Functional Wild-Type and Carboxy-Terminal-Tagged Rat Substance P Receptors Expressed in Baculovirus-Infected Insect Sf9 Cells |
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Authors: | J. Schreurs,R. Yamamoto, J. Lyons, S. Munemitsu,L. Conroy, R. Clark,&dagger Y. Takeda,&dagger J. E. Krause, M. Innis |
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Affiliation: | Departments of Pharmacology and; Molecular Biology, Cetus/Chiron Corporation, Emeryville, California;and; Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri, U.S.A. |
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Abstract: | Abstract: 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 ( K D = 360 p M ) 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 ( K D = 405 p M ), and that of the Glu-tagged receptor was slightly lower ( K D = 1,082 p M ). 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. |
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Keywords: | Tachykinin Neurokinin Phosphatidylinositol Calcium flux Expression vector Heterologous expression Recombinant protein |
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