Integrins Flashcards
Integrins
A large family of transmembrane, heterodimeric glycoprotein receptors
Major metazoan receptors for cell adhesion - evolutionarily conserved, present in all multicellular organisms
Integrin subunits
Alpha and beta
760-1000 AA
18 alpha and 8 beta currently known
Assemble to form the 24 known integrin heterodimers
Knockout studies have revealed each of the 24 integrins has a specific, non-redundant function
Integrin dimers
Each dimer has distinctive properties and functions
The same integrin molecule in different cell types can have different ligand-binding specificities!
How can integrins be grouped?
According to their ligand-binding properties i.e. the specific amino acid sequences they bind to in ECM proteins/cell surface proteins
RGD
Canonical integrin-binding motif
Present in fibronectin and other ECM proteins
LDV
Fibronectin and other ECM proteins
GFOGER
Collagen
Collagen-binding motif
GFOGER
Integrin structure
Short C-terminal intracellular tails
Large N-terminal extracellular globular heads that can project >20 nm from the lipid bilayer
Subunits have a modular, multi-domain structure
Modular composition provides inherent structural flexibility
Alpha subunit
Determines specificity of ligand binding
H2N-beta propeller domain-Thigh-Calf1-Calf2-mem-COOH
Ligand binding is mediated by an Mg2+ ion in the “metal-ion-dependent adhesion site” (MIDAS) in the I domain
I domain is within the beta-propeller domain
Ca2+ can bind to 5, 6 and 7 domains within the beta-propeller domain and allosterically regulate ligand binding
Cytoplasmic tail contains a conserved GFFKR motif, but otherwise little homology in cytoplasmic region
What affects the binding of integrins to their ECM ligands?
The extracellular concentration of Mg2+ and Ca2+
Beta subunit
H2N-PSI-hybrid-IEGF-mem-COOH
Smaller extracellular domain than alpha subunit
PSI domain = Plexin-Semaphorin-Integrin
Hybrid domain contains I domain, MIDAS domain and ADMIDAS domain
4 I-EGF domains = Cys-rich EGF repeat domains
Beta-subunit cytoplasmic tails exhibit considerable homology, most contain two NPxY motifs (Y can be phosphorylated, critical for scaffolding/adaptor molecule recruitment)
ADMIDAS domain
Inhibitory metal-ion-binding site
Occupied by Ca2+ when integrin is inactive and displaced by Mn2+ during activation
Function of I domain in beta subunit
Involved in ligand binding in integrins where the alpha subunit lacks its own I domain e.g. aIIb
Integrins exist in…
…an equilibrium between 2 conformations
A low affinity, bent, inactive form and a high affinity, extended, active form
Inactive form bound to integrin inactivators
Extended, fully activated integrin coupled to actin and the ECM
Inactive integrin
99.75 % of the time
Bent
Extracellular domains folded into a compact structure that only allows for low affinity ligand binding
Cytoplasmic tails closely bound via an inhibitory, non-covalent association (salt bridge) that prevents their interactions with cytoskeletal linker proteins
Active integrin
Straight
Conformational change in ligand-binding site allows for higher affinity binding
Cytoplasmic tails separate
Integrin activation
Involves a major conformational change that simultaneously exposes the intra- and extracellular ligand-binding sites
Can be induced from both the inside and outside of the cell
“Outside-in” integrin activation
Can occur due to external forces in the ECM or binding of a ligand e.g. collagen, fibronectin, laminin
This forces the integrin into a straighter conformation that separates the cytoplasmic tails
This exposes the binding sites for talin and other cytoplasmic adaptor proteins on the cytoplasmic tail of the beta-subunit, allowing the binding of these adaptor proteins
“Inside-out” integrin activation
Generally depends on intracellular signals that promote the ability of talin and other cytoplasmic adaptor proteins to bind to the beta-subunit of the integrin
Talin competes with the alpha-subunit for its binding on the beta-subunit cytoplasmic tail
“Inside-out” integrin activation in platelets
- Thrombin binds to and activates the thrombin receptor PAR1, resulting in the activation of IP3 and DAG (Gaq coupled)
- This leads to the activation of CALDAG-GEFI, which converts Rap1-GDP (inactive) to Rap1-GTP (active)
- Rap1-GTP recruits its effector RIAM and its binding partner talin to the plasma membrane
- Talin binds to the beta-subunit tail of the aIIb/b3 integrin
(kindlin also plays a crucial role in this process) - Active talin can interact with further proteins (e.g. adaptor protein vinculin), resulting in the formation of an actin linkage
Rap1
Ras-related protein 1 (GTPase)
RIAM
Rap1-GTP-interacting adaptor molecule
How do talin and kindlin promote integrin activation?
