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Integrin trafficking ata glance
Rebecca E. Bridgewater1, Jim C.Norman2 and Patrick T. Caswell1,*1Wellcome Trust Centre for Cell Matrix Research,Faculty of Life Sciences, University of Manchester,Manchester M13 9PT, UK2Beatson Institute for Cancer Research, GarscubeEstate, Glasgow G61 1BD, UK
*Author for correspondence
Journal of Cell Science 125, 3695–3701
� 2012. Published by The Company of Biologists Ltd
doi: 10.1242/jcs.095810
Integrins are heterodimeric transmembrane
receptors for extracellular matrix (ECM)
components and they link the intracellular
actin cytoskeleton to the cellular environment
(Humphries et al., 2006; Hynes, 2002). The
a- and b-subunits of integrin heterodimers
are type I membrane proteins that typically
have a large extracellular domain and a
short intracellular tail. Through different
combinations of the 18 a- and eight b-
subunits, 24 distinct integrin heterodimers
exist in mammals. The integrin family thus
comprises an array of cell surface receptors,
which are expressed in a cell- and tissue-
specific manner, for a plethora of soluble and
insoluble ECM ligands, including collagens,
laminins, fibronectin and vitronectin
(Humphries et al., 2006). As a result,
integrins are able to control diverse cellular
processes, including proliferation, apoptosis,
differentiation and cell migration and thus
have key roles in development, immune
responses and the progression of diseases
such as cancer (Legate et al., 2009). Integrin
heterodimers can adopt a bent or closed
conformation that has a low affinity for ligand
(‘inactive’) or an extended or open
conformation that has a high affinity for
ligand (‘active’). As a consequence of this
conformational switching, integrins are
able to signal bidirectionally across the
membrane: ligand binding elicits signalling
responses within the cell (‘outside-in’
signalling), but binding of intracellular
proteins such as talin and kindlins to
integrins regulates the activation of integrins
to promote ligand-binding (‘inside-out’
signalling) (Legate et al., 2009).
In addition, the control of integrin
availability at the plasma membrane is
key to their function. However, although
we have known for over 20 years that
integrins undergo an exocytic–endocytic
cycle, these pathways have only been
studied in detail relatively recently. In
this Cell Science at a Glance article, we
will describe the mechanisms that control
integrin endocytosis and recycling, and
discuss the insight that understanding these
mechanisms has provided into the role of
trafficking in integrin function and vice
versa. Furthermore, we draw attention to
two emerging features of integrin
trafficking: first, the specific nature of the
mechanisms that control aspects of integrin
trafficking, and, second, the role that
integrin trafficking has in intracellular
signalling.
(See poster insert)
Cell Science at a Glance 3695
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Mechanisms of integrin endocytosisIntegrin endocytosis has been studied
extensively in the context of viral entry
(Pellinen and Ivaska, 2006), but mechanisms
that regulate integrin internalisation in the
absence of pathogens have been identified.
Integrins can be internalised through the
major endocytic routes (Box 1), including
the formation of circular dorsal ruffles
(CDRs) during macropinocytosis (Gu
et al., 2011), and clathrin-dependent and
-independent endocytic pathways. Direct
recruitment of endocytic regulators to
integrins constitutes a general mechanism
that can drive integrin endocytosis.
Examples of this include the recruitment of
HS1 associated protein X-1 (HAX-1) to the
cyoplasmic tail of integrin b6 (Ramsay et al.,
2007) and the recruitment of Rab21 to the
cytoplasmic tail of integrin a subunits
(Pellinen et al., 2006). Protein kinase C
alpha (PKCa) controls caveolar endocytosis
(Smart et al., 1995), and can itself bind to b1
integrins (i.e. integrins that contain a b1
subunit and any a subunit) and regulate
integrin internalisation (Ng et al., 1999). In
endothelial cells, recruitment of active a5b1
integrin to the neuropilin–GIPC1 (for GAIP
C-terminus-interacting protein) complex
regulates endocytosis of this integrin
directly from fibrillar adhesions (Valdembri
et al., 2009). Xenopus GIPC1 (also known
as kermit2) regulates a5b1 integrin
internalisation into Rab21-positive vesicles
(Spicer et al., 2010), which suggests that
mechanisms that regulate integrin
internalisation are conserved.
