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 ce lls by infection with recombinant baculovirus. The receptor bound SP w ith high affinity (K-D = 360 pM) and had a rank order of affinity of S P > neurokinin A. neurokinin B. Ligand activation of the receptor resu lted in an increase in both inositol lipid hydrolysis and intracellula r Ca2+ concentration ([Ca2+](i)). However, high-level expression of th e receptor, in the absence of ligand, was correlated with increased ba sal turnover of inositol lipids and an elevated rate of Ca2+ influx. T hese results demonstrate that the Sf9 cells provide a suitable environ ment for the high-level expression of a functionally active SPR. Two c arboxy-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-typ e receptor (K-D = 405 pM), and that of the Glu-tagged receptor was sli ghtly lower (K-D = 1,082 pM).The high-affinity SP binding site of all three receptors was sensitive to guanosine 5'-O-(3-thiotriphosphate) p retreatment. The maximal signal-transducing ability of the epitope-tag ged 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, 88-1 00%). These data show that heterologous expression in the baculovirus system results in high expression of functional wild-type and tagged r eceptors.