© 1999 Macmillan Magazines Ltd letters to nature 436 NATURE | VOL 398 | 1 APRIL 1999 | www.nature.com Xenopus embryo and oocyte microinjection. hWIF-1 was expressed from the pCS2 + expression vector. RNA synthesis and microinjection into Xenopus embryos have been described 15 . Armadillo stabilization assays. Drosophila clone-8 cells, seeded one day earlier and grown to 80% con¯uence, were incubated with control or Wg- containing conditioned medium from S2 cells. Before incubation with clone-8 cells, the S2 conditioned medium (0.4 ml) was pre-incubated for 25 min at 4 8C with 0.4 ml DMEM/F-12 medium from transfected or control 293 cells. After 3 h at 25 8C, clone-8 cells were collected, washed in 1 3 PBS, 5 mM EDTA, and lysed in 80 ml hypotonic buffer (10 mM Tris, pH 7.5, 0.2 mM MgCl 2 ) contain- ing protease inhibitors. After addition of 20 ml 1.25 M sucrose, a membrane- free cytoplasmic fraction was prepared by centrifugation at 100,000g for 30 min at 4 8C, resolved by SDS±PAGE, immunoblotted and analysed for Armadillo (mAb N2-7A1; ref. 16), actin (Amersham) and HSP-70 (Sigma). Solution binding assay for Wg and XWnt8±Myc. 200 ml 293-cell conditioned medium containing WIF-1±IgG, WD±IgG, or IgG (each adjusted by ultra®ltration to 60 nM) was incubated with protein A±Sepharose beads at 4 8C for 1 h, after which the beads were washed 3 times with PBS and then incubated with 400 ml Wg or XWnt8±Myc conditioned medium at 4 8C for 2 h. The beads were separated from unbound material by low-speed centrifugation and washed ®ve times with PBS. Co-precipitates were analysed by SDS±PAGE and immunoblotted with af®nity-puri®ed rabbit anti-Wg antibodies or anti- Myc mAb 9E10 (ref. 17). Quantitative binding of XWnt8±AP and hWIF-1. Conditioned medium (100 ml) containing WIF-1 (10 mg ml -1 ), WIF-1±IgG (5 mg ml -1 ), or IgG (4 mg ml -1 ) was used to coat 96-well plate at 4 8C overnight, followed by incubation at 4 8C for 4 h with 200 ml 2 mg ml -1 BSA in binding buffer (Hank's balanced salt, 20 mM HEPES, pH 7.0). 150 ml XWnt8±AP diluted in 2 mg ml -1 BSA in binding buffer was applied to each well and incubated at 4 8C for 24 h. After 5 washes with 200 ml each of binding buffer, bound XWnt8±AP was quanti®ed by measuring alkaline phosphatase activity spectrophotometrically. A plot of alkaline phosphatase activity, representing the concentration of bound XWnt8±AP relative to the total concentration of XWnt8, was ®tted to the simple binary binding model (A  B $ A×B). Received 17 December 1998; accepted 26 January 1999. 1. Wodarz, A. & Nusse, R. Mechanisms of Wnt signaling in development. Annu. Rev. Cell Dev. Biol. 14, 59±88 (1998). 2. Moon, R. T., Brown, J. D. & Torres, M. 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Piccolo, S., Sasai, Y., Lu, B. & DeRobertis, E. M. Dorsoventral patterning in Xenopus: inhibition of ventral signals by direct binding of chordin to BMP-4. Cell 86, 589±598 (1996). 9. Zimmerman, L. B., De Jesus-Escobar, J. M. & Harland, R. M. The Spemann organizer signal noggin binds and inactivates bone morphogenetic protein 4. Cell 86, 599±606 (1996). 10. Hsu, D. R., Economides,A. N., Wang, X., Eimon, P. M. & Harland, R. M. The Xenopus dorsalizing factor gremlin identi®es a novel family of secreted proteins that antagonize BMP activities. Mol. Cell 1, 673±683 (1998). 11. Cadigan, K. M., Fish, M. P., Rulifson, E. J. & Nusse, R. Wingless repression of Drosophila frizzled2 expression shapes the Wingless morphogen gradient. Cell 93, 767±777 (1998). 12. Finch, P. W. et al. Puri®cation and molecular cloning of a secreted frizzled-related antagonist of Wnt signaling. Proc. Natl Acad. Sci. USA 94, 6770±6775 (1997). 13. Aruffo, A., Stamenkovic, I., Melnick, M., Underhill, C. B. & Seed, B. CD44 is the principal cell surface receptor for hyaluronate. Cell 61, 1303±1313 (1990). 14. Rebagliati, M. R., Toyoma, R., Haffter, P. & Dawid, I. B. Cyclops encodes a nodal-related factor involved in midline signaling. Proc. Natl Acad. Sci. USA 95, 9932±9937 (1998). 15. He, X., Saint-Jeannet, J.-P., Woodgett, J. R., Varmus, H. E. & Dawid, I. B. Glycogen synthase kinase-3 and dorsoventral patterning in Xenopus embryos. Nature 374, 617±622 (1995). 16. Peifer, M., Orsulic, S., Sweeton, D. & Wieschaus, E. A role for the Drosophila segment polarity gene armadillo in cell adhesion and cytoskeletal integrity during oogenesis. Development 118, 1191±1207 (1993). 