By stabilising the extended, active conformation
Talin
“Master regulator of integrins” - most important integrin-interacting protein
Adaptor protein that links integrins to actin filaments
Structure of talin
Can be divided into 2 domains linked via a flexible 80 AA linker
- N-terminal globular head domain containing a linear FERM domain
- C-terminal rod domain composed of 62 alpha-helices that form 13 amphipathic alpha-helical bundles and a dimerisation domain
N-terminal globular head domain of talin
FERM domain directs membrane localisation
Composed of 3 structural modules (F1, F2, F3) that form a compact, clover-shaped structure (talin FERM domain also has additional F0 domain)
F3 domain responsible for binding to first membrane-proximal NPxY motif in beta-subunit via PTB fold - this binding disrupts the salt. bridge between the alpha and beta subunits
FERM
4.1 Ezrin Radixin Moesin
C-terminal rod domain of talin
Responsible for interacting with cytoskeletal proteins e.g. actin, vinculin
Provides a direct link between integrins and the cytoskeleton
Vinculin binding sites (11 in total) are cryptic but become progressively exposed as the rod domain is extended by tension in the actin cytoskeleton (i.e. mechanosensitive)
Auto-inhibition of talin
F3 FERM domain can bind to specific residues in the talin rod which masks the primary integrin binding site in the F3 domain
Auto-inhibition is alleviated by binding of RIAM and/or PIP2 to globular had
Kindlin
Co-activator of integrins
Enhances talin-induced integrin activation but cannot activate integrins in the absence of talin
Believed to promote clustering of talin-activated integrins - essential for formation of focal-adhesions
Structure of kindlin
Smaller than talin
Significant structural homology with talin globular head domain - contains PTB fold, responsible for binding to second NPxY motif in beta-subunit tail
No extended rod domain
Dimerises in vivo which serves to fully activate integrin clusters
What modulates integrin activation status?
Competition/balance between cytosolic activators and inactivators modulates the dynamics of integrin activation status
What can inhibit the recruitment of talin and kindlin?
Certain proteins binding to the cytoplasmic tails of integrins
Phosphorylation events
Examples of integrin activators and inactivators
Phosphorylation of filamin (actin-binding protein) on Ser2152 by RSK2 promotes the binding of filamin to the cytoplasmic tail of the beta-subunit
KRIT1 binds to and sequesters ICAP1, thus preventing ICAP1 binding to the b1 cytoplasmic tail. Free ICAP1 normally binds to the b1 cytoplasmic tail and causes integrin dissociation (inactivation)
Phosphorylation of Y in the NPxY motif by Src enhances the binding of DOK1, which outcompetes talin
Focal adhesions
Dynamic, multi-protein complexes that form in association with the cytoplasmic tails of integrins following ligand binding
Sites at which integrins bridge the cytoskeleton and the ECM
“Dense plaques”
Functions of focal adhesions
Mechanosensing e.g. the nature, amplitude and direction of mechanical loading
Mechanotransmission (bidirectionally between the ECM and actin cytoskeleton)
Mechanotransduction (i.e the translation of mechanical stimuli into a biochemical, cellular response)
The protein composition of focal adhesions is…
…reorganised in response to mechanical force
Protein composition of immature focal adhesions
Force-insensitive,
Force-sensitive (positive and negative regulation) and
Force-responsive (positive and negative regulation)
proteins
Transmit specific integrin-mediated signals in a coordinated manner
Protein composition of mature focal adhesions
In response to mechanical force, the abundance of negative force-sensitive and force-responsive proteins decreases while the abundance of positive force-sensitive proteins increases
Force-insensitive proteins have a similar abundance in both mature and immature focal adhesions
Focal adhesion layers
Focal adhesions can be divided into 3 functional layers, each with a distinct molecular composition
- Integrin signalling layer - integrins are activated via the binding of the talin head domain to the beta cytoplasmic tail in the ISL. FAK and paxillin also present in this layer
- Force transduction layer - rich in vinculin and contains rod domain of talin. This layer contains an abundance of proteins involved in mechanotransduction
- Actin regulators layer - contains actin and alpha-actinin
Focal adhesion downstream signalling events
Can be divided into 3 temporal stages
- Immediate effects (0-10min): up-regulation of lipid kinase activity, leading to increase in PIP2 and PIP3. Also rapid phosphorylation of substrates, particularly at nascent adhesion itself
- Short term effects (10-60min): activation of Rho GTPases and other actin-regulatory proteins to drive reorganisation of the actin cytoskeleton
- Long term effects (>60min): activation of proliferation and survival pathways, control of cell morphology
Focal adhesions downstream signalling diagram
(draw)
FAK
(PTK2)
Focal adhesion kinase
