Characterization of antibodies to the rat substance P (NK-1) receptor and to a chimeric substance P receptor expressed in mammalian cells

Abstract

Antibodies to neuropeptide receptors can be used to localize and characterize the receptors in tissues and cell lines. Two strategies were used to study the rat substance P receptor (SPR, NK-1) by immunological methods. First, a polyclonal antiserum was raised by immunizing rabbits with a peptide corresponding to the 15 amino acid residues (KTMTESSSFYSNMLA, SPR393-407) at the intracellular C-terminus of the rat SPR coupled to bovine thyroglobulin. An antiserum was obtained with a titer for half- maximal binding of 125I-SPR393-407 of 1:70,000. Nonradioactive SPR393-407 inhibited 50% of binding at a concentration of 10 pM. Binding of 125I-SPR393-407 to the antiserum was also displaced in a parallel manner by membrane proteins from tissues expressing high levels of the SPR (brain and submaxillary gland). Second, a chimeric SPR construct of a hydrophilic Flag peptide (DYKDDDDK) genetically engineered in sequence with the extracellular N-terminus of rat SPR was generated by polymerase chain reaction. The Flag-SPR chimera was expressed in rat kidney epithelial cells (KNRK) and judged to be fully functional, assessed by binding of 125I- substance P (apparent K(d) of 5.63 nM) and calcium mobilization in response to substance P (EC50 of 0.66 nM). Antibodies to SPR393-407 and the Flag peptide stained the plasma membrane of KNRK cells expressing the native SPR or the Flag-SPR chimera. Staining was abolished by preincubation with SPR393-407 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 transfected with vector alone. The signal was quenched by preincubation of the antiserum with SPR393-407. By immunohistochemistry, the SPR antiserum was found to bind to neurons in the dorsal horn of the rat spinal cord and to ganglion cells in the myenteric plexus of the rat ileum near substance P-immunoreactive nerve fibers. Staining was abolished by preabsorption of the antiserum with SPR393-407. These antibodies can be used to localize the SPR in tissues and cells and to examine the function of the receptor in cell lines.