Clathrin-dependent endocytosis
The clathrin adaptors disabled homologue 2
(DAB2) and NUMB interact, through their
phosphotyrosine-binding (PTB) domains,
with the NPxY-motifs in the b-integrin
cytoplasmic tails (Calderwood et al., 2003),and several lines of evidence support thenotion that clathrin-adaptors regulate
integrin endocytosis. Microtubule-inducedfocal adhesion (FA) disassembly hasbeen shown to require clathrin-dependentendocytosis (CDE) (Chao and Kunz, 2009;
Ezratty et al., 2009). Indeed clathrin andDAB2 localise to FAs in the mid-region ofmoving cells and regulate a5b1 integrin
internalisation to facilitate migration.NUMB binds to b1 and b3 integrins, andlocalises around focal contacts at the
leading edge of migrating cells togetherwith clathrin. Phosphorylation of NUMBdownstream of the PAR3 (also known as
PARD3)–atypical PKC (aPKC) complexregulates integrin internalisation at thesesites to promote migration (Nishimura andKaibuchi, 2007). Inactive integrins are also
internalised in a clathrin-dependent manner:DAB2 and AP2 regulate endocytosis ofa1b1, a2b1 and a3b1 (but not a5b1)
integrins from the dorsal surface of thecell, and the rate of DAP2–AP2-mediatedendocytosis correlates positively with
migration specifically towards ligands forthese integrins (Teckchandani et al., 2009).
Clathrin-independent endocytosis
Accumulating evidence indicates thatintegrins can follow clathrin-independentendocytic routes. Indeed, b1 integrins arefound within clathrin-independent carriers
(CLICs, see Box 1) (Howes et al.,2010a). Rab21 overexpression circumventsthe requirement for CDE to internalise b1
integrins in dividing cells by driving a formof clathrin-independent endocytosis (CIE)(Pellinen et al., 2008). Cholesterol depletion
experiments have shown that integrins canenter the cell in a raft-dependent fashion(Fabbri et al., 2005), and a5b1 and avb3
integrins are thought to associate withcaveolin-1 (Galvez et al., 2004; Wickstromet al., 2002). More direct evidence supports arole for caveolae in the internalisation of
a5b1 in myofibroblasts (Shi and Sottile,2008), a2b1 in an osteosarcoma cell line(Upla et al., 2004) and active b1 integrins in
bone marrow mesenchymal stem cells(BMMSCs; Du et al., 2011).
Regulation of integrin endocytosis bycell–matrix adhesion
Although integrin endocytosis can beconstitutive and ligand-independent, cell
adhesion to the ECM through otheradhesion receptors can promote integrinendocytosis. For instance, myelin-associated
Box 1. Endocytosis
Endocytosis regulates membrane lipid and protein composition, thereby allowing cellular
functions to be controlled. Many distinct routes of internalisation have been identified, and
these are typically characterised by the mechanism employed, the cargo transported and/or
the morphology of the internalising structure (Hansen and Nichols, 2009). Generally,
endocytic processes are classified as clathrin-dependent endocytosis (CDE) or clathrin-
independent endocytosis (CIE), whereby CIE describes several distinct endocytic routes
(Doherty and McMahon, 2009).
Clathrin-dependent endocytosis
CDE is the best-characterised internalisation route and involves the recruitment of clathrin
triskelia to the membrane, where they are assembled to form a clathrin-coated pit (CCP). This
is preceded by a nucleation step, wherein factors such as phosphatidylinositol 4,5-
diphosphate [PtdIns(4,5)P2], epidermal growth factor receptor pathway substrate 15
(EPS15), intersectins and FCH domain only (FCHO) proteins define the site of CCP
formation. This ‘nucleation module’ recruits the clathrin-adaptor AP-2, along with cargo-
specific adaptor proteins that mediate cargo selection, and, at the same time, clathrin
assembles to form the CCP. The GTPase dynamin is subsequently recruited to the neck of
the CCP, where it polymerises. GTP hydrolysis by dynamin finally leads to membrane
scission (McMahon and Boucrot, 2011).
Clathrin-independent endocytosis
In general, less is known about the mechanisms involved in CIE, and, in particular, how
cargoes are recruited. Caveolae are flask-shaped invaginations of the membrane that are
50–80 nm in diameter and enriched in cholesterol, sphingolipids and oligomeric caveolin-1.
