Molecular and pharmacological characterisation of the MSH-R alleles in Swiss cattle breeds

The coat colour in mammals is determined by the relative amounts of eumelanin (black/brown) and phaeomelanin (red/yellow), produced in melanocytes, which are controlled by melanocyte stimulating hormone receptor (MSH-R). Melanocyte stimulating hormone receptor is activated by alpha-melanocyte-stimulating hormone (alpha-MSH). Stimulated MSH-R activates adenylyl cyclase (AC), thereby increasing the amount of cyclic AMP in the cell, which activates the enzyme tyrosinase resulting in eumelanin synthesis. In this study the complete coding sequences of five alleles of the MSH-R gene found in Holstein, Red Holstein, Simmental, and Brown Swiss cattle were cloned into a mammalian expression vector and transfected into human embryonic kidney (HEK) 293 cells. The expressed receptors were analyzed for their ability to increase intracellular cAMP in response to stimulation by alpha-MSH. The recessive red allele (e) found in Red Holstein and Simmental and the dominant black allele (ED) found in Holstein were unresponsive to a wide range of alpha-MSH concentrations. Two alleles from Brown Swiss (E(d1), E(d2)) and one allele found in the Simmental breed (e(f)) responded to stimulation by alpha-MSH in a dose-dependent manner. When compared to E(d1) and E(d2), the cells transfected with the e(f) MSH-R allele, however, reached the corresponding intracellular cAMP concentrations at a 10-fold higher concentration of alpha-MSH. In conjunction with the mode of inheritance of coat colour, the results indicate that the e MSH-R allele is a non-functional receptor, E(D) is constitutively activated receptor, and E(d1) and E(d2) are hormonally activated receptors. The delay in e(f) MSH-R response may explain the similarity between the e and e(f) phenotypes.