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 [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.
Mulheron
J G
JG
Department of Medicine, Nephrology and Gastroenterology, Duke University Medical Center, Durham, North Carolina 27710.
Casañas
S J
SJ
Arthur
J M
JM
Garnovskaya
M N
MN
Gettys
T W
TW
Raymond
J R
JR
D000595:Amino Acid Sequence; D000813:Anilides; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002478:Cells, Cultured; D003001:Cloning, Molecular; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009036:Moths; D011985:Receptors, Serotonin / Q000235:genetics / Q000378:metabolism*
D000813:Anilides; D006150:Guanine Nucleotides; D011985:Receptors, Serotonin; D019204:GTP-Binding Proteins