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 (N K1-R), an antibody to NK1-R was generated by using a linear peptide se quence from the deduced third extracellular region (ECR) corresponding to the seven transmembrane rat brain NK1-R. The ECR-3 peptide was cou pled 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 W estern blot analysis of SDS-PAGE-separated rat brain membranes, a sing le band with an apparent molecular weight (MW) of 53-54 kDa. An affini ty 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 [I-125]Bolten-Hunter SP binding to intact cell s with a K-d = 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 anti body could inhibit [I-125]Bolten-Hunter SP binding to intact C6 astroc ytes 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 c ould be generated upon immunization with an NK1-R peptide.