At the sites of vascular injury bioactive molecules such as thrombin, ADP, collagen, fibrinogen and thrombospondin are generated, secreted or exposed. These stimuli activate platelets, converting the major platelet integrin alphaIIbbeta3 from a resting state to an active conformation, in a process termed integrin priming or 'inside-out signalling'. Integrin activation refers to the change required to enhance ligand-binding activity. The activated alphaIIbbeta3 interacts with the fibrinogen and links platelets together in an aggregate to form a platelet plug. AlphaIIbbeta3 bound to fibrin generates more intracellular signals (outside-in signalling), causing further platelet activation and platelet-plug retraction. In the resting state the alpha and beta tails are close together. This interaction keeps the membrane proximal regions in a bent conformation that maintains alphaIIbbeta3 in a low affinity state. Integrin alphaIIbbeta3 is released from its inactive state by interaction with the protein talin. Talin interacts with the beta3 cytoplasmic domain and disrupts the salt bridge between the alpha and beta chains. This separation in the cytoplasmic regions triggers the conformational change in the extracellular domain that increases its affinity to fibrinogen. Much of talin exists in an inactive cytosolic pool, and the Rap1 interacting adaptor molecule (RIAM) is implicated in talin activation and translocation to beta3 integrin cytoplasmic domain.
View original pathway at:Reactome.
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Upon the production of activated Rap1A at the plasma membrane, RIAM interacts with Rap1A-GTP with its N-ter RA domain, and with its C-ter PH domain it interacts with PIP2.
As integrins do not have an intrinsic catalytic activity, the signals initiated by the ECM-integrin interactions are transduced into cells through the integrin bound protein-tyrosine kinases. Focal adhesion kinase 1 (FADK1, FAK) is one of the protein tyrosine kinases that plays a prominent role in integrin signaling. FAK has been implicated in controlling cell motility and transmitting a cell survival signal from ECM.
FAK is recruited to sites of integrin clustering by directly binding to integrin associated c-Src.
The co-localization of FAK with integrins in focal adhesions and the actin cytoskeleton is essential for the activation and phosphorylation of FAK. FAK has six tyrosine phosphorylation sites and tyrosine 397 is the main auto-phosphorylation site present upstream of the kinase domain.
The interaction between talin and integrin alphaIIb beta3 breaks the putative salt bridge between the alphaIIb (R995) and beta3 (D723) integrin chains and induces conformational changes in their external domains increasing their affinity for fibrinogen and other ECM ligands. Breaking of this salt bridge is necessary but not sufficient for full activation. The Talin F3 subdomain of the FERM domain has a phosphotyrosine binding (PTB) domain fold. This domain interacts with the membrane-proximal (MP) region within the integrin beta3 chain. The primary function of this interaction is to provide an initial strong linkage between talin and integrin and this interaction holds the key to the molecular recognition required for activation. In platelets SRC kinase and its negative regulator CSK associates constitutively with integrin alphaIIbbeta3. SRC is involved in alphaIIbbeta3 dependent activation of SYK, and both SRC and SYK are required to initiate cytoskeletal events responsible for platelet spreading on fibrinogen.
Phosphorylated tyrosine 925 in the FAT domain of FADK1 creates a docking site for the SH2 domain of GRB2 and recruits the GRB2/SOS complex. FADK1 may use this mechanism to activate Ras and the MAP kinase pathway.
Talin is one of the major cytoskeletal proteins involved in integrin activation and linking the resulting focal adhesion (FA) with cytoskeleton. Talin comprises an N-ter head region and a flexible rod domain. The head region has the FERM domain (subdivided into F1, F2 and F3 subdomains), which has the binding sites for beta integrin cytoplasmic regions and actin binding sites close to the C-terminal rod domain. Talin exists in closed inactive conformation, where the head region interacts with the rod domain masking the integrin binding sites. At the plasma membrane the RIAM bound to active Rap1 recruits talin to form the integrin activation complex. This interaction exposes the integrin-binding site in talin F3 domain leading to integrin activation.
The recruitment of FADK1 to active SRC leads to the efficient tyrosine phosphorylation of multiple additional sites on FADK1. SRC trans-phosphorylates FADK1 within the kinase doman activation loop (Y576 and Y577) and within the FADK1 C-terminal domain (Y861 and Y925).
