Targeting melanocortin receptors: An approach to treat weight disorders and sexual dysfunction. Nat Rev Drug Discov 7 307-323 (2008).

2 Lasaga, M., Debeljuk, L., Durand, D., et al. Role of α-melanocyte stimulating hormone and melanocortin 4 receptor in brain inflammation. Peptides 29 1825-1835 (2008).

3 Zhang, Y., Kilroy, G.E., Henagan, T.M., et al. Targeted deletion of melanocortin receptor subtypes 3 and 4, but not CART, alters nutrient partitioning and compromises behavioral and metabolic responses to leptin. FASEB J 19 1482-1491 (2005).

4 MacKenzie, R.G. Obesity-associated mutations in the human melanocortin-4 receptor gene. Peptides 27 395-403 (2006).

5 Govaerts, C., Srinivasan, S., Shapiro, A., et al. Obesity-associated mutations in the melanocortin 4 receptor provide novel insights into its function. Peptides 26 1909-1919 (2005).

6 Van der Ploeg, L.H.T., Martin, W.J., Howard, A.D., et al. A role for the melanocortin 4 receptor in sexual function. Proc Natl Acad Sci USA 99(17) 11381-11386 (2002).

7 Caquineau, C., Leng, G., Guan, X.M.M., et al. Effects of α-melanocyte-stimulating hormone on magnocellular oxytocin neurones and their activation at intromission in male rats. J Neuroendocrinol 18 685-691 (2006).

Govaerts, C., Srinivasan, S., Shapiro, A., et al. Obesity-associated mutations in the melanocortin 4 receptor provide novel insights into its function. Peptides 26 1909-1919 (2005).

MacKenzie, R.G. Obesity-associated mutations in the human melanocortin-4 receptor gene. Peptides 27 395-403 (2006).

Caquineau, C., Leng, G., Guan, X.M.M., et al. Effects of α-melanocyte-stimulating hormone on magnocellular oxytocin neurones and their activation at intromission in male rats. J Neuroendocrinol 18 685-691 (2006).

Lasaga, M., Debeljuk, L., Durand, D., et al. Role of α-melanocyte stimulating hormone and melanocortin 4 receptor in brain inflammation. Peptides 29 1825-1835 (2008).

Zhang, Y., Kilroy, G.E., Henagan, T.M., et al. Targeted deletion of melanocortin receptor subtypes 3 and 4, but not CART, alters nutrient partitioning and compromises behavioral and metabolic responses to leptin. FASEB J 19 1482-1491 (2005).

Van der Ploeg, L.H.T., Martin, W.J., Howard, A.D., et al. A role for the melanocortin 4 receptor in sexual function. Proc Natl Acad Sci USA 99(17) 11381-11386 (2002).

Wikberg, J.E.S., Mutulis, F. Targeting melanocortin receptors: An approach to treat weight disorders and sexual dysfunction. Nat Rev Drug Discov 7 307-323 (2008).

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