Molecular cloning of a resiniferatoxin-binding protein.
Capsaicin and resiniferatoxin are neurotoxins which act on a sensory neuron membrane-associated receptor. In order to identify sensory neuron capsaicin binding proteins, expressed fusion proteins encoded by a directionally-cloned rat neonatal dorsal root ganglion library in lambda Zap-II were photoaffinity-labelled with the potent resiniferatoxin and capsaicin-like agonist resiniferanol-9,13,14-orthophenylacetate-20-(3-azido, 4-methoxyphenyl) acetate. Four clones encoding possible binding proteins were detected 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 encode a putative 235 amino acid protein of molecular weight 26,000 which we named RBP-26. In vitro translation of transcribed cRNA resulted in the synthesis of radiolabelled protein of the predicted molecular weight. In situ hybridisation showed that the mRNA encoding this protein was present in sensory neuron cell bodies. Both expressed bacterial fusion proteins and cytoplasmic fractions from COS cells transfected with an expression vector encoding RTX-42 showed [3H]resiniferatoxin binding activity (IC50 approximately 10 nM). RBP-26 is expressed in non-neuronal 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 established.
Ninkina
N N
NN
Sandoz Institute for Medical Research, London, UK.
Willoughby
J J
JJ
Beech
M M
MM
Coote
P R
PR
Wood
J N
JN
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D005727:Ganglia, Spinal / Q000378:metabolism; D015723:Gene Library; D005815:Genetic Code; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011956:Receptors, Cell Surface / Q000235:genetics*; D012680:Sensitivity and Specificity
D009419:Nerve Tissue Proteins; C087847:Phax protein, rat; D011956:Receptors, Cell Surface