J Thromb Haemost. 2007 Dec;5(12):2476-83. Epub 2007 Sep 24.

hTRPC1-associated alpha-actinin, and not hTRPC1 itself, is tyrosine phosphorylated during human platelet activation.External 2231691f894ba696de1310221b0a0dbbb31a7251e75115c265587c3d9d5f507c

Redondo, P. C., Harper, A. G., Harper, M. T., Brownlow, S. L., Rosado, J. A., Sage, S. O.,
["Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK."]
BACKGROUND: Canonical transient receptor potential channels (TRPCs), which are regulated by several processes, including tyrosine phosphorylation, are candidates for the conduction of store-operated Ca(2+) entry (SOCE). OBJECTIVES: To assess hTRPC phosphotyrosine content upon platelet stimulation. METHODS: A new protein complex immunological separation assay (ProCISA) was developed to allow assessment of isolated hTRPC tyrosine phosphorylation by Western blotting. RESULTS: Classical immunoprecipitation suggested that thrombin (Thr) evoked an initial decrease in hTRPC1 phosphotyrosine content, which reached a minimum at 1 s, and then increased again, exceeding basal levels after 3 min. However, TRPC isolation from protein complexes using ProCISA revealed that hTRPC1, 4 and 5 were not tyrosine phosphorylated at rest or after Thr stimulation. Stimulation with Thr for 3 min increased the phosphotyrosine content of alpha-actinin, which shows similar electrophoretic properties to hTRPCs and coimmunoprecipitates with hTRPC1. Thr-evoked alpha-actinin tyrosine phosphorylation was increased by inhibiting the alpha-actinin phosphatase, SHP-1, which enhanced phosphorylation of the TRPC complex and SOCE. Inhibition of tyrosine phosphorylation impaired the interaction between hTRPC1 and the intracellular Ca(2+) sensor STIM1. CONCLUSIONS: hTRPC1, 4 and 5 are not tyrosine phosphorylated during SOCE in human platelets although tyrosine phosphorylation is important for SOCE. The results obtained using ProCISA caution the use of classical immunoprecipitation for the determination of the tyrosine phosphorylation state of a given protein, where the presence of other proteins with similar electrophoretic mobilities may give misleading results.
PMID: 17892531External 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
TRPC1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ơ-actinin-1 Inference Prediction 17892531
(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
TRPC1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 ơ-actinin-1 Co-immunoprecipitation Human platelets 17892531
TRPC1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 STIM1 Co-immunoprecipitation Human platelets 17892531
TRPC1 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 TRPC4 Co-immunoprecipitation Human platelets 17892531
TRPC4 Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4 TRPC1 Co-immunoprecipitation Human platelets 17892531
(Link 2bd4d11adb659cddf58197a94e201f0a44c55d8d7cb427c624971b42e122c0a4: click the arrow icon to show interactions only between the corresponding TRP channel and the interactor)
TRP / Interactor

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