Endocytosis through caveolae requires the function of dynamin (Mayor and Pagano, 2007). A
second type of lipid-raft-associated CIE involves clathrin- and dynamin-independent carriers
(CLICs) or glycosylphosphatidylinositol (GPI)-enriched early endosomal compartments
(GEECs), which are endomembrane compartments that are formed by clathrin- and
dynamin-independent endocytosis. It is thought that BAR domain proteins such as the Rho
GTPase activating protein 26 (ARHGAP26, also known as GRAF) confer membrane
curvature that might be sufficient for scission. The cargoes of CLIC and GEEC pathways
include GPI-anchored proteins, but given that many transmembrane proteins (including
flotillins) are found within CLIC and GEEC structures it is likely that these pathways constitute
a general internalisation route for a variety of proteins (Howes et al., 2010b; Hansen and
Nichols, 2009). Finally, macropinocytosis produces large endocytic vesicles (with a diameter
.500 nm), and this process is often associated with fluid uptake as well as internalisation of
membrane-associated proteins that are recruited into the macropinocytic cup. Growth-factor
stimulation promotes actin polymerisation and the formation of circular dorsal ruffles that is
associated with the initiation of macropinocytosis. However, little is known about the precise
mechanisms that govern scission and internalisation (Doherty and McMahon, 2009).
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glycoprotein (MAG) inhibits axonal growth
in neurons and repels growth cones by
increasing the local Ca2+ concentration,
thereby stimulating clathrin-dependent
endocytosis of b1 integrins to promote
growth cone turning (Hines et al., 2010).
Syndecan-4 can act as a co-receptor for
fibronectin (alongside integrins). In addition,
syndecan-4 engagement controls the
availability of a5b1 at the plasma
membrane in fibroblasts through a
mechanism that is consistent with caveolar
endocytosis of the integrin (Bass et al., 2011).
Post-endocytic trafficking andrecyclingInternalised integrins are rapidly trafficked
to early endosomes (EEs), where cargoes
are sorted for degradation or recycling
(Box 2; Caswell and Norman, 2006). In the
presence of fibronectin ligand, a5b1
integrin can be routed to late endosomes
and degraded (Dozynkiewicz et al., 2012;
Lobert et al., 2010; Tiwari et al., 2011), but
the majority of internalised integrins are
rapidly recycled back to the plasma
membrane (Bretscher, 1989; Bretscher,
1992). Integrins can follow the major
recycling routes of other cargoes, such as
the transferrin receptor (TfnR, also known
as TFRC) (Box 2) (Caswell and Norman,
2006; Caswell et al., 2009; Pellinen and
Ivaska, 2006). However, although there are
shared mechanisms between these two
functionally distinct cargoes, there are
also integrin-specific elements that
provide spatial and temporal control of
adhesion receptor availability at the plasma
membrane.
Rab4-dependent recycling
avb3 integrins recycle through the Rab4-
dependent ‘short-loop’ pathway (Box 2),
which returns the heterodimers from EEs
back to the plasma membrane without
transiting through the perinuclear recycling
compartment (PNRC) (Roberts et al., 2001;
Jones et al., 2009; di Blasio et al., 2010).
PKD1 (for protein kinase D1), when
autophosphorylated on Ser916 in response
to growth factor stimulation, interacts with
the extreme C-terminus of integrin b3, and
this interaction is required for Rab4-
dependent recycling of this integrin
(Woods et al., 2004; White et al., 2007; di
Blasio et al., 2010). Rab4-dependent
trafficking of avb3 is key to the control of
directionally persistent migration (di Blasio
et al., 2010; White et al., 2007; Woods et al.,2004) and branching morphogenesis of
endothelial vessels (Jones et al., 2009).Although a5b1 does not follow theRab4 recycling pathway in platelet-derivedgrowth factor (PDGF)-stimulated fibroblasts
(Roberts et al., 2001), recent studies haveindicated that b1 integrins can follow a Rab4-dependent route to the plasma membrane:
epidermal growth factor (EGF) promotes b1integrin recycling in HeLa cells in a mannerthat requires the raft-associated membrane
protein supervillin (Fang et al., 2010), andrecycling of inactive b1 integrins is Rab4-dependent in breast cancer cell lines (Arjonenet al., 2012).
