Functional wild-type and carboxyl-terminal-tagged rat substance P receptors expressed in baculovirus-infected insect Sf9 cells.

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 (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.

journal_name

J Neurochem

authors

Schreurs J,Yamamoto R,Lyons J,Munemitsu S,Conroy L,Clark R,Takeda Y,Krause JE,Innis M

doi

10.1046/j.1471-4159.1995.64041622.x

subject

Has Abstract

pub_date

1995-04-01 00:00:00

pages

1622-31

issue

4

eissn

0022-3042

issn

1471-4159

journal_volume

64

pub_type

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