CHARACTERIZATION OF ANTIBODIES TO THE RAT SUBSTANCE-P (NK-1) RECEPTORAND TO A CHIMERIC SUBSTANCE-P RECEPTOR EXPRESSED IN MAMMALIAN-CELLS

Antibodies to neuropeptide receptors can be used to localize and chara cterize the receptors in tissues and cell lines. Two strategies were u sed to study the rat substance P receptor (SPR, NK-1) by immunological methods. First, a polyclonal antiserum was raised by immunizing rabbi ts with a peptide corresponding to the 15 amino acid residues (KTMTESS S-FYSNMLA, SPR(393-407)) at the intracellular C-terminus of the rat SP R coupled to bovine thyroglobulin. An antiserum was obtained with a ti ter for half-maximal binding of I-125-SPR(393-497) of 1:70,000. Nonrad ioactive SPR(393-407) inhibited 50% of binding at a concentration of 1 0 pM. Binding of I-125-SPR(393-407) to the antiserum was also displace d in a parallel manner by membrane proteins from tissues expressing hi gh levels of the SPR (brain and submaxillary gland). Second, a chimeri c SPR construct of a hydrophilic Flag peptide (DYKDDDDK) genetically e ngineered in sequence with the extracellular N-terminus of rat SPR was generated by polymerase chain reaction. The Flag-SPR chimera was expr essed in rat kidney epithelial cells (KNRK) and judged to be fully fun ctional, assessed by binding of I-125-substance P (apparent K-d of 5.6 3 nM) and calcium mobilization in response to substance P (EC(50) of 0 .66 nM). Antibodies to SPR(393-407) and the Flag peptide stained the p lasma membrane of KNRK cells expressing the native SPR or the Flag-SPR chimera. Staining was abolished by preincubation with SPR(393-407) or the Flag peptide. Cells transfected with vector alone were unstained. The SPR antiserum recognized a broad protein band on Western blots of membranes prepared from cells expressing SPR but not from cells trans fected with vector alone. The signal was quenched by preincubation of the antiserum with SPR(393-407). By immunohistochemistry, the SPR anti serum was found to bind to neurons in the dorsal horn of the rat spina l cord and to ganglion cells in the myenteric plexus of the rat ileum near substance P-immunoreactive nerve fibers. Staining was abolished b y preabsorption of the antiserum with SPR(393-407). These antibodies c an be used to localize the SPR in tissues and cells and to examine the function of the receptor in cell lines.