The overall shape of integrins is that of a globular 'head' supported by two rod like legs. The ligand-binding pocket is formed by the combination of A-domain or beta-I domain on the beta3 subunit and the putative beta-propeller fold on the alphaIIb subunit in the head regions. The binding of ligand to integrin is also dependent on divalent cations (usually Mn++ or Mg++or Ca++). A conserved motif, the metal ion-dependant adhesion site (MIDAS) is located in the alpha and the beta chains that coordinate the divalent cation at the top of the domain. Active integrin alphaIIb beta3 interacts with a variety of plasma proteins such as fibrinogen, vWF, thrombin, thrombospondin, and fibronectin. The ability of alphaIIbbeta3 to bind fibrinogen plays a crucial role in platelet aggregation and hemostasis. Most of these matrix proteins have integrin binding sites of 3-6 amino acids length, of which the best known are the 'RGD' and 'KGD' motifs. The alpha and beta integrin subunits are both required for ligand binding.
Son of sevenless protein homolog 1(SOS) is an activating nucleotide exchange factor for H-Ras (p21Ras). SOS is brought close to the H-Ras by interacting with the FADK1 bound GRB2.
Signals from agonist receptors (such as GPVI) trigger the production of PIP3, DAG, cAMP and elevated Ca++ levels. This leads to the activation and translocation of active Rap1-GTP to the plasma membrane. Rap-GEFs stimulate the replacement of GDP for GTP, activating Rap1. Several Rap1 GEFs have been identified enabling Rap1 to respond to diverse stimuli. CalDAG-GEFs activate Rap1 in response to calcium and DAG, downstream of Phospholipase C. EPAC (exchange proteins directly activated by cAMP) GEFs are activated by binding cAMP.
SH3-mediated binding of p130Cas to FAK is linked to enhanced tyrosine phosphorylation of p130Cas at multiple sites. The Cas substrate domain contains 15 separate YxxP motifs, a main site of tyrosine phosphorylation on the CAS molecule. Once phosphorylated, this domain serves as a docking site for the SH2 domains of Crk or Nck adaptor proteins that affect the downstream MAPK signalling pathway, resulting in cell survival and increased motility.
Crk is an adaptor protein with one SH2 and two SH3 domains. It is involved in integrin mediated signalling and is recruited to the focal adhesion complexes by interacting with p130Cas or paxillin through its SH2 domain.
p130Cas (Crk-associated substrate) is an adaptor protein that promotes protein-protein interactions, leading to the multiprotein complexes. The interaction of p130Cas with other proteins modulates cell motility, survial and proliferation. P130Cas is one of the main phosphorylation targets of the FAK/Src complex. The p130Cas SH3 domain binds to PR1 and PR2 (‘PxxP’) domains in the FAK C-terminal domain.
Clustering of Integrin alphaIIb beta3 complexes results in the trans auto-phosphorylation of SRC tyrosine residue 419 (often referred to as 418 in the literature, as the initiating methionine is cleaved in the mature peptide) in SRC's kinase activation loop.
The fibrinogen-bound integrin alphaIIb beta3 clusters platelets together to form a platelet plug and generates intracellular signals (outside-in) causing further platelet activation and platelet plug retraction. Intracellular integrin alphaIIb beta3 clustering brings SRCs bound to integrin beta3 chains into proximity. SRC associates constitutively with integrin alphaIIb beta3. In unstimulated cells this SRC is inactive, auto-inhibited by an internal interaction between phosphorylated Y530 and the SH2 domain. CSK is selective for the Y530 residue and prevents access to SRC of PTP1B, a protein tyrosine phosphatase that is capable of de-phosphorylating Y530.
The integrin alphaIIb beta3:Inactive SRC complex recruits PTP1B protein tyrosine phosphatase resulting in the dephosphorylation of SRC tyrosine 530. The phosphorylated tail of SRC tail interacts with the SH2 domain thereby repressing kinase activity; removal of phosphorylation activates SRC kinase activity.
CSK bound to integrin alphaIIb beta3 negatively regulates SRC by phosphorylating the Tyr-530. Platelet adhesion to fibrinogen causes the disassociation of CSK from alphaIIb beta3 complex.
Integrin alphaIIb beta3 'outside-in' signalling involves multiple proteins including SRC, SYK, SLP-76 and PLCgamma2. SRC is constitutively associated with the C-terminal tail of integrin beta 3. SYK is recruited to the beta3 tail and subsequently activated by SRC.
