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In addition to their structural role, integrins also regulate intracellular signalling

pathways that modulate cellular functions. These signalling pathways are induced

follow ing binding o f integrins to their ligands. Integrin ligands are generally

immobilised, consequently the signalling is usually localised to discrete regions o f the

plasma membrane. Integrins are able to signal through the cell membrane in either

and Ruoslahti, 1999). The extracellular ligand binding activity o f integrins, i.e.

adhesive affinity is modulated in response to cellular physiology from the inside o f

the cell (inside-out signal transduction). In this process, there is interaction o f

specific intracellular proteins with the integrin cytoplasmic tail to promote a structural

change in the integrin conformation that is transmitted across the plasma membrane

to the extracellular domain, causing altered ligand-binding affinity. Alternatively, the

binding o f the ECM ligand may elicit signals that are transmitted into the cell to

induce changes in protein activities or gene expression (outside-in signal

transduction).

As integrins cooperatively bind to the multivalent ECM ligands, they become

clustered in the plane o f the cell membrane at distinct sites known as focal contacts or

focal adhesions. These contain ECM proteins, integrins and cytoskeletal proteins,

which assemble into aggregates on each side o f the membrane and provide a link

between the ECM proteins and the cytoskeletal-signalling com plex (Burridge and

Chrzanowska-Wodnicka, 1996). The different actin-associated proteins present

within this complex include, a-actinin, vinculin, tensin and paxillin, which promote

assembly o f actin filaments figure 1.4). The reorganization o f actin filaments into

larger stress fibres, in turn causes more integrin clustering thus enhancing matrix

ECM p r o t e i n s E x tr a c e ll u la r M atrix C y t o s k e l e t o n talin a-actinin paxillin vinculin tensin actin

Figure 1.4 Transmembrane connections between the extracellular matrix

(ECM) and the cytoskeleton. Ligand activation o f integrins by ECM proteins

induce the recruitment o f cytoskeletal proteins which promote the assembly o f actin filaments, in turn inducing integrin aggregation at the cell membrane.

The cytoplasmic tails o f integrins provide anchors for the actin cytoskeleton and are

generally short and always devoid o f enzymatic features, therefore integrins require

an association with adaptor proteins that connect the integrin to the cytoskeleton,

cytoplasmic kinases, and trans-membrane growth factor receptors in order to

transduce signals (Giancotti and Ruoslahti, 1999). The detailed sequence o f events

following ECM binding to the integrin is unclear but it is thought that integrins

undergo a conformational change, which allows the intracellular P-subunit

cytoplasmic domain to interact directly with focal-adhesion proteins such as talin and

a-actinin, which then interact with both vinculin and paxillin (figure 1.4, Liu et al,

2000). However, it is now becoming clear that, like binding to the ECM, integrin

signalling is determined by both a and p subunits (Giancotti, 2000).

Many, perhaps all integrins except a6 p 4 , use a core signalling machinery that can

regulate the actin cytoskeleton and activate the mitogen-activated protein kinase

(MAPK) pathway (Howe et al, 1998). Subsets o f integrins and even individual

integrins recruit specific signalling components. The protein tyrosine kinase, focal

adhesion kinase (FAK) is central to many integrin-mediated intracellular signalling

events, regulating cell adhesion and/or migration (Schlaepfer and Hunter, 1998).

Following attachment to the ECM, FAK localises to focal adhesions where it is

tyrosine phosphorylated. It then combines with Src-family kinases (Src or Fyn),

which phosphorylate paxillin and p i 30 cas. Both o f these m olecules are then able to

initiate a signalling cascade by recruiting various adaptors and signalling

activation. These include small GTPases, protein tyrosine kinases, Src family kinases

and adaptor proteins (Table 1.1). The mechanism by which these proteins are

activated, how they couple with each other, and how their activation by integrins

affects different cell functions are still under investigation (Miranti and Brugge,

2002).

Sm all G TPases Rho, Rac, cdc42

Protein tyrosine kinases FAK

Src fam ily kinases Abl, Syk/ZAP, Csk, Ras, Raf, Mek,

Erk, PKC, Cbl, Pyk2, protein kinase A,

Etk, Ack2, EAR, PEST

A daptor proteins Crk, Nek, Grb-2

Table 1.1 Signalling Proteins linked to Initegrin A ctivation.

Integrins often synergise with growth factor receptors to enhance their activity since

they have many common elements in their signalling pathways. In addition, they are

able to regulate sodium-proton antiporters and protein kinase C has been shown to

associate with integrin-containing focal adhesions. Integrins are also able to inhibit

or activate other integrins, resulting in local modulation o f cell adhesiveness.

Therefore, there is the potential for a broad range o f integrin signals however, if

uncontrolled they m ay result in tumourigenesis. The MAPK pathway is particularly

important in promoting cancer growth

in vivo,

highly activated during the late progression o f colorectal cancer (Licato et al, 1997).

High frequencies o f MAP kinase activation have also been observed in a large study

o f primary tumours o f diverse origins (Hoshino et al, 1999) and very significantly, a

highly potent inhibitor o f MAP kinase activation has been identified which is capable

o f inhibiting human cancer growth in immune-deficient m ice (Sebolt-Leopold et al,

1999).

The MAP kinase pathway is initiated by activation o f Ras leading to the sequential

stimulation o f the protein kinase Raf. This is then able to activate one o f the three

separate M APK pathways: activation o f MEK followed by the extracellular signal

regulated kinases, ERKl and ERK2, the p38 MAPK pathway and finally the Janus

kinase (JNK) pathway. A number o f integrin and growth-factor signals converge at

multiple points within these pathways, suggesting that there is a great deal o f

com plexity within integrin signalling. This complexity, where multiple and possibly

parallel and intersecting pathways activate specific signalling proteins is best

illustrated for ERK activation. Although FAK is capable o f activating ERK through

the recruitment o f Grb2, She or Src, other mechanisms that result in ERK activation

have also been described. These include integrin coupling with caveolin leading to

the recruitment o f Fyn resulting in She phosphorylation and transactivation o f the

epidermal growth factor (EOF) receptor by integrins (Schwartz and Ginsberg, 2002,

Giancotti and Ruoslahti, 1999, Howe et al, 1998, Hynes, 2002). Figure 1.5 shows a

Cytokines

EGF Receptor Tyrosine Kinase Substrate Rac/ cdc42 Grb2 PKCa Sos MEKK5 Ras _MEKK PLCy GTP Raf MKK3 JNKK/SEK MEK P38 / RK JN K/SAPK MAPK MAPKAP2 Elk1 ATF2 C-Jun NUCLEUS

Figure 1.5 Integrin Mediated Signalling of the MAP Kinase Pathways

A range o f stimuli via integrins can activate the three intracellular MAP Kinase signalling pathways. This leads to the activation o f transcription factors within the nucleus resulting in regulation o f gene expression and function.