Expression of the human 5-hydroxytryptamine1A receptor in Sf9 cells. Reconstitution of a coupled phenotype by co-expression of mammalian G protein subunits.
The possibility that Spodoptera frugiperda (Sf9) cells can provide an intact cell setting for reconstitution of the human 5-hydroxytryptamine1A (5-HT1A) receptor with mammalian G protein subunits was explored. The 5-HT1A receptor was found to assume an uncoupled phenotype when expressed alone in Sf9 cells at relatively high levels (5-34 pmol of receptor/mg of membrane protein), i.e. agonist-binding to the receptor was characterized by a relatively high Kd and an insensitivity to GTP. Co-expression of the receptor with members of the alpha i "family" together with various combinations of beta 1 and gamma subunits increased the affinity for agonists to that observed for the coupled form of receptor in mammalian cells, concomitant with conferrance of guanosine 5'-(beta,gamma-imino)triphosphate sensitivity. The agonists employed were [3H]8-hydroxy-N,N-dipropyl-2-aminotetralin ([3H]8-OH-DPAT) and [125I]R(+)-trans-8-hydroxy-2-[N-n-propyl-N-(3'-iodo-2'-propenyl) amino]tetralin ([125I]8-OH-PIPAT). The binding of an antagonist, [125I]4-(2'-methoxyphenyl)-1-[2'-[N-(2"- pyridinyl)-p-iodobenzamido]ethyl]piperazine ([125I]p-MPPI), was unaffected by co-expression of G protein subunits. Both alpha and beta gamma subunits were required for optimal coupling. No differences were evident among alpha i1, alpha i2, alpha i3, alpha o, and alpha z when expressed with beta 1 gamma 2 in this regard, nor among most permutations of beta 1 gamma subunits when expressed with alpha i1 (beta 1 gamma 2 approximately beta 1 gamma 3 approximately beta 1 gamma 5 > beta 1 gamma 1). Alpha s and alpha q expressed with beta 1 gamma 2 did not participate in coupling. These data support the conclusion that normal interactions between a mammalian receptor and a select array of G proteins can be established in intact Sf9 cells, and extend previous observations of 5-HT1A receptor coupling to G(o) and the pertussis toxin-insensitive G protein Gz.
Butkerait
P
P
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia 19104-6084, USA.
Zheng
Y
Y
Hallak
H
H
Graham
T E
TE
Miller
H A
HA
Burris
K D
KD
Molinoff
P B
PB
Manning
D R
DR
D017371:8-Hydroxy-2-(di-n-propylamino)tetralin / Q000378:metabolism; D000595:Amino Acid Sequence; D000631:Aminopyridines / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology; D010879:Piperazines / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011985:Receptors, Serotonin / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D044263:Receptors, Serotonin, 5-HT1; D011994:Recombinant Proteins / Q000096:biosynthesis; D015398:Signal Transduction; D018411:Spodoptera / Q000166:cytology
D000631:Aminopyridines; D010446:Peptide Fragments; D010879:Piperazines; D011985:Receptors, Serotonin; D044263:Receptors, Serotonin, 5-HT1; D011994:Recombinant Proteins; C087205:4-(2'-methoxyphenyl)-1-(2'-(N-(2''-pyridinyl)-4-iodobenzamido)ethyl)piperazine; D006165:Guanylyl Imidodiphosphate; D017371:8-Hydroxy-2-(di-n-propylamino)tetralin; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins