Association of Fyn and Lyn with the Proline-rich Domain of Glycoprotein VI Regulates Intracellular Signaling

Abstract

The glycoprotein VI (GPVI)-Fc receptor (FcR)␥The adhesion and activation of platelets by subendothelial collagen fibers initiates aggregate formation at sites of vessel damage. Glycoprotein (GP) 1 VI plays a critical role in the activatory events induced by collagen as shown by the lack of response to collagen in human and mice platelets deficient in the glycoprotein (1, 2). A collagen-related peptide and a snake venom toxin, convulxin, interact specifically with GPVI and mimic many of the responses to collagen (3-5).Because of the physiological importance of GPVI, the mechanism of the GPVI-mediated signaling system has been extensively investigated (6 -8). GPVI is present as a complex with Fc receptor (FcR) ␥-chain in the platelet membrane (8 -10). The Src family kinases, Fyn and Lyn, are associated with GPVIFcR ␥-chain complex in platelets and initiate activation through phosphorylation of the immunoreceptor tyrosinebased activation motif (ITAM) in the FcR ␥-chain leading to binding and activation of the tyrosine kinase Syk. A series of adapter molecules including LAT and SLP76 orchestrate a carefully regulated signaling network leading to activation of PLC␥2, phosphoinositol 3-kinase, and small molecular weight G proteins, leading to platelet activation (6, 7).The cloning of GPVI (11-14) has revealed it to be a member of the immunoglobulin (Ig) superfamily, showing close homology to Fc␣RI. GPVI has a charged arginine residue in its transmembrane domain. This arginine, together with elements within the cytoplasmic domain, is crucial for association of GPVI with FcR ␥-chain and GPVI-mediated signal transduction (15,16). In addition, the cytoplasmic tail of GPVI has a cluster of 6 proline residues of unknown function (11)(12)(13)(14). This sequence of GPVI, RPLPPLPPLP, contains a consensus Src family kinase-SH3 recognition motif (RPLPPLP) (17,18), and provides a potential site of interaction with Fyn and Lyn via their SH3 domains.In this study, we demonstrate that depletion of the prolinerich domain in GPVI abolishes the association with Fyn and Lyn and prevents tyrosine phosphorylation of FcR ␥-chain and downstream responses. From these findings, we suggest that Fyn/Lyn directly bind the proline-rich domain of GPVI and that this association is necessary for phosphorylation of the ITAM and downstream signals.

Delineation of the Region in the Glycoprotein VI Tail Required for Association with the Fc Receptor γ-Chain

“…This demonstrates that the proline-rich sequence in the GPVI cytosolic tail is required for optimal signaling by convulxin in transfected RBL cells. This is consistent with our previous observation of a role for this region of GPVI in mediating activation through the recruitment of the Src kinases Fyn and Lyn to GPVI⅐FcR␥-chain complex (15).…”

“…The oligonucleotide sequence used for ⌬316GPVI was 5Ј-ctgccgcccctcccgtctagagactacaag-3Ј; for ⌬309GPVI, 5Ј-gtgcagaggccgctttctagagactacaagg-3Ј; for ⌬294GPVI, 5Ј-gactggcacagccggtctagagac-tacaagg-3Ј; for ⌬basicGPVI, 5Ј-gaggactggcacagccggcacaggggcagg-3Ј; for ⌬juxtaGPVI, 5Ј-gcggggtttctggcaaggaggcgcctgcgg-3Ј; for E289A/D290A GPVI, 5Ј-gggtttctggcagcggcctg gcacagccgg-3Ј; for W291A/H292A/S293A, 5Ј-gcagaggacgccgcagcacggaggaagcgc-3Ј; for W291A, 5Ј-gcagaggacgcgcacagccggaggaagcgc-3Ј; for H292AGPVI, 5Ј-ggcagaggactgggccagccggaggaagcg-3Ј and for S293A GPVI, 5Ј-gcagaggactggcacgcccggaggaagcgc-3Ј. The constructs ⌬288GPVI and R272AGPVI have been described previously (12,15). All mutated sequences were verified by sequencing.…”

“…Dose-response curves to convulxin are shown as mean Ϯ S.E. Src family kinases, Fyn and Lyn (15). ⌬294GPVI lacks additional 15 amino acids covering the basic region, which has been shown to mediate association with calmodulin.…”

“…1A) (13). We have shown previously that the proline-rich sequence mediates association with Fyn and Lyn via their Src homology 3 domains (15) and is required for tyrosine phosphorylation of the ITAM in transfected COS-7 cells. The basic region contains a sequence that has been shown to mediate association with calmodulin (16).…”

Platelet Interactions in Thrombosis

“…First, a membrane-proximal positively-charged consensus motif within the cytoplasmic domain of GPVI binds calmodulin (35,36). Second, a proline-rich sequence immediately Cterminal of the calmodulin-binding site is a consensus motif for binding SH3 (src homology-3) domains of src-family kinases, and binds to fyn and lyn (36,37). Third, an arginine residue within the transmembrane domain and additional elements within the cytoplasmic tail mediate the association of GPVI with FcRg (36,38).…”