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Journal of Neuroscience, Vol 14, 834-845, Copyright © 1994 by Society for Neuroscience
Characterization of antibodies to the rat substance P (NK-1) receptor and to a chimeric substance P receptor expressed in mammalian cells
SR Vigna, JJ Bowden, DM McDonald, J Fisher, A Okamoto, DC McVey, DG Payan and NW Bunnett
Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710.
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 Kd 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.
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