17. Evan, G. I., Lewis, G. K., Ramsay, G. & Bishop, M. J. Isolation of monoclonal antibodies speci®c for human c-myc proto-oncogene product. Mol. Cell. Biol. 5, 3610±3616 (1985). Acknowledgements. We thank R. Moon for Xwnt8-Myc cDNA, J. Flanagan for the alkaline phosphatase plasmid, J. Corden for the mouse RNA polymerase II clone, B. Appel, L. Roman and D. Grunwald for cDNA libraries, and P. Bhanot and I. Munoz-Sanjuan for comments on the manuscript. Supported by the Howard Hughes Medical Institute (J.-C.H., A.R., P.M.S., C.H.S., R.N., J.N.). Correspondence and requests for materials should be addressed to J.N. (e-mail: jnathans@jhmi.edu). Genbank accession numbers for WIF-1 are: human, AF122922; mouse, AF122923; Xenopus, AF122924; zebra®sh, AF122925. A capsaicin-receptor homologue with a high threshold for noxious heat Michael J. Caterina*, Tobias A. Rosen*², Makoto Tominaga*², Anthony J. Brake* & David Julius* * Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California 94143-0450, USA ² These authors contributed equally to this work ......................................................................................................................... Pain-producing heat is detected by several classes of nociceptive sensory neuron that differ in their thermal response thresholds 1±3 . The cloned capsaicin receptor, also known as the vanilloid receptor subtype 1 (VR1), is a heat-gated ion channel that has been proposed to mediate responses of small-diameter sensory neurons to moderate (43 8C) thermal stimuli 4,5 . VR1 is also activated by protons, indicating that it may participate in the detection of noxious thermal and chemical stimuli in vivo. Here we identify a structurally related receptor, VRL-1, that does not respond to capsaicin, acid or moderate heat. Instead, VRL-1 is activated by high temperatures, with a threshold of ,52 8C. Within sensory ganglia, VRL-1 is most prominently expressed by a subset of medium- to large-diameter neurons, making it a candidate receptor for transducing high-threshold heat responses in this class of cells. VRL-1 transcripts are not restricted to the sensory nervous system, indicating that this channel may be activated by stimuli other than heat. We propose that responses to noxious heat involve these related, but distinct, ion-channel subtypes that together detect a range of stimulus intensities. To identify new proteins involved in the detection of noxious stimuli by sensory neurons, we searched the GenBank database for sequences related to VR1. Most of the expressed sequence tag (EST) sequences identi®ed appeared to encode human and mouse ortho- logues of the same protein, which we named vanilloid-receptor-like protein 1 (VRL-1). Using this information we isolated full-length VRL-1 complementary DNAs from rat brain and the CCRF-CEM human myeloid cell line (Fig. 1). These clones encode proteins of 761 amino acids and 764 amino acids, respectively, which share 78.4% identity and 86.2% similarity with one another. By com- parison, rat and human VRL-1 are roughly 49% identical and 66% similar to rat VR1. Like VR1, VRL-1 is predicted to contain six transmembrane domains, a putative pore-loop region, a cytoplasmic amino termi- nus with three ankyrin-repeat domains, and a cytoplasmic carboxy terminus. This overall architecture is characteristic of a family of ion channels de®ned by the transient receptor potential (TRP) and TRP-like (TRPL) channels of the Drosophila phototransduction pathway and which is now known to include vertebrate and nematode homologues 6±8 . Of the TRP-related sequences reported so far, the one that resembles VR1 and VRL-1 most closely is that of the OSM-9 protein, which is required for osmosensation and the detection of some odorants in Caenorhabditis elegans 8 . Even OSM- 9, however, is only 23% identical to rat VR1 and 24% identical to rat VRL-1, indicating that these latter molecules may form a distinct subgroup within this growing family of proteins. VR1 is activated by capsaicin (the main pungent ingredient in `hot' chilli peppers) and by its very potent analogue, resiniferatoxin (from Euphorbia plants). These vanilloid compounds elicit pain by evoking non-selective cationic currents in small-diameter nocicep- tive neurons (nociceptors) 4,9±11 . Even at very high concentrations, however, neither capsaicin (Fig. 2a, d) nor resiniferatoxin (not shown) evoked currents in Xenopus oocytes or HEK293 human embryonic kidney cells expressing VRL-1. Thus, VRL-1 does not