Rab11- and Arf6-dependent recycling
Integrins can also follow a second, ‘long-
loop’ recycling pathway through the PNRC(Box 2), and this is dependent on theGTPases Rab11 and Arf6 and is linked to
cell migration (Powelka et al., 2004;Caswell and Norman, 2006; Roberts et al.,2004). For b1 integrins, many of themechanistic details are shared with other
cargoes. EHD1 (for EH-domain containing1), which is recruited to tubulesemanating from the PNRC by MICAL-like
1 (MICALL1) and phosphoinositides,regulates the recycling of both b1 integrinsand the TfnR (Jovic et al., 2007; Jovic et al.,
2009; Naslavsky and Caplan, 2011; Sharmaet al., 2009). In addition, Myotubularin, aphosphoinositide phosphatase, is required
for the exit of integrins from intracellularcompartments in Drosophila (Ribeiro et al.,2011). SNARE function is crucial indocking and fusion of vesicles throughout
the endosomal system, and several Q-SNAREs (e.g. syntaxin 6, SNAP29,syntaxin 4 and SNAP23) and R-SNAREs
(e.g. VAMP2 and VAMP3) have beenshown to regulate b1 integrin trafficking(Veale et al., 2010; Tiwari et al., 2011;
Skalski and Coppolino, 2005; Rapaportet al., 2010; Hasan and Hu, 2010).
Although integrins share much of thebasic machinery for Rab11-dependent
recycling with the TfnR, stages of thelong-loop recycling pathway that arespecifically required for integrin recycling
have been identified. Rab21 binds to aconserved motif within the a-subunits ofb1 integrin heterodimers and specifically
promotes their internalisation andtrafficking through early endosomes tothe PNRC (Pellinen et al., 2008; Pellinen
et al., 2006). Here, p120RasGAP (alsoknown as RASA1) displaces Rab21,thereby promoting the release of the
Box 2. Rab and Arf GTPases control endocytic recycling
Small GTPases are thought of as molecular switches that cycle between a GTP-bound ‘on’
state, and a GDP-bound ‘off’ state, in response to the activities of guanine nucleotide
exchange factors (GEFs) and GTPases activating-proteins (GAPs), respectively. This switch
controls the ability of the small GTPases to interact with their effector molecules. There are
more than 60 Rab proteins and six Arf proteins that localise to distinct membrane
compartments, and these families of GTPases control most of the known intracellular
trafficking events in eukaryotic cells (Grant and Donaldson, 2009; Stenmark, 2009).
The function of early endosomes, where internalised cargoes are sorted, is dependent on
Rab5. Early endosomal cargo can be directed to late endosomes through a maturation
process that involves the exchange of Rab5 for Rab7 on the endosomal membrane (Rink et
al., 2005). Late endosomal proteins are either lysosomally degraded or recycled through
alternative pathways [e.g. the Rab9–retromer pathway (Seaman et al., 1998; Lombardi et al.,
1993) or the Rab25–CLIC3 (for chloride intracellular channel 3) pathway (Dozynkiewicz et al.,
2012)]. Early endosomal cargoes can also be recycled through ‘short-loop’ (fast) or ‘long-
loop’ (slow) recycling pathways. The short-loop recycling pathway is regulated by Rab4 and
Rab35, and is characterised by cargoes that exit the early endosome to recycle to the plasma
membrane. Long-loop recycling involves trafficking of cargo through the perinuclear recycling
compartment (PNRC) and is thought to primarily require the activity of Rab11 and Arf6, but
roles for Rab8, Rab10 and Rab22a have also been described (Grant and Donaldson, 2009;
Stenmark, 2009).
Rab and Arf proteins act together to coordinate the main steps of membrane trafficking. For
example, Rab9 mediates vesicle budding by recruiting sorting adaptors to promote cargo
selection into budding recycling vesicles on late endosomes (Carroll et al., 2001). Rab
GTPases can also regulate tethering, docking and fusion events by recruiting tethering
factors, SNAREs and the exocyst complex, and they can promote vesicle transport by
recruiting both microtubule- and actin-based motor proteins directly or through additional
effectors (Grant and Donaldson, 2009; Stenmark, 2009). By contrast, Arf6 regulates
phospholipid signalling, which is important in many trafficking steps, but can also provide links
to microtubule motors kinesin and dynein through interactions with scaffold proteins and
provide a link to vesicle transport (Donaldson and Jackson, 2011).
