Recognition of neurokinin 1 receptor (NK1-R): an antibody to a peptide sequence from the third extracellular region binds to brain NK1-R.
Substance P (SP) can produce cytokine-like responses by astrocytes and mononuclear cells. In an effort to identify neurokinin-1-receptors (NK1-R), an antibody to NK1-R was generated by using a linear peptide sequence from the deduced third extracellular region (ECR) corresponding to the seven transmembrane rat brain NK1-R. The ECR-3 peptide was coupled to keyhole-limpet hemocyanin and the antisera produced in rabbits was purified by binding to a peptide-affinity matrix. The specificity for the anti-peptide antibody was shown by its reactivity to the ECR-3 peptide by ELISA. The anti-ECR-3 peptide antibody could detect, by Western blot analysis of SDS-PAGE-separated rat brain membranes, a single band with an apparent molecular weight (MW) of 53-54 kDa. An affinity matrix made from the anti-ECR-3 antibody was used to isolate NK1-R from rat brain membranes which exhibited two products on SDS-PAGE with apparent MW of 54 and 44 kDa. The C6 astrocytes were shown to express NK1-R as determined by [125I]Bolten-Hunter SP binding to intact cells with a Kd = 0.32 nM. These C6 cells did not co-express either NK2-R or NK3-R when analyzed at the mRNA level. The anti-ECR-3 peptide antibody could inhibit [125I]Bolten-Hunter SP binding to intact C6 astrocytes and CHO cells expressing NK1-R by greater than 95% when compared to normal rabbit IgG which failed to inhibit radiolabeled SP binding. Thus, an antibody which recognizes surface determinants to the NK1-R could be generated upon immunization with an NK1-R peptide.
van Ginkel
F W
FW
Department of Microbiology, University of Alabama at Birmingham 35294-2170, USA.
Pascual
D W
DW
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D000915:Antibody Affinity; D001254:Astrocytoma; D001483:Base Sequence; D001666:Binding Sites, Antibody / Q000276:immunology*; D001667:Binding, Competitive / Q000276:immunology; D015153:Blotting, Western; D001923:Brain Chemistry / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D007457:Iodine Radioisotopes; D008969:Molecular Sequence Data; D015288:Neurokinin A / Q000737:chemistry / Q000276:immunology; D010446:Peptide Fragments / Q000276:immunology; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000032:analysis; D011817:Rabbits; D051381:Rats; D018040:Receptors, Neurokinin-1 / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D013373:Substance P / Q000737:chemistry / Q000276:immunology; D015320:Tachykinins / Q000737:chemistry / Q000276:immunology; D014407:Tumor Cells, Cultured / Q000737:chemistry / Q000276:immunology / Q000378:metabolism
D000906:Antibodies; D007457:Iodine Radioisotopes; D010446:Peptide Fragments; D012333:RNA, Messenger; D018040:Receptors, Neurokinin-1; D015320:Tachykinins; D013373:Substance P; D015288:Neurokinin A