Human 5-HT1A receptor expressed in insect cells activates endogenous G(o)-like G protein(s)

• Published in: The Journal of biological chemistry Vol. 269; no. 17; pp. 12954 - 12962
• Main Authors: Mulheron, J.G., Casañas, S.J., Arthur, J.M., Garnovskaya, M.N., Gettys, T.W., Raymond, J.R.
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 29.04.1994
• Subjects: Amino Acid Sequence; Anilides; Animals; Baculoviridae - genetics; Cells, Cultured; Cloning, Molecular; GTP-Binding Proteins - metabolism; Guanine Nucleotides - metabolism; Humans; Molecular Sequence Data; Moths; Receptors, Serotonin - genetics; Receptors, Serotonin - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(18)99968-0

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 [3H]8-hydroxy-2-(di-n-propylamino)-1,2,3,4-tetranaphthalene). Peak expression (approximately 150 fmol/mg of membrane protein) was attained approximately 72-96 h post-infection. 5-HT-increased covalent binding of [32P]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 Gs 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.