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.