Antibodies to the rat substance P receptor: production and characterization.
1. A protein A-rat substance P receptor (SPR) fusion protein was genetically engineered and used as an immunogen to raise a polyclonal antiserum to the SPR. The fusion protein was expressed in Escherichia coli driven by the heat-inducible lambda promoter (lambda Pr). 2. The fusion protein was purified using an IgG-Sepharose column, which specifically binds proteins containing the protein A moiety. The IgG fraction obtained after the immunization was cleaved to produce Fab fragments, which were subsequently purified using a fusion protein affinity column. The serum (anti-SPR Fab serum) was analyzed by fluorescence-activated cell sorting (FACS) and immunohistochemistry on both a constitutive cell line for the SPR (AR42J) and a cell line transfected with the SPR (KNRKSPR). 3. Specificity of the antiserum for SPR was confirmed by immunohistochemistry on cells using antiserum that had been preincubated with the protein A fusion protein (blocked). 4. The Ca2+ signal normally observed on stimulation of SPR with SP in AR42J cells and SP binding to KNRKSPR cells was shown to be diminished in the presence of anti-SPR Fab serum. SPR from both cell lines was immunoprecipitated using the anti-SPR Fab serum. The antiserum itself did not induce intracellular Ca2+ mobilization normally observed when cells were incubated with SP. 5. This specific SPR antiserum will be a useful tool to investigate further the mechanisms of SP/SPR interactions.
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology* / Q000302:isolation & purification; D000918:Antibody Specificity; D002118:Calcium / Q000494:pharmacology; D002460:Cell Line; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D008969:Molecular Sequence Data; D011817:Rabbits; D051381:Rats; D018040:Receptors, Neurokinin-1; D017981:Receptors, Neurotransmitter / Q000276:immunology* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000276:immunology; D013373:Substance P / Q000378:metabolism
D000906:Antibodies; D018040:Receptors, Neurokinin-1; D017981:Receptors, Neurotransmitter; D011993:Recombinant Fusion Proteins; D013373:Substance P; D002118:Calcium