Am J Physiol Cell Physiol. 2007 May;292(5):C1746-58. Epub 2007 Jan 24.

Regulation of TRP channel TRPM2 by the tyrosine phosphatase PTPL1.External 2231691f894ba696de1310221b0a0dbbb31a7251e75115c265587c3d9d5f507c

Zhang, W., Tong, Q., Conrad, K., Wozney, J., Cheung, J. Y., Miller, B. A.,
["Department of Pediatrics, The Pennsylvania State University College of Medicine, Milton S. Hershey Medical Center, PO Box 850, Hershey, PA 17033, USA."]
TRPM2, a member of the transient receptor potential (TRP) superfamily, is a Ca(2+)-permeable channel, which mediates susceptibility to cell death following activation by oxidative stress, TNFalpha, or beta-amyloid peptide. We determined that TRPM2 is rapidly tyrosine phosphorylated after stimulation with H(2)O(2) or TNFalpha. Inhibition of tyrosine phosphorylation with the tyrosine kinase inhibitors genistein or PP2 significantly reduced the increase in [Ca(2+)](i) observed after H(2)O(2) or TNFalpha treatment in TRPM2-expressing cells, suggesting that phosphorylation is important in TRPM2 activation. Utilizing a TransSignal PDZ domain array blot to identify proteins which interact with TRPM2, we identified PTPL1 as a potential binding protein. PTPL1 is a widely expressed tyrosine phosphatase, which has a role in cell survival and tumorigenesis. Immunoprecipitation and glutathione-S-transferase pull-down assays confirmed that TRPM2 and PTPL1 interact. To examine the ability of PTPL1 to modulate phosphorylation or activation of TRPM2, PTPL1 was coexpressed with TRPM2 in human embryonic kidney-293T cells. This resulted in significantly reduced TRPM2 tyrosine phosphorylation, and inhibited the rise in [Ca(2+)](i) and the loss of cell viability, which follow H(2)O(2) or TNFalpha treatment. Consistent with these findings, reduction in endogenous PTPL1 expression with small interfering RNA resulted in increased TRPM2 tyrosine phosphorylation, a significantly greater rise in [Ca(2+)](i) following H(2)O(2) treatment, and enhanced susceptibility to H(2)O(2)-induced cell death. Endogenous TRPM2 and PTPL1 was associated in U937-ecoR cells, confirming the physiological relevance of this interaction. These data demonstrate that tyrosine phosphorylation of TRPM2 is important in its activation and function and that inhibition of TRPM2 tyrosine phosphorylation reduces Ca(2+) influx and protects cell viability. They also suggest that modulation of TRPM2 tyrosine phosphorylation is a mechanism through which PTPL1 may mediate resistance to cell death.
PMID: 17251321External 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
TRPM2 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 PTPL1 Inference Prediction 17251321
(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
TRPM2 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 PTPL1 Co-immunoprecipitation Human monocytic U937-ecoR cell 17251321
TRPM2 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 PTPL1 Co-immunoprecipitation HEK293T Human Full-length Human Full-length 17251321
(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
TRPM2 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 PTPL1 Fusion protein-pull down assay Human Not determined Human 1099-1199 17251321
TRPM2 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 PTPL1 Fusion protein-pull down assay Human Not determined Human 1888-1990 17251321
(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
TRPM2 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 PTPL1 In vivo PTM assay Dephosphorylation 17251321
TRPM2 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 PTPL1 Calcium measurement Inhibition 17251321
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
TRP / Interactor

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