Dual expression of mouse and rat VRL-1 in the dorsal root ganglion derived cell line F-11 and biochemical analysis of VRL-1 after heterologous expression.
The vanilloid-like TRP-channel VRL-1 (TRPV2) is a nonselective cation channel expressed by primary sensory neurons and non-neuronal tissues [Caterina, M.J., Rosen, T.A., Tominaga, M., Brake, A.J and Julius, D. (1999) Nature 398, 436-441]. It is one of the six members of the vanilloid-like TRP-channel family which is now termed the TRPV family [Montell, G., Birnbaumer, L., Flockerzi, V., Bindels, R.J., Brutford, E.A., Caterina, M.J., Clapham, D.E., Harteneck, C., Heller, S., Julius, D., Kojima, I., Mori, Y., Penner, R., Prawitt, D., Scharenberg, A.M., Schultz, G., Shimizu, N. and Zhu, M.X. (2002) Mol. Cell 2, 229-231]. As it is a temperature-gated channel, VRL-1 appears to be functionally related to VR1. In contrast to VR1, VRL-1 is activated at a higher temperature threshold and it does not respond to capsaicin or protons. Here we describe the expression of VRL-1 in the rat dorsal root ganglion-derived cell line F-11, a hybridoma of mouse neuroblastoma (N18TG2) and rat dorsal root ganglion cells. We found by RT-PCR that F-11 cells express not only the rat VRL-1, but also its mouse orthologue in a single cell. The F-11 parental cell line N18TG2 also expressed murine VRL-1. Due to its neuronal character, the DRG-derived F-11 cell line provides an experimental system for the study of VRL-1 biochemistry. However, one has to be aware that both the mouse and the rat protein are expressed simultaneously. Furthermore we cloned VRL-1 from rat brain and analyzed its glycosylation and localization in comparison to the endogenously expressed protein in F-11 cells. In contrast to the endogenous VRL-1 the overexpressed protein is glycosylated. Similar to VR1 the glycosylation is N-linked as shown by an deglycosylation assay. Immunofluorescence analysis of the endogenous VRL-1 in F-11 cells gives only weak signals in the cytoplasm whereas the overexpressed rat VRL-1 appears mainly at the plasma membrane.
Jahnel
Ricarda
R
Arbeitsgruppe Neurochemie, Institut für Chemie-Biochemie, Freie Universität Berlin, Germany.
Bender
Olaf
O
Münter
Lisa M
LM
Dreger
Mathias
M
Gillen
Clemens
C
Hucho
Ferdinand
F
D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000378:metabolism; D002460:Cell Line; D018076:DNA, Complementary; D005727:Ganglia, Spinal / Q000166:cytology / Q000378:metabolism*; D006031:Glycosylation; D007473:Ion Channels; D051379:Mice; D008969:Molecular Sequence Data; D051381:Rats; D011955:Receptors, Drug / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D050916:TRPV Cation Channels
D018076:DNA, Complementary; D007473:Ion Channels; D011955:Receptors, Drug; D011994:Recombinant Proteins; D050916:TRPV Cation Channels; C476722:Trpv2 protein, rat