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integrin-containing vesicles from theperinuclear region and their recycling to
the plasma membrane (Mai et al., 2011). Incarcinoma cells, Rab-coupling protein(RCP), a member of the Rab11-familyinteracting proteins (RAB11FIPs), which
act as Rab11 effectors, has a crucial role inregulating integrin recycling but isdispensable for TfnR recycling through
Rab11 recycling endosomes (Caswell et al.,2008; Muller et al., 2009). Both a5b1 andavb3 integrins follow a Rab11-dependent
route in unstimulated fibroblasts. Thisrequires the inactivation of glycogensynthase kinase 3 beta (GSK3b) throughphosphorylation by AKT (also known as
PKB) (Roberts et al., 2004). Serumstimulation can also promote traffickingof b1 integrins through this route, and
AKT activity is required to phosphorylateACAP1 (for ArfGAP with coiled-coil,ankyrin repeat and PH domains 1), which
recruits b1 integrins to a recycling coatcomplex that contains clathrin (Powelkaet al., 2004; Li et al., 2005; Li et al., 2007).
PKCe phosphorylates vimentin, a type IIIintermediate filament protein, and releasesb1-integrin-containing vesicles fromintermediate filaments in the perinuclear
region to allow recycling of b1 integrins(Ivaska et al., 2005; Ivaska et al., 2002).The events regulated by AKT and PKCeare specific to integrin trafficking, and arenot required for recycling of TfnR.
Cellular functions of integrintraffickingMany of the studies described above providecompelling evidence that integrin trafficking
contributes directly to cellular processessuch as cell migration. Endocyticmechanisms can contribute to the turnover
of adhesion complexes, and, in this way,directly regulate interactions with the ECMand proteins involved in adhesion signalling
[e.g. focal adhesion kinase (FAK)phosphorylation (Ezratty et al., 2005;Ezratty et al., 2009)]. It is unclear,however, whether trafficking pathways can
influence the activation status of integrins orthe ability of integrins to mediate adhesivecontacts with the substratum, and in some
cases this is evidently not the case (Caswellet al., 2007; Caswell et al., 2008). Trackingintegrin trafficking using antibody surface-
labelling approaches has indicated thatinternalised integrins can subsequentlylocalise to focal complexes and focal
adhesions, but only after extended periods(i.e. hours) following antibody exposure (Guet al., 2011). Given that the t1/2 of integrin
recycling is often ,15 minutes, this could
indicate that integrins do not recycle directly
to focal adhesions (Caswell and Norman,
2006). Accumulating evidence suggests that
instead integrin trafficking influences
intracellular signalling, either directly or by
influencing trafficking of other cargoes.
Integrin trafficking dictates Rho GTPase
signalling
Integrin trafficking pathways can directly
impact on Rho GTPase signalling. Rab21-
mediated recycling of a5b1 is required for
the activation of RhoA at the cleavage
furrow and, hence, the completion of
cytokinesis (Pellinen et al., 2008). Rho
GTPases are master regulators of the
cytoskeleton, and balancing RhoA and Rac
activities is crucial for controlling the
directional persistence of migrating cells
(Danen et al., 2005). In fibroblasts, Rab4-
dependent recycling of avb3, and the
activity of this integrin, suppresses
recycling of a5b1, thereby promoting
directionally persistent migration that is
characterised by a broad leading
lamellipodium (Caswell et al., 2008; White
et al., 2007). Inhibition of avb3 recycling or
ligand binding enhances recycling of a5b1
and signalling through the ROCK (RhoA–
Rho-associated, coiled-coil containing
protein kinase) pathway, which, in turn,
leads to phosphorylation of the downstream
effector cofilin and rapid random migration
(White et al., 2007).
Integrin trafficking promotes tumour cell
invasion and metastasis
Integrin trafficking influences the ability
of cells to move in three-dimensional
matrices, as well as on two-dimensional
substrates. Hypoxia-driven invasion
through a laminin-rich matrix requires
Rab11, and there is evidence that this
involves the Rab11-dependent recycling
of a6b4 (Yoon, 2005). avb6 promotes
invasion in squamous cell carcinoma, and
the interaction of this integrin with HAX1
allows its clathrin-dependent endocytosis,
which in turn promotes carcinoma cell
motility in organotypic culture (Ramsay
et al., 2007).