SYK activation in integrin signalling is associated with increased tyrosine phosphorylation. SYK activation and phosphorylation of SYK targets can be blocked by SRC inhibitors or expression of dominant negative SRC mutants.
The beta 3 integrin cytoplasmic tail binds SH2-containing protein (SHC), an adapter in Ras signaling. Phosphorylation of Y785 may be necessary for binding; phosphorylation of T779 inhibits SHC binding. Mice expressing a mutated beta 3 where Y773 and Y785 have been mutated to F exhibit rebleeding from tail wounds and subtle defects in clot retraction and platelet aggregation.
The binding of SHC to integrin alpha IIb beta 3 is blocked by phosphorylation of beta 3 at Thr-779, or by substitution of this residue for Asp. PDK1 and Akt1/PKB-alpha both specifically target Thr-779 in in vitro assays.
In a mechanism that is presumed to be analagous to signaling of SHC downstream of the insulin and TrkA receptors, SHC becomes phosphorylated and dissociates from the integrin alphaIIb beta3 complex.
In a mechanism that is presumed to be analagous to signaling of SHC downstream of the insulin and TrkA receptors, SHC becomes phosphorylated and dissociates from the integrin alphaIIb beta3 complex.
Signals from agonist receptors (such as GPVI) trigger the production of PIP3, DAG, cAMP and elevated Ca++ levels. This leads to the activation and translocation of active Rap1-GTP to the plasma membrane. Rap-GEFs stimulate the replacement of GDP for GTP, activating Rap1. Several Rap1 GEFs have been identified enabling Rap1 to respond to diverse stimuli. CalDAG-GEFs activate Rap1 in response to calcium and DAG, downstream of Phospholipase C. EPAC (exchange proteins directly activated by cAMP) GEFs are activated by binding cAMP.
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DataNodes
alpha IIb beta
3:p-Y530-SRC:CSK:Talin:RIAM complex:p-Y427-SHCalphaIIb beta
3:p(Y530)-SRC:CSK:Talin:RIAM complex:SHCalphaIIb
beta3:p-Y530-SRC:CSK:Talin:RIAM complexalphaIIb
beta3:p-Y530-SRC:CSKp130Cas in complex
with FADK1complex:ECM ligands: 2X(Integrin alphaIIb beta3:Inactive
(p-Y530)-SRC)complex:ECM ligands: 2X(Integrin alphaIIb
beta3:p(Y530)-SRC:CSK)complex:ECM ligands: 2X(Integrin
alphaIIb beta3:SRC)complex:ECM ligands: Integrin alphaIIb beta3:Active
(p-Y419)-SRC:p(Y397,407,576,577,861,925)-FADK1complex:ECM ligands: alphaIIb beta3:Active
(p-Y419)-SRC:p(Y397)-FADK1complex:ECM ligands:2X(Integrin alphaIIb beta3:Active
(p-Y419)-SRC)ECM ligands:Integrin alphaIIb beta3:Active
(p-Y419)- SRC:FADK1ligands:2X (Integrin alphaIIb beta3:Active
(p-Y419)-SRC):SYKligands:2X(Integrin alphaIIb beta3:Active
(p-Y419)-SRC):p(Y)-SYKAnnotated Interactions
alpha IIb beta
3:p-Y530-SRC:CSK:Talin:RIAM complex:p-Y427-SHCalpha IIb beta
3:p-Y530-SRC:CSK:Talin:RIAM complex:p-Y427-SHCalphaIIb beta
3:p(Y530)-SRC:CSK:Talin:RIAM complex:SHCalphaIIb beta
3:p(Y530)-SRC:CSK:Talin:RIAM complex:SHCalphaIIb
beta3:p-Y530-SRC:CSK:Talin:RIAM complexalphaIIb
beta3:p-Y530-SRC:CSK:Talin:RIAM complexalphaIIb
beta3:p-Y530-SRC:CSK:Talin:RIAM complexalphaIIb
beta3:p-Y530-SRC:CSK:Talin:RIAM complexalphaIIb
beta3:p-Y530-SRC:CSKalphaIIb
beta3:p-Y530-SRC:CSKp130Cas in complex
with FADK1p130Cas in complex
with FADK1FAK is recruited to sites of integrin clustering by directly binding to integrin associated c-Src.
FAK has six tyrosine phosphorylation sites and tyrosine 397 is the main auto-phosphorylation site present upstream of the kinase domain.
