EXPRESSION OF FUNCTIONAL MELANOCORTIN-1 RECEPTORS IN INSECT CELLS

We expressed epitope-tag,oed human melanocortin 1 receptor (MC1R) in i nsect cells using two different recombinant baculovirus constructs; on e of which encoded MC1R with an N-terminal Flag epitope and a C-termin al polyHis tag, while the other encoded the MC1R with a C-terminal Myc tag. The constructs were used to infect Sf9 insect cells. For both co nstructs, immunoblotting with tag-specific antibodies demonstrated the presence of the receptor in the infected cells. The infected Sf9 cell s were characterized by radioligand binding using [I-125][Nle(4), D-Ph e(7)]alpha-MSH. Both saturation and competition analysis, using alpha- , beta-, and gamma(1)-MSH on the tagged MC1R expressed in the insect c ells, gave binding constants and potency orders that were undistinguis hable from those obtained on MC1R expressed in COS cells. The expressi on level obtained (in the order of pmoles of binding sites per mg of p rotein) will now facilitate attempts to purify the receptor. This is t he first report that demonstrates a functional expression of recombina nt melanocortin receptor in nonmammalian cells. (C) 1996 Academic Pres s, Inc.