Inhibition of avb3, or expression of
cancer-associated mutant forms of p53,
promotes a5b1 recycling in carcinoma cell
lines (Caswell et al., 2008; Muller et al.,
2009). This requires the interaction of b1
integrin with RCP (Caswell et al., 2008),
which is itself upregulated in breast cancer
(Zhang et al., 2009). The actin nucleation
promoting factor (NPF) WASH (for WAS
protein family homologue) has a role in
a5b1 recycling through this pathway,
presumably by remodelling actin on
endosomes (Zech et al., 2011). RCP-
dependent a5b1 trafficking promotes
invasive migration in three-dimensional
matrices, which is characterised by the
extension of invasive pseudopods. The
production of phosphatidic acid within
the pseudopod tip is required to localise
RCP and permit integrin recycling
(Rainero et al., 2012). Rather than
influence a5b1 activity, a5b1 and RCP
recruit EGF receptor 1 (EGFR1) and
coordinate its recycling, which,
in turn, potentiates EGFR activation and
downstream signalling to promote invasion
(Caswell et al., 2008; Muller et al., 2009).
In this context, integrins and RCP act as
key components of a ‘recyclosome’
complex, which recruits receptor tyrosine
kinases (RTKs), including EGFR1, ErbB2
and Met, promotes their recycling and
potentiates signalling to induce metastasis
(Muller et al., 2009; Caswell et al.,
2008; Muller et al., 2012; P. T. Caswell,
unpublished observations). Interestingly, in
neuronal axons, Rab11 and RCP can
control trafficking of b1 integrins to
promote axonal extension (Eva et al.,
2010), indicating that this mechanism is
not unique to cancer cells.
Overexpression of Rab25 (also known
as Rab11c) is associated with aggressive
ovarian cancers (Cheng et al., 2004).
Rab25 specifically regulates trafficking of
a5b1 by directly interacting with b1
integrin (Caswell et al., 2007). Inactive
integrins that become internalised into
Rab25 vesicles at the front of invasive
cancer cells are recycled back to the
plasma membrane within this subcellular
region, thus promoting their spatial
restriction (Caswell et al., 2007; Caswell
and Norman, 2008; Caswell et al., 2009).
Those integrins that remain in the active
conformation following internalisation,
however, are sorted through Rab25-
positive late endosomes to lysosomes,
moving from the front of the cell towards
the rear. CLIC3 regulates the subsequent
recycling of active a5b1 from lysosomes
to the plasma membrane. This allows
Rab25 to control pseudopod extension at
the front of the cell and coordinate
retraction of the cell body by trafficking
active integrins towards the rear of the cell
to promote integrin signalling and forward
movement (Dozynkiewicz et al., 2012).
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Integrin trafficking and signallingregulates endocytic transport ofother cargoes
Integrin trafficking pathways can indirectlyinfluence intracellular signalling bycontrolling endocytic trafficking of other
cell surface receptors. In BMMSCs, softsubstrates promote endocytosis of b1integrins through caveolae, and this is
required for the internalisation of bonemarrow morphogenetic protein (BMP)receptor IA (BMPRIA). BMPRIA
colocalises with integrins in an intracellularcompartment, which implies that theseendocytic cargoes co-internalise, and thatthis, in turn, inhibits BMP-induced SMAD
signalling, which allows cells to differentiatealong the neuronal rather than osteogenic ormyogenic lineage (Du et al., 2011).
avb3 integrin controls receptortrafficking, suppressing the recycling ofa5b1 integrin, and thereby leads to changes
in Rho GTPase signalling in fibroblasts andpromotes receptor-tyrosine kinase traffickingand signalling in carcinoma cells asdiscussed above. Inhibiting avb3 with
small molecule inhibitors such asCilengitide in endothelial cells drives Rab4-dependent vascular endothelial growth factor
receptor 2 (VEGFR2) recycling, protectingthis receptor from degradation in thepresence of VEGF ligand and increasing its
levels on the cell surface (Reynolds et al.,2009). This promotes VEGF-drivenendothelial cell migration, sprouting of
aortic explants and tumour angiogenesis invivo, which ultimately leads to enhancedtumorigenesis.
