Functional wild-type and carboxyl-terminal-tagged rat substance P receptors expressed in baculovirus-infected insect Sf9 cells.
The rat substance P (SP) receptor (SPR) was expressed in insect Sf9 cells by infection with recombinant baculovirus. The receptor bound SP with high affinity (KD = 360 pM) and had a rank order of affinity of SP > neurokinin A > neurokinin B. Ligand activation of the receptor resulted in an increase in both inositol lipid hydrolysis and intracellular Ca2+ concentration ([Ca2+]i). However, high-level expression of the receptor, in the absence of ligand, was correlated with increased basal turnover of inositol lipids and an elevated rate of Ca2+ influx. These results demonstrate that the Sf9 cells provide a suitable environment for the high-level expression of a functionally active SPR. Two carboxy-terminal epitope-tagged receptors (SPR-KT3 = SPR-TPPPEPET, COOH; SPR-Glu = SPR-EEEEYMPME, COOH) were also expressed. The affinity of the KT3-tagged receptor for ligand was similar to that of the wild-type receptor (KD = 405 pM), and that of the Glu-tagged receptor was slightly lower (KD = 1,082 pM). The high-affinity SP binding site of all three receptors was sensitive to guanosine 5'-O-(3-thiotriphosphate) pretreatment. The maximal signal-transducing ability of the epitope-tagged receptors was comparable to that of the wild-type receptor ([Ca2+]i rise as a percentage of wild-type: SPR-KT3, 80-100%; SPR-Glu, 88-100%). These data show that heterologous expression in the baculovirus system results in high expression of functional wild-type and tagged receptors.
Schreurs
J
J
Department of Pharmacology, Cetus/Chiron Corporation, Emeryville, California 94608-2916.
Yamamoto
R
R
Lyons
J
J
Munemitsu
S
S
Conroy
L
L
Clark
R
R
Takeda
Y
Y
Krause
J E
JE
Innis
M
M
D000818:Animals; D001424:Bacterial Infections / Q000378:metabolism*; D016367:Baculoviridae; D001483:Base Sequence; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D007313:Insecta / Q000166:cytology; D015335:Molecular Probes / Q000235:genetics; D008969:Molecular Sequence Data; D010716:Phosphatidylinositols / Q000378:metabolism; D011869:Radioligand Assay; D051381:Rats; D018040:Receptors, Neurokinin-1 / Q000235:genetics / Q000378:metabolism*; D016324:Sequence Tagged Sites; D015398:Signal Transduction
D015335:Molecular Probes; D010716:Phosphatidylinositols; D018040:Receptors, Neurokinin-1; D002118:Calcium