Functional Wild‐Type and Carboxy‐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 (K D =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 Ca 2+ concentration ([Ca 2+ ] 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 Ca 2+ 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 pM), and that of the Glu-tagged receptor was slightly lower (K D =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 ([Ca 2+ ] 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