Clearly, integrins are cargoes of
intracellular trafficking pathways;however, the relationship betweenintegrins and endocytic traffic is complex
because integrins themselves govern signalsthat control endocytic flux. In someinstances adhesion negatively regulates
endocytosis: engagement of integrin b1 byligand can slow the dynamics of clathrin-coated structures, and adhesion of cells tofibronectin decreases the rate of TfnR
endocytosis (Batchelder and Yarar, 2010).By contrast, b1 integrin engagementpromotes endocytosis of the platelet-
derived growth factor receptor (PDGFR)during fibroblast chemotaxis. In thisscenario, integrins are required to maintain
the stability of the actin NPF neuralWiscott–Aldrich syndrome protein (N-WASP), which controls the formation of
CDRs and the internalisation of PDGFthrough macropinocytosis (King et al.,2011). Signalling from b1 integrins is also
required for the fusion of vesicle-associated
membrane protein 7 (VAMP7) vesicleswith the plasma membrane in extendingneurites on laminin substrates (Gupton and
Gertler, 2010).
Integrin engagement also regulates thetrafficking of caveolae and/or lipid rafts.Loss of adhesion triggers internalisation of
lipid rafts, which is controlled by the shiftof phosphorylated caveolin from adhesioncomplexes to caveolae (del Pozo et al.,
2005; del Pozo et al., 2004). Following re-adhesion, lipid rafts recycle to the plasmamembrane from the PNRC in an Arf6-dependent manner (Balasubramanian et al.,
2007). RalA regulates exocytosis throughthe exocyst complex (Balasubramanianet al., 2010), which provides a platform
for the activation of Rac. Keratinocytesthat lack integrin b1, or the adhesionsignalling protein integrin-linked kinase
(ILK), also show a defect in themembrane targeting of caveolae. Caveolarendocytosis occurs independently of b1
integrins and ILK, but integrin signallingpromotes the recruitment of a complexcontaining IQGAP (IQ motif containingGTPase activating protein) and mDia1
(also known as DIAPH1) to the plasmamembrane, which in turn locally capturesand stabilises microtubules to permit re-
exocytosis of caveolin-1 (Wickstrom et al.,2010).
Future perspectivesIntegrin trafficking is a dynamic processthat is important for the regulation ofcellular processes, including migration
and cytokinesis. Technical limitations havehindered our understanding of integrintrafficking in vivo, but these are beingovercome in some model systems (Yuan
et al., 2010; Ribeiro et al., 2011), andfurthering these studies to directly visualisetrafficking events in vivo is a priority.
Trafficking pathways can promotespatial restriction or en masse movementof integrins, and, interestingly, some
regulators, such as Rab25, have a role inboth (Caswell et al., 2007; Dozynkiewiczet al., 2012). Further studies are necessary to
determine the sorting steps that controlthe decision to recycle integrins to themembrane from which they were initiallyinternalised or to more distal sites. It will
also be important to determine how thedirectional movement of integrins interfaceswith downstream signalling pathways.
Unlike most cargoes, integrins are almostuniquely positioned in that they are cargoesfor pathways that they themselves regulate.
Further investigation is needed to determinewhether integrins form central componentsof multi-cargo trafficking complexes that
are controlled by adhesion signalling. Thisalso raises an interesting question: areintegrins able to elicit signals when they
are transiting the endosomal system?The presence of active integrins onendomembrane compartments has nowbeen noted by several investigators, and
the recent identification of the integrininactivator sharpin (Rantala et al., 2011)suggests that integrins could remain active
on intracellular compartments untilinactivated. Endomembranes are surfacesthat provide a scaffold for signalling
pathways such as the AKT (Schenck et al.,2008) and ROCK–myosin light chain 2(MLC2) pathways (Sturge et al., 2006). This
opens up the exciting possibility thatintegrins could associate with downstreamsignalling components on endosomes topropagate signals from a lipid and protein
environment distinct from the adhesionplaque.
Funding
The work of our laboratories is supported by
the Wellcome Trust (P.T.C.) and Cancer
Research UK (J.C.N). R.E.B is supported by
a BBSRC studentship and a Research Impact
Scholarship from the ‘Your Manchester’
Fund.
A high-resolution version of the poster is available for
downloading in the online version of this article at
jcs.biologists.org. Individual poster panels are available
as JPEG files at http://jcs.biologists.org/lookup/suppl/
doi:10.1242/jcs.095810/-/DC1.
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