Phosphatidylinositol 3,4,5-trisphosphate regulates Ca(2+) entry via btk in platelets and megakaryocytes without increasing phospholipase C activity.
The role of phosphatidylinositol 3,4,5-trisphosphate (PI3,4,5P(3)) and Btk in signalling by the collagen receptor glycoprotein VI was investigated. PI3,4,5P(3) was increased in platelets from mice deficient in the SH2 domain-containing inositol 5-phosphatase (SHIP), in response to collagen related peptide (CRP). Tyrosine phosphorylation and activation of phospholipase Cgamma2 (PLCgamma2) were unaltered in SHIP(-/-) platelets, whereas Btk was heavily tyrosine phosphorylated under basal conditions and maximally phosphorylated by low concentrations of CRP. There was an increase in basal Ca(2+), maximal expression of P-selectin, and potentiation of Ca(2+) and aminophospholipid exposure to CRP in SHIP(-/-) platelets in the presence of Ca(2+) (1 mM). Microinjection of PI3,4, 5P(3) into megakaryocytes caused a 3-fold increase in Ca(2+) in response to CRP, which was absent in X-linked immunodeficiency (Xid) mice, which have a mutation in the PH domain of Btk. There was a corresponding partial reduction in the sustained level of intracellular Ca(2+) in response to CRP in Xid mice but no change in PLC activity. These results demonstrate a novel pathway of Ca(2+) entry that involves PI3,4,5P(3) and Btk, and which is independent of increased PLC activity.
Pasquet
J M
JM
Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, UK.
Quek
L
L
Stevens
C
C
Bobe
R
R
Huber
M
M
Duronio
V
V
Krystal
G
G
Watson
S P
SP
D000077329:Agammaglobulinaemia Tyrosine Kinase; D000818:Animals; D001692:Biological Transport; D001792:Blood Platelets / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D003029:Coagulants / Q000032:analysis; D008040:Genetic Linkage; D006801:Humans; D007153:Immunologic Deficiency Syndromes / Q000235:genetics; D008533:Megakaryocytes / Q000378:metabolism*; D051379:Mice; D019007:P-Selectin; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics; D010766:Phosphorylation; D010980:Platelet Membrane Glycoproteins / Q000819:agonists; D011498:Protein Precursors / Q000032:analysis; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D016511:Severe Combined Immunodeficiency; D010738:Type C Phospholipases / Q000378:metabolism*; D014960:X Chromosome / Q000235:genetics
D003029:Coagulants; D019007:P-Selectin; D018129:Phosphatidylinositol Phosphates; D010980:Platelet Membrane Glycoproteins; D011498:Protein Precursors; C060974:phosphatidylinositol 3,4,5-triphosphate; C098341:platelet membrane glycoprotein VI; D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase; C000625949:BTK protein, human; C000625951:Btk protein, mouse; D010744:Phosphoric Monoester Hydrolases; C098706:INPPL1 protein, human; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases; D010738:Type C Phospholipases; D002118:Calcium