MOLECULAR-CLONING OF A RESINIFERATOXIN-BINDING PROTEIN

Capsaicin and resiniferatoxin are neurotoxins which act on a sensory n euron membrane-associated receptor. In order to identify sensory neuro n capsaicin binding proteins, expressed fusion proteins encoded by a d irectionally-cloned rat neonatal dorsal root ganglion library in lambd a Zap-II were photoaffinity-labelled with the potent resiniferatoxin a nd capsaicin-like agonist 14-orthophenylacetate-20-(3-azido,4-methoxyp henyl) acetate. Four clones encoding possible binding proteins were de tected with rabbit anti-resiniferanotoxin antiserum and sequenced. Two clones were homologous and hybridised on Northern blots with a 1.6 kb transcript enriched in dorsal root ganglia, but also present in other non-neuronal tissues. The full-length sequence corresponding to this transcript (RTX-42) was verified using primer extension and found to e ncode a putative 235 amino acid protein of molecular weight 26,000 whi ch we named RBP-26. In vitro translation of transcribed cRNA resulted in the synthesis of radiolabelled protein of the predicted molecular w eight. In situ hybridisation showed that the mRNA encoding this protei n was present in sensory neuron cell bodies. Both expressed bacterial fusion proteins and cytoplasmic fractions from COS cells transfected w ith an expression vector encoding RTX-42 showed [H-3]resiniferatoxin b inding activity (IC50 similar to 10 nM). RBP-26 is expressed in non-ne uronal and capsaicin-insensitive neuronal tissues, and shows distinct binding characteristics from the resiniferatoxin binding site defined on DRG membranes. The functional role of RBP-26 thus remains to be est ablished.