The Talin F3 subdomain of the FERM domain has a phosphotyrosine binding (PTB) domain fold. This domain interacts with the membrane-proximal (MP) region within the integrin beta3 chain. The primary function of this interaction is to provide an initial strong linkage between talin and integrin and this interaction holds the key to the molecular recognition required for activation. In platelets SRC kinase and its negative regulator CSK associates constitutively with integrin alphaIIbbeta3. SRC is involved in alphaIIbbeta3 dependent activation of SYK, and both SRC and SYK are required to initiate cytoskeletal events responsible for platelet spreading on fibrinogen.
Talin exists in closed inactive conformation, where the head region interacts with the rod domain masking the integrin binding sites. At the plasma membrane the RIAM bound to active Rap1 recruits talin to form the integrin activation complex. This interaction exposes the integrin-binding site in talin F3 domain leading to integrin activation.
Active integrin alphaIIb beta3 interacts with a variety of plasma proteins such as fibrinogen, vWF, thrombin, thrombospondin, and fibronectin. The ability of alphaIIbbeta3 to bind fibrinogen plays a crucial role in platelet aggregation and hemostasis. Most of these matrix proteins have integrin binding sites of 3-6 amino acids length, of which the best known are the 'RGD' and 'KGD' motifs. The alpha and beta integrin subunits are both required for ligand binding.
Intracellular integrin alphaIIb beta3 clustering brings SRCs bound to integrin beta3 chains into proximity. SRC associates constitutively with integrin alphaIIb beta3. In unstimulated cells this SRC is inactive, auto-inhibited by an internal interaction between phosphorylated Y530 and the SH2 domain. CSK is selective for the Y530 residue and prevents access to SRC of PTP1B, a protein tyrosine phosphatase that is capable of de-phosphorylating Y530.
complex:ECM ligands: 2X(Integrin alphaIIb beta3:Inactive
(p-Y530)-SRC)complex:ECM ligands: 2X(Integrin alphaIIb beta3:Inactive
(p-Y530)-SRC)complex:ECM ligands: 2X(Integrin alphaIIb
beta3:p(Y530)-SRC:CSK)complex:ECM ligands: 2X(Integrin alphaIIb
beta3:p(Y530)-SRC:CSK)complex:ECM ligands: 2X(Integrin
alphaIIb beta3:SRC)complex:ECM ligands: 2X(Integrin
alphaIIb beta3:SRC)complex:ECM ligands: Integrin alphaIIb beta3:Active
(p-Y419)-SRC:p(Y397,407,576,577,861,925)-FADK1complex:ECM ligands: Integrin alphaIIb beta3:Active
(p-Y419)-SRC:p(Y397,407,576,577,861,925)-FADK1complex:ECM ligands: Integrin alphaIIb beta3:Active
(p-Y419)-SRC:p(Y397,407,576,577,861,925)-FADK1complex:ECM ligands: Integrin alphaIIb beta3:Active
(p-Y419)-SRC:p(Y397,407,576,577,861,925)-FADK1complex:ECM ligands: alphaIIb beta3:Active
(p-Y419)-SRC:p(Y397)-FADK1complex:ECM ligands: alphaIIb beta3:Active
(p-Y419)-SRC:p(Y397)-FADK1complex:ECM ligands:2X(Integrin alphaIIb beta3:Active
(p-Y419)-SRC)complex:ECM ligands:2X(Integrin alphaIIb beta3:Active
(p-Y419)-SRC)complex:ECM ligands:2X(Integrin alphaIIb beta3:Active
(p-Y419)-SRC)complex:ECM ligands:2X(Integrin alphaIIb beta3:Active
(p-Y419)-SRC)ECM ligands:Integrin alphaIIb beta3:Active
(p-Y419)- SRC:FADK1ECM ligands:Integrin alphaIIb beta3:Active
(p-Y419)- SRC:FADK1ECM ligands:Integrin alphaIIb beta3:Active
(p-Y419)- SRC:FADK1ligands:2X (Integrin alphaIIb beta3:Active
(p-Y419)-SRC):SYKligands:2X (Integrin alphaIIb beta3:Active
(p-Y419)-SRC):SYKligands:2X (Integrin alphaIIb beta3:Active
(p-Y419)-SRC):SYKligands:2X(Integrin alphaIIb beta3:Active
(p-Y419)-SRC):p(Y)-SYK