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Please use this identifier to cite or link to this item: http://hdl.handle.net/1959.13/39937
- The spectrin cytoskeleton influences the surface expression and activation of human transient receptor potential channel 4 channels
Odell, Adam F.;
van Helden, Dirk F.;
Scott, Judith L.
- The University of Newcastle. Faculty of Health, School of Biomedical Sciences and Pharmacy
- 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, αII- and βV-spectrin, were identified as binding partners. The interaction of hTRPC4 with αII-spectrin and βV-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 Ca²⁺/calmodulin- and IP₃R-binding region (CIRB domain). This region is deleted in the proposed δhTRPC4 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 αII-spectrin. Furthermore, depletion of αII-spectrin by small interference RNA reduces the basal surface expression of αhTRPC4 and prevents the enhanced membrane insertion in response to EGF application. Importantly, depletion of αII-spectrin did not affect the expression of the δ 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.
- Journal of Biological Chemistry Vol. 283, Issue 7, p. 4395-4407
- Publisher Link
- American Society for Biochemistry and Molecular Biology
transient receptor potential channel (TRPC);
epidermal growth factor (EGF);
- Resource Type
- journal article
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