Agouti protein and
Agouti-related protein (Agrp) are paracrine-signaling molecules that normally regulate pigmentation and
body weight, respectively. These
proteins antagonize the effects of
alpha-melanocyte-stimulating hormone (
alpha-MSH) and other
melanocortins, and several alternatives have been proposed to explain their biochemical mechanisms of action. We have used a sensitive bioassay based on Xenopus melanophores to characterize pharmacologic properties of recombinant
Agouti protein, and have directly measured its cell-surface binding to mammalian cells by use of an
epitope-tagged form (HA-Agouti) that retains biologic activity. In melanophores,
Agouti protein has no effect in the absence of
alpha-MSH, but its action cannot be explained solely by inhibition of
alpha-MSH binding. In 293T cells, expression of the Mc1r confers a specific, high-affinity binding site for HA-Agouti. Binding is inhibited by
alpha-MSH, or by Agrp, which indicates that
alpha-MSH and
Agouti protein bind in a mutually exclusive way to the Mc1r, and that the similarity between
Agouti protein and Agrp includes their binding sites. The effects of Agouti and the Mc1r in vivo have been examined in a sensitized background provided by the chinchilla (Tyrc-ch) mutation, which uncovers a phenotypic difference between overexpression of Agouti in lethal yellow (Ay/a) mice and loss of Mc1r function in recessive yellow (Mc1re/Mc1re) mice. Double and triple mutant studies indicate that a functional Mc1r is required for the pigmentary effects of Agouti, and suggest that
Agouti protein can act as an agonist of the Mc1r in a way that differs from
alpha-MSH stimulation. These results resolve questions regarding the biochemical mechanism of
Agouti protein action, and provide evidence of a novel signaling mechanism whereby
alpha-MSH and
Agouti protein or Agrp function as independent
ligands that inhibit each other's binding and transduce opposite signals through a single receptor.