HUMAN 5-HT1A RECEPTOR EXPRESSED IN INSECT CELLS ACTIVATES ENDOGENOUS G(O)-LIKE G-PROTEIN(S)

Insect cell expression systems are used to characterize signaling components such as G protein-coupled receptors. As such, one must know whether endogenous G proteins couple to non-native receptors. We examined G protein linkages after infection of Sporodoptera frugiperda (Sf9) cells with a baculovirus encoding the 5-HT1A receptor. Receptor expression 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 covalent binding of [P-32]GTP-azidoanilide to a 40 kDa band, which was identified as a G protein by nucleotide blocking, Mg2+ dependence, and immunoblot and immunoprecipitation studies. The band comigrated with 1) pertussis toxin substrate(s), and 2) a band recognized by two G(o alpha) antisera and one common to heterotrimeric G protein alpha-subunits, but 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 labeling 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.