J Biol Chem. 2008 Feb 15;283(7):4395-407. Epub 2007 Nov 29.

The spectrin cytoskeleton influences the surface expression and activation of human transient receptor potential channel 4 channels.External 2231691f894ba696de1310221b0a0dbbb31a7251e75115c265587c3d9d5f507c

Odell, A. F., Van Helden, D. F., Scott, J. L.,
["School of Biomedical Sciences, Faculty of Health, University of Newcastle, Level 5, MSB, University Drive, New South Wales 2308, Australia. a.f.odell@leeds.ac.uk"]
Despite over a decade of research, only recently have the mechanisms governing transient receptor potential channel (TRPC) channel function begun to emerge, with an essential role for accessory proteins in this process. We previously identified a tyrosine phosphorylation event as critical in the plasma membrane translocation and activation of hTRPC4 channels following epidermal growth factor (EGF) receptor activation. To further characterize the signaling events underlying this process, a yeast-two hybrid screen was performed on the C terminus of hTRPC4. The intracellular C-terminal region from proline 686 to leucine 977 was used to screen a human brain cDNA library. Two members of the spectrin family, alphaII- and betaV-spectrin, were identified as binding partners. The interaction of hTRPC4 with alphaII-spectrin and betaV-spectrin was confirmed by glutathione S-transferase pulldown and co-immunoprecipitation experiments. Deletion analysis identified amino acids 730-758 of hTRPC4 as critical for the interaction with this region located within a coiled-coil domain, juxtaposing the Ca(2+)/calmodulin- and IP(3)R-binding region (CIRB domain). This region is deleted in the proposed deltahTRPC4 splice variant form, which failed to undergo both EGF-induced membrane insertion and activation, providing a genetic mechanism for regulating channel activity. We also demonstrate that the exocytotic insertion and activation of hTRPC4 following EGF application is accompanied by dissociation from alphaII-spectrin. Furthermore, depletion of alphaII-spectrin by small interference RNA reduces the basal surface expression of alphahTRPC4 and prevents the enhanced membrane insertion in response to EGF application. Importantly, depletion of alphaII-spectrin did not affect the expression of the delta variant. Taken together, these results demonstrate that a direct interaction between hTRPC4 and the spectrin cytoskeleton is involved in the regulation of hTRPC4 surface expression and activation.
PMID: 18048348External 2231691f894ba696de1310221b0a0dbbb31a7251e75115c265587c3d9d5f507c
Screening Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
  Experimental screening Non-experimental screening Reference
TRP channel construct Interactor source
TRP channel Interactor Method Species Region Species Organ/tissue Sample type
TRPC4 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ƢV-spectrin Yeast two-hybrid Human 686-977 Human Brain cDNA library 18048348
TRPC4 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 NHERF1 Yeast two-hybrid Human 686-977 Human Brain cDNA library 18048348
TRPC4 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ơII-spectrin Yeast two-hybrid Human 686-977 Human Brain cDNA library 18048348
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
Validation: In vitro validation Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
  Assay with recombinant proteins Reference
TRP channel construct Interactor construct
TRP channel Interactor Method Species Region Expression system Species Region Expression system
TRPC4 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ƢV-spectrin Fusion protein-pull down assay Human Full-length HEK293 lysates Human 3232-3674 E. coli 18048348
TRPC4 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ơII-spectrin Fusion protein-pull down assay Human Full-length HEK293 lysates Human 1998-2472 E. coli 18048348
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
Validation: In vivo validation Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
  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
TRPC4 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 FYN Co-immunoprecipitation Rat brain microsome 18048348
TRPC4 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 NHERF1 Co-immunoprecipitation HEK293 Mouse Full-length Not used 18048348
TRPC4 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ơII-spectrin Co-immunoprecipitation HEK293 Mouse Full-length Not used 18048348
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
Characterization Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
  Binding region mapping Stoichiometry Affinity (Kd) Reference
TRP channel Interactor
TRP channel Interactor Method Species Region Species Region
TRPC4 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ƢV-spectrin Yeast two-hybrid Human 686-977 Human 3232-3674 18048348
TRPC4 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ƢV-spectrin Fusion protein-pull down assay Human 686-977 Human 3232-3674 18048348
TRPC4 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ơII-spectrin Yeast two-hybrid Human 686-859 Human 1998-2472 18048348
TRPC4 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ơII-spectrin Fusion protein-pull down assay Human 730-758 Human 2202-2313 18048348
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
Functional consequence Toggle 893349bafcc528f8346c51dc3420151d67b0126b2c122dd1017121c03fa0f69b
TRP channel Interactor Method Post-translational modification Subcellular trafficking Activity Reference
TRPC4 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ơII-spectrin Cell surface biotinylation Increase in plasma membrane level 18048348
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
TRP / Interactor

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