J Biol Chem. 2005 May 13;280(19):19393-400. Epub 2005 Mar 9.
MxA, a member of the dynamin superfamily, interacts with the ankyrin-like repeat domain of TRPC.
Lussier, M. P., Cayouette, S., Lepage, P. K., Bernier, C. L., Francoeur, N., St-Hilaire, M., Pinard, M., Boulay, G.,
["Department of Pharmacology, Universite de Sherbrooke, Sherbrooke, Quebec J1H 5N4, Canada."]
["Department of Pharmacology, Universite de Sherbrooke, Sherbrooke, Quebec J1H 5N4, Canada."]
Mammalian transient receptor potential canonical channels have been proposed as the molecular entities associated with calcium entry activity in nonexcitable cells. Amino acid sequence analyses of TRPCs revealed the presence of ankyrin-like repeat domains, one of the most common protein-protein interaction motifs. Using a yeast two-hybrid interaction assay, we found that the second ankyrin-like repeat domain of TRPC6 interacted with MxA, a member of the dynamin superfamily. Using a GST pull-down and co-immunoprecipitation assay, we showed that MxA interacted with TRPC1, -3, -4, -5, -6, and -7. Overexpression of MxA in HEK293T cells slightly increased endogenous calcium entry subsequent to stimulation of G(q) protein-coupled receptors or store depletion by thapsigargin. Co-expression of MxA with TRPC6 enhanced agonist-induced or OAG-induced calcium entry activity. GTP binding-defective MxA mutants had only a minor potentiating effect on OAG-induced TRPC6 activity. However, a MxA mutant that could bind GTP but that lacked GTPase activity produced the same effect as MxA on OAG-induced TRPC6 activity. These results indicated that MxA interacted specifically with the second ankyrin-like repeat domain of TRPCs and suggested that monomeric MxA regulated the activity of TRPC6 by a mechanism requiring GTP binding. Additional results showed that an increase in the endogenous expression of MxA, induced by a treatment with interferon alpha, regulated the activity of TRPC6. The study clearly identified MxA as a new regulatory protein involved in Ca2+ signaling.
PMID: 15757897

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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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MxA | Inference | Prediction | 15757897 | |||||
TRPC3 |
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MxA | Inference | Prediction | 15757897 | |||||
TRPC4 |
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MxA | Inference | Prediction | 15757897 | |||||
TRPC5 |
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MxA | Inference | Prediction | 15757897 | |||||
TRPC6 |
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MxA | Yeast two-hybrid | Human | 3-259 | Human | Fetal brain | cDNA library | 15757897 | |
TRPC7 |
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MxA | Inference | Prediction | 15757897 |
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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 | ||
TRPC3 |
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MxA | Fusion protein-pull down assay | Human | 1-201 | In vitro translation | Human | 572-662 | E. coli | 15757897 |
TRPC4 |
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MxA | Fusion protein-pull down assay | Human | 3-183 | In vitro translation | Human | 572-662 | E. coli | 15757897 |
TRPC5 |
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MxA | Fusion protein-pull down assay | Human | 1-183 | In vitro translation | Human | 572-662 | E. coli | 15757897 |
TRPC6 |
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MxA | Fusion protein-pull down assay | Human | 95-243 | In vitro translation | Human | 572-662 | E. coli | 15757897 |
TRPC7 |
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MxA | Fusion protein-pull down assay | Human | 3-195 | In vitro translation | Human | 572-662 | E. coli | 15757897 |
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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 | ||||
TRPC1 |
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MxA | Co-immunoprecipitation | HEK293T | Human | Full-length | Human | Full-length | 15757897 | |
TRPC3 |
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MxA | Co-immunoprecipitation | HEK293T | Human | Full-length | Human | Full-length | 15757897 | |
TRPC4 |
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MxA | Co-immunoprecipitation | HEK293T | Human | Full-length | Human | Full-length | 15757897 | |
TRPC6 |
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MxA | Co-immunoprecipitation | HEK293T | Human | Full-length | Human | Full-length | 15757897 |
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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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MxA | Fusion protein-pull down assay | Human | 1-201 | Human | 572-662 | 15757897 | ||
TRPC4 |
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MxA | Fusion protein-pull down assay | Human | 3-183 | Human | 572-662 | 15757897 | ||
TRPC5 |
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MxA | Fusion protein-pull down assay | Human | 1-183 | Human | 572-662 | 15757897 | ||
TRPC6 |
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MxA | Yeast two-hybrid | Human | 3-259 | Human | 542-662 | 15757897 | ||
TRPC6 |
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MxA | Fusion protein-pull down assay | Human | 95-243 | Human | 572-662 | 15757897 | ||
TRPC7 |
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MxA | Fusion protein-pull down assay | Human | 3-195 | Human | 572-662 | 15757897 |
(
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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 | ||||||
TRPC6 |
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MxA | Calcium measurement | Activation | 15757897 |
(
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click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
