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Beta-1 integrin variants in myogenesis and cytoskeletal signaling

van der Fliers, A.

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Citation for published version (APA):van der Fliers, A. (2001). Beta-1 integrin variants in myogenesis and cytoskeletal signaling.

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CHAPTERR 1

Generall introduction

--

GeneralGeneral introduction

Generall introductio n

Duringg development multicellular organisms and tissues are formed and cells are held togetherr by adhesion molecules. Adhesion between cells and between cells and the extracellular matrixx is mediated by different members of the integrin, cadherin, immunoglobulin, and selectin familiess [1]. Besides their role in the maintenance of tissue structure and polarity, cell adhesion moleculess are also involved in the regulation of cell proliferation and differentiation [2, 3].

Thee work described in this thesis focuses on a variant of the cytoplasmic domain of the p 1 integrinn subunit. We determined the expression pattern and cytoskeletal interactions specific for this variant.. Furthermore, we studied the epithelial-mesenchymal-like transition that follows upon (51 expressionn in epithelial cells that lack endogenous pi. In this chapter, the integrin protein family andd their splice variants will be introduced. Several aspects of the biological function of integrins in generall and in myogenesis in particular will be addressed.

Integri nn protein family, extracellular matrix and ligands Integrinss are a family of glycosylated, heterodimeric transmembrane adhesion receptors that

consistt of a non-covalently bound a- and p-subunit. Most integrins bind to extracellular matrix (ECM)) components while others bind to receptors on other cells. The name integrin refers to their functionn in the integral linkage of the cells' exterior (ECM) to the cells' interior (cytoskeleton). By reasonn of their fundamental role in cell adhesion, migration and signal transduction, integrins play ann important role in various processes such as immune function, platelet aggregation, tissue repair, invasionn and metastasing of cancer cells. Integrins are highly conserved in the taxonomy from spongess to humans, although the complexity of variants and their redundancy increases later in taxonomyy [4, 5]. To date 18 a and 8 P-subunits have been identified in vertebrates, which form at leastt twenty-four different heterodimers. (Table I) (see for recent review [6]). The combination of thee a and P-subunit determines the ligand specificity of the integrins. Many integrins have binding specificitiess for the same ligands (see Table I) and it is the combination of the integrin expression/activationn pattern and the available ligand that determines the in vivo binding.

Thee extracellular matrix (ECM) is a complex network of high molecular weight proteins (e.g. laminins,, collagens, fibronectin) and polysaccharides, which are deposited by the surrounding cells [7].. The ECM provides structural support for cells but also acts as a physical barrier or selective filterr to soluble molecules (e.g. basement membrane). Finally, ECMs sequester growth factors and aree critical for the differentiation and growth of a number of cell types. This latter phenomenon is alsoo known as anchorage-dependent growth.

Integrinss containing the a4, a5, a8, allb and av subunits bind to ECM components that containn the RGD (Arg-Gly-Asp) sequence as present in fibronectin. Other integrins bind to laminins andd collagens, of which the cryptic RGD sites are thought to be normally inaccessible for binding. Inn general, integrins from haematopoietic cells bind to receptors on other cells (e.g. ICAMs and VCAMs)) or plasma proteins that are deposited at sites of injury (e.g. fibrinogen, von Willebrand factor)) and complement factors (e.g. iC3b). Several pathogenic agents bind to integrins, such as snakee venoms (disintegrins) which prevent platelet-mediated blood plug formation. Furthermore, micro-organismss (Lyme disease spirochete, Borrelia burgdorferi; Yersinia invasin protein) and virusess (echo-; adeno-; papilloma-; foot-and-mouth-virus; HIV Tat protein) use the integrins as their

9 9

ChapterChapter 1

Tablee I Integrins s

Integrin n

all pi a22 pi

a33 pi

a44 pi

P7 7

a55 pi

a66 pi p4 4

a77 pi a88 pi a99 pi

alOpl l a l l p l l

avv pi

P3 3

P5 5 p6 6 P8 8

andd their ligands

ECM M Co;; Ln Co;; Ln

Ln;; Co;

Fn n

Fn n

Fn n

Ln n Ln n Ln n Fn;; Tn; Tn;Tn; Op; Co;Ln n Co o Co o Fn;; Vn

Vn;; Tn,

Rn n

Nn n

Fn;; Tsp; Op

Vn n Fn;; Tn Co;; Ln; Fn n

Soluble e

------pp-vWF, , tTG;; FXIII

--

tTG G

--------pp-vWF; ; tTG;; FXIII

------Fg;; vWF

--TGFP-LAP P

--

Ligand d Cell-cell l

------VCAM-1 1

VCAM-1; ; MadCAM M ADAMM 15 ADAM1,2,9 9

------VCAM-1; ; ADAM-12,15 5

------ADAM-15, , MMP2 2

------

Pathogen n

. . echoviruss 1 YersiniaYersinia (invastn) YersiniaYersinia (invasin)

--

Yersinia;Yersinia; Borrelia; Shigella (Ipa)

papillomaa virus; Yersinia (invasin) . . . . _ _ . .

_ _ --echoviruss 22 snakee venoms, disintegrins;

adenovirus;; HIV Tat

adenoviruss 2, HIV Tat foot-and-mouthh disease virus

--

a l lbp33 Vn; Fn

aLL P2 a M p2 2 aXX p2 a D p2 2 aEE p7

Fg;; vWF

ICAM1-6 6 Fg;iC3b;; FX ICAM1; VCAM 6 Fg;; iC3b

ICAM3,6;; VCAM1 E-cadherin n

Borrelia;Borrelia; disintegrins; Ticks;; Leech (decorsin, ornatin)

Candida;Candida; Borrel ia.

ADAM == A disintegin And Metalloprotease, Co = collagen, Fg = fibrinogen, Fn = fibronectin, FX=coagulation factor X,, FXIII = coagulation factor XIII , iC3b=inactivated complement component C3b, [CAM = Intra-Cellular Adhesion Molecule,, Ln = laminin, MMP = Matrix Metallo Protease, Nn = Nephronectin, Op = osteopontin, pp-vWF= prepro von Willebrandd Factor, Rn = reelin, TGF0-LAP= TGFp latency-associated peptide, Tn = tenascin, Tsp = thrombospondin, tTG== tissue transglutaminase, VCAM = Vascular Cell Adhesion Molecule, Vn = vitronectin, vWF = von Willebrand Factor. .

receptorss (reviewed in [8]). Interaction between a6pl on the egg cell and ADAM-1 and -2 (A

Disintegrinn And Metallo-protease) on sperm cells has been implicated in egg-sperm cell fusion [9,

10]] and other ADAMs have also been shown to interact with integrins.

Despitee the overlap in binding specificity of many of the integrins, the loss of nearly each

integrinn a- or p-subunit leads to biological defects as shown in genetically modified mice. These

genee knock-out studies show that the loss of most integrin subunits is lethal at embryonic stages or

shortlyy after birth (reviewed in [11-13]).

10 0


Integrin ss in disease Mutationss in the integrin p2-subunit lead to leukocyte adhesion deficiency (LAD1), and

mutationss in either the allb or p3-subunit result in the bleeding disease Glanzmann's thrombastheniaa [14]. Junctional epidermolysis bullosa associated with pyloric atresia (PA-JEB), a skinn blistering disease, is due to mutations in genes encoding either the a.6 or p4 integrin subunit [15-17].. Loss of the a6p4 integrin results in rudimentary hemidesmosomