Proc Natl Acad Sci U S A. 2006 Jan 10;103(2):335-40. Epub 2006 Jan 3.
Role of Src in C3 transient receptor potential channel function and evidence for a heterogeneous makeup of receptor- and store-operated Ca2+ entry channels.
Kawasaki, B. T., Liao, Y., Birnbaumer, L.,
["Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA."]
["Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA."]
Receptor-operated Ca2+ entry (ROCE) and store-operated Ca2+ entry (SOCE) are known to be inhibited by tyrosine kinase inhibitors and activation of C-type transient receptor potential channel (TRPC) isoform 3 (TRPC3), a cation channel thought to be involved in SOCE and/or ROCE, was recently shown to depend on src tyrosine kinase activity. What is not known is the step at which src acts on TRPC3 and whether the role for tyrosine kinases in ROCE or SOCE is a general phenomenon. Using in vitro and in cell protein-protein interaction assays we now report that src phosphorylates TRPC3 at Y226 and that formation of phospho-Y226 is essential for TRPC3 activation. This requirement is unique for TRPC3 because (i) mutation of the cognate tyrosines of the closely related TRPC6 and TRPC7 had no effect; (ii) TRPC6 and TRPC7 were activated in src-, yes-, and fyn-deficient cells; and (iii) src, but not yes or fyn, rescued TRPC3 activation in src-, yes-, and fyn-deficient cells. The Src homology 2 domain of src was found to interact with either the N or the C termini of all TRPCs, suggesting that other tyrosine kinases may play a role in ion fluxes mediated by TRPCs other than TRPC3. A side-by-side comparison of the effects of genistein (a general tyrosine kinase inhibitor) on endogenous ROCE and SOCE in mouse fibroblasts, HEK and COS-7 cells, and ROCE in HEK cells mediated by TRPC3, TRPC6, TRPC7, and TRPC5 showed differences that argue for ROCE and SOCE channels to be heterogeneous.
PMID: 16407161

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Screening
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Experimental screening | Non-experimental screening | Reference | ||||||||
TRP channel construct | Interactor source | |||||||||
TRP channel | Interactor | Method | Species | Region | Species | Organ/tissue | Sample type | |||
TRPC1 |
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SRC | Inference | Prediction | 16407161 | |||||
TRPC2 |
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SRC | Inference | Prediction | 16407161 | |||||
TRPC3 |
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SRC | Inference | Prediction | 16407161 | |||||
TRPC5 |
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SRC | Inference | Prediction | 16407161 | |||||
TRPC6 |
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SRC | Inference | Prediction | 16407161 |
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)

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Validation: In vitro validation
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Assay with recombinant proteins | Reference | |||||||||
TRP channel construct | Interactor construct | |||||||||
TRP channel | Interactor | Method | Species | Region | Expression system | Species | Region | Expression system | ||
TRPC1 |
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SRC | Fusion protein-pull down assay | Mouse | 1-341 | In vitro translation | Chicken | Full-length | E. coli | 16407161 |
TRPC2 |
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SRC | Fusion protein-pull down assay | Mouse | 1-616 | In vitro translation | Chicken | Full-length | E. coli | 16407161 |
TRPC3 |
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SRC | Fusion protein-pull down assay | Mouse | 1-340 | In vitro translation | Chicken | Full-length | E. coli | 16407161 |
TRPC4 |
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SRC | Fusion protein-pull down assay | Mouse | 626-974 | In vitro translation | Chicken | Full-length | E. coli | 16407161 |
TRPC5 |
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SRC | Fusion protein-pull down assay | Mouse | 630-975 | In vitro translation | Chicken | Full-length | E. coli | 16407161 |
TRPC5 |
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SRC | Fusion protein-pull down assay | Mouse | 1-321 | In vitro translation | Chicken | Full-length | E. coli | 16407161 |
TRPC6 |
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SRC | Fusion protein-pull down assay | Mouse | 1-396 | In vitro translation | Chicken | Full-length | E. coli | 16407161 |
(
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)

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Validation: In vivo validation
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Assay with endogenous proteins | Assay with overexpressed proteins | Reference | ||||||||
Cell or tissue | Cell or tissue | TRP channel construct | Interactor construct | |||||||
TRP channel | Interactor | Method | Species | Region | Species | Region | ||||
TRPC3 |
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SRC | Co-immunoprecipitation | COS-7 | Mouse | Full-length | Chicken | Full-length | 16407161 |
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)

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Characterization
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Binding region mapping | Stoichiometry | Affinity (Kd) | Reference | |||||||
TRP channel | Interactor | |||||||||
TRP channel | Interactor | Method | Species | Region | Species | Region | ||||
TRPC3 |
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SRC | Co-immunoprecipitation | Mouse | 1-370 | Chicken | SH2 domain | 16407161 |
(
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)

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Functional consequence
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TRP channel | Interactor | Method | Post-translational modification | Subcellular trafficking | Activity | Reference | ||||||
TRPC3 |
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SRC | In vivo PTM assay | Phosphorylation (Tyr-226) | 16407161 | |||||||
TRPC3 |
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SRC | Calcium measurement | Activation | 16407161 |
(
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
