Jg. Mulheron et al., HUMAN 5-HT1A RECEPTOR EXPRESSED IN INSECT CELLS ACTIVATES ENDOGENOUS G(O)-LIKE G-PROTEIN(S), The Journal of biological chemistry, 269(17), 1994, pp. 12954-12962
Insect cell expression systems are used to characterize signaling comp
onents such as G protein-coupled receptors. As such, one must know whe
ther endogenous G proteins couple to non-native receptors. We examined
G protein linkages after infection of Sporodoptera frugiperda (Sf9) c
ells with a baculovirus encoding the 5-HT1A receptor. Receptor express
ion was confirmed by immunoblot. Some of the receptors were functional
, showing guanine nucleotide-sensitive binding to the specific agonist
ligand [H-3]8-hydroxy-2-(di-n-propylamino)-1,2,3,4- tetranaphthalene)
. Peak expression (approximate to 150 fmol/mg of membrane protein) was
attained approximate to 72-96 h post-infection. 5-HT-increased covale
nt binding of [P-32]GTP-azidoanilide to a 40 kDa band, which was ident
ified as a G protein by nucleotide blocking, Mg2+ dependence, and immu
noblot and immunoprecipitation studies. The band comigrated with 1) pe
rtussis toxin substrate(s), and 2) a band recognized by two G(o alpha)
antisera and one common to heterotrimeric G protein alpha-subunits, b
ut not by sera specific for G(s alpha) or G(i alpha). Labeled species
could be precipitated with a G(o alpha) antiserum. 5-HT-increased labe
ling of the band was prevented by preincubation with pertussis toxin.
These studies suggest that the 5-HT1A receptor couples effectively to
native insect cell G(o) like proteins.