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


OBJECTIVE
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.
Redondo
P C
PC
Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK.
Harper
A G S
AG
Harper
M T
MT
Brownlow
S L
SL
Rosado
J A
JA
Sage
S O
SO
D000185:Actinin / Q000378:metabolism*; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D015153:Blotting, Western; D020013:Calcium Signaling / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000378:metabolism; D046912:Multiprotein Complexes / Q000378:metabolism; D009363:Neoplasm Proteins / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D015539:Platelet Activation / Q000187:drug effects; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6 / Q000037:antagonists & inhibitors / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011804:Quinolines / Q000494:pharmacology; D000071737:Stromal Interaction Molecule 1; D050052:TRPC Cation Channels / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism; D013997:Time Factors; D014443:Tyrosine / Q000378:metabolism
D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D046912:Multiprotein Complexes; C512715:NSC-87877; D009363:Neoplasm Proteins; D011804:Quinolines; C103530:STIM1 protein, human; D000071737:Stromal Interaction Molecule 1; D050052:TRPC Cation Channels; C489571:TRPC4 ion channel; C491780:TRPC5 protein, human; C096351:transient receptor potential cation channel, subfamily C, member 1; D000185:Actinin; D019000:Phosphotyrosine; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D013917:Thrombin