Non-receptor tyrosine kinase
Bi-functional - has catalytic and scaffolding functions
Rapidly recruited to the ISL of nascent focal adhesions by talin
Associates with the beta-cytoplasmic tail - this association is mediated by its interaction with PIP2 and other scaffolding proteins e.g. talin, paxillin
Activated FAK is a critical and early effector of outside-in signalling events
Structure of FAK
Central kinase domain surrounded by a FERM homology domain at the N-terminus and a FAT domain at the C-terminus
Both terminal domains are separated from the kinase domain by a proline-rich linker
FAT domain
Focal adhesion targeting domain
Integrin-mediation FAK activation
- The adhesion of cells to the ECM via integrins results in integrin clustering and the recruitment of focal adhesion proteins such as FAK, talin, vinculin, paxillin and PIP5KIy that form adhesion structures to link integrins to the actin cytoskeleton
- Recruitment of PIP5KIy leads to a localised increase in PIP2 levels in focal adhesions
- PIP2 binds to the FERM domain of FAK, resulting in FAK clustering at the membrane
- This PIP2-induced FAK clustering causes ‘conformational relaxation’ and allows for the autophosphorylation of FAK, leading to the recruitment of Src via SH2 domains
- Recruited Src phosphorylates Tyr residues in the activation loop of FAK, resulting in its full activation and release of the kinase from the membrane-clustered FERM domain
Therapeutic rationale for targeting FAK in diverse solid tumours
Solid tumours commonly show an increased expression/activity of FAK
FAK is a prognostic marker of a solid tumour malignancy and poor patient survival (characteristic of a more aggressive tumour)
How does FAK drive cancer progression?
Through kinase-dependent and -independent pathways as a result of its bifunctional role (catalytic and scaffolding)
FAK activates pro-proliferative and anti-apoptotic pathways, rendering tumour cells competent to proliferate and survive
FAK also promotes metastasis thought regulating processes involved in cell motility and invasion
Why is regulation of integrin function essential?
Strict temporospatial integrin regulation is essential throughout life
GOF and LOF analyses have been performed in mice for all alpha and beta integrin genes, with phenotypes ranging from mild to severe to embryonically lethal
Inherited human diseases associated with faulty integrin genes
Human Leukocyte Adhesion Deficiency (LAD) type 1 = rare, recurrent bacterial/fungal soft tissue infections
Epidermal Bullosa with Pyloric Atresia (EB-PA) = involves a6 and b4, skin is fragile and blisters easily
Glantzmann’s thrombasthenia = extremely rare, involves aIIb and b3 (i.e. those involved in platelets), bleeding disorder
What do av integrins activate?
Latent (matrix-bound) TGFb
How is ECM structure and function controlled?
By feedback loops
Changes in ECM stiffness or in external/internal mechanical loading affect the cellular responses that lead to either the negative feedback loop (homeostatic regulation of ECM) or the positive feedback loop (fibrotic conditions)
What drives tissue fibrosis?
Increased ECM stiffness and increased signalling via integrins and TGFb
How is TGFb secreted from cells?
As an inactive dimer held together by disulfide bonds
Furin proteolytically cleaves the sequence to generate LAP and TGFb dimers that remain non-covalently associated
Latent TGFb (TGFb-LAP) is stored in the ECM together with LTBP
Features of the amino acid sequence encoded by the TGFb gene
Cys33 forms a disulfide linkage to LTBPs
AAs 42-59 form an alpha helix
Cys223 and Cys225 form inter-chain disulfide linkages responsible for LAP homodimer formation
RGD motif near C-terminal of LAP region
LTBPs
Latent TGFb-binding proteins
LAP
Latency associated peptide
How is latent TGFb activated?
Actomyosin-mediated cell contraction force is transmitted to an RGD binding site in LAP, inducing a conformational change that liberates TGFb and allowing TGFb to interact with its receptor
Integrins involved in tissue fibrosis
av integrins
b1, b3, b5 and b8 on fibroblasts
b6 on epithelia
These integrins activate TGFb through interaction with the RGD motif on LAP
Role of TGFb in driving tissue fibrosis
Once activated, TGFb can activate resident fibroblasts and induce their transformation into a-SMA +ve myofibroblasts
These myofibroblasts are responsible for the production and deposition of collagen and fibronectin, resulting in an increasingly stiff and fibrotic ECM
These myofibroblasts can also simultaneously produce and activate latent TGFb as a result of their increased contractility and enhanced av integrin expression
The presence of myofibroblasts is a classical feature of established IPF, because these cells are incredibly active in the secretion of collagen and fibronectin
Why can TGFb not be targeted directly for treatment of IPF?
Because TGFb is ubiquitous
Plays an important immunomodulatory role in dampening down inflammatory states
Therefore integrins are targeted instead because of the role they play in activating TGFb
