CHARACTERIZATION OF MELANOCORTIN RECEPTOR SUBTYPE EXPRESSION IN MURINE ADIPOSE TISSUES AND IN THE 3T3-L1 CELL-LINE

It has been known for many years that adipocytes express high affinity -ACTH and alpha-melanocyte stimulating hormone (MSH) binding sites, an d that ACTH, alpha-MSH, and beta-lipotropin are potent lipolytic hormo nes. We show here that the adipocyte response to the melanocortin pept ides results from the expression of both the MC2 (ACTH) receptor as we ll as the newly discovered MC5 receptor. Using RT-PCR and Northern blo t hybridization, high levels of MC2 receptor messenger RNA (mRNA) were found in all adipose tissues examined in the mouse, whereas MC5 recep tor mRNA was found in a subset of these. Both receptor mRNAs were also found in the 3T3-L1 cell line but only after the cells had been induc ed to differentiate into adipocytes. This cell line was then used to c haracterize the pharmacological properties of the MC2 and MC5 receptor sites in situ. The MC2 receptor exhibits properties similar to the AC TH receptor characterized in adrenocortical cells, coupling to activat ion of adenylyl cyclase with an EC(50) of approximately 1 nM. An MSH b inding site characterized in these cells is presumably the MC5 recepto r, based on the observation that this is the only other melanocortin r eceptor mRNA detected in these cells. The MC5 receptor in the 3T3-L1 a dipocyte activated adenylyl cyclase in response to cr-MSH stimulation. Interestingly, Nle(4), D-Phe(7)-alpha-MSH (NDP-MSH), a commonly used synthetic alpha-MSH agonist, was a potent antagonist of the MC5 recept or expressed in the 3T3-L1 cell line. Although the agouti signaling pe ptide is a potent antagonist of NDP-MSH binding to the MC1 and MC4 mel anocortin receptors, agouti was unable to block NDP-MSH binding in the 3T3-L1 adipocyte.