Translocation of calcium-permeable TRPV2 channel to the podosome: Its role in the regulation of podosome assembly.
The present study was conducted to investigate localization and function of TRPV2 channel in a mouse macrophage cell line, TtT/M87. We infected an adenovirus vector encoding TRPV2 tagged with c-Myc in the extracellular domain. Immunoreactivity of c-Myc epitope exposed to the cell surface formed a ring structure, which was colocalized with markers of the podosome, namely β-integrin, paxillin and Pyk2. The ring structure was also observed in TRPV2-GFP-expressing cells using total internal reflection fluorescent microscopy. Addition of formyl-Met-Leu-Phe (fMLP) increased the number of podosome and increased the intensity of the TRPV2 signal associated with the podosome. Measurement of subplasmalenmal free calcium concentration ([Ca(2+)](pm)) revealed that [Ca(2+)](pm) was elevated around the podosome. fMLP further increased [Ca(2+)](pm) in this region, which was abolished by a TRPV2 inhibitor ruthenium red. Phosphorylated Pyk2 was detected in fMLP-treated cells, and knockdown of TRPV2 reduced the expression of phospho-Pyk2. Introduction of dominant-negative Pyk2 or knockdown of TRPV2 increased the number of podosome. Conversely, elevation of [Ca(2+)](pm) by the addition of ionomycin reduced the number of podosome. These results indicate that TRPV2 is localized abundantly in the podosome and increases [Ca(2+)](pm) by the podosome. The elevation of [Ca(2+)](pm) is critical to regulate assembly of the podosome.
Department of Cell Biology, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Japan.
Masahiro
M
Nagasawa
Itaru
I
Kojima
D000256:Adenoviridae; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D051418:Focal Adhesion Kinase 2 / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D039641:Integrin beta Chains / Q000235:genetics / Q000378:metabolism; D008264:Macrophages / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D051419:Paxillin / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects / Q000235:genetics; D021381:Protein Transport / Q000187:drug effects / Q000235:genetics; D050916:TRPV Cation Channels / Q000235:genetics / Q000378:metabolism*
D015220:Calcium Channels; D039641:Integrin beta Chains; D051419:Paxillin; D050916:TRPV Cation Channels; C485717:Trpv2 protein, mouse; D009240:N-Formylmethionine Leucyl-Phenylalanine; D051418:Focal Adhesion Kinase 2; C496053:Ptk2b protein, mouse; D002118:Calcium