Association of Fyn and Lyn with the proline-rich domain of glycoprotein VI regulates intracellular signaling.
The glycoprotein VI (GPVI)-Fc receptor (FcR) gamma-chain complex, a key activatory receptor for collagen on platelet surface membranes, is constitutively associated with the Src family kinases Fyn and Lyn. Molecular cloning of GPVI has revealed the presence of a proline-rich domain in the sequence of GPVI cytoplasmic tail which has the consensus for interaction with the Src homology 3 (SH3) domains of Fyn and Lyn. A series of in vitro experiments demonstrated the ability of the SH3 domains of both Src kinases to bind the proline-rich domain of GPVI. Furthermore, depletion of the proline-rich domain in GPVI (Pro(-)-GPVI) prevented binding of Fyn and Lyn and markedly reduced phosphorylation of FcR gamma-chain in transiently transfected COS-7 cells, but did not affect the association of the gamma-chain with GPVI. Jurkat cells stably transfected with wild type GPVI show robust increases in tyrosine phosphorylation and intracellular Ca2+ in response to the snake venom convulxin that targets GPVI. Importantly, convulxin is not able to activate cells transfected with Pro(-)-GPVI, even though the association with the immunoreceptor tyrosine-based activation motif-containing chains is maintained. These findings demonstrate that the proline-rich domain of GPVI mediates the association with Fyn/Lyn via their SH3 domain and that this interaction initiates activation signals through GPVI.
Suzuki-Inoue
Katsue
K
Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, United Kingdom. katsue.inoue@pharm.ox.ac.uk
Tulasne
David
D
Shen
Yang
Y
Bori-Sanz
Teresa
T
Inoue
Osamu
O
Jung
Stephanie M
SM
Moroi
Masaaki
M
Andrews
Robert K
RK
Berndt
Michael C
MC
Watson
Steve P
SP
D000818:Animals; D001792:Blood Platelets / Q000378:metabolism; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005434:Flow Cytometry; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry*; D011233:Precipitin Tests; D011392:Proline / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051076:Proto-Oncogene Proteins c-fyn; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D018909:src Homology Domains; D019061:src-Family Kinases / Q000378:metabolism*
D018076:DNA, Complementary; D008024:Ligands; D010980:Platelet Membrane Glycoproteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; C098341:platelet membrane glycoprotein VI; D011392:Proline; D005982:Glutathione Transferase; C494957:FYN protein, human; D051076:Proto-Oncogene Proteins c-fyn; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases; D002118:Calcium