Expression of functional melanocortin 1 receptors in insect cells.
We expressed epitope-tagged human melanocortin 1 receptor (MC1R) in insect cells using two different recombinant baculovirus constructs; one of which encoded MC1R with an N-terminal Flag epitope and a C-terminal 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 constructs, immunoblotting with tag-specific antibodies demonstrated the presence of the receptor in the infected cells. The infected Sf9 cells were characterized by radioligand binding using [125I][Nle4,D-Phe7]alpha-MSH. Both saturation and competition analysis, using alpha-, beta-, and gamma 1-MSH on the tagged MC1R expressed in the insect cells, gave binding constants and potency orders that were undistinguishable from those obtained on MC1R expressed in COS cells. The expression level obtained (in the order of pmoles of binding sites per mg of protein) will now facilitate attempts to purify the receptor. This is the first report that demonstrates a functional expression of recombinant melanocortin receptor in nonmammalian cells.
Schiöth
H B
HB
Department of Pharmaceutical Pharmacology, Uppsala University, Sweden.
Kuusinen
A
A
Muceniece
R
R
Szardenings
M
M
Keinänen
K
K
Wikberg
J E
JE
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D006801:Humans; D008969:Molecular Sequence Data; D018043:Receptors, Corticotropin / Q000235:genetics* / Q000378:metabolism; D044101:Receptors, Melanocortin; D018411:Spodoptera
D018043:Receptors, Corticotropin; D044101:Receptors, Melanocortin