The structure-activity relationship and signal transduction properties of the pro-opiomelanocortin (POMC)-derived gamma-MSH peptides in the GH3 cell line was compared with that described for the known melanocortin receptors (MCRs). Single alanine replacements showed that, unlike the classical MCRs, the His(5)-Phe(6)-Arg(7)-Trp(8) sequence in gamma2-MSH is not a core sequence for activating the gamma-MSH receptor in GH3 cells, whereas Met(3) is essential. gamma2-MSH increased binding of [35S]GTPgammaS to membrane preparations of GH3 cells. Blockade of protein kinase A abolished the [Ca(2+)](i) responses to gamma3-MSH, and low nanomolar doses of gamma3-MSH increased intracellular cAMP levels, which could be blocked by pertussis toxin (PTX). We conclude that the putative novel gamma-MSH receptor in GH3 cells is a GPCR, but with structure-activity and signal transduction features different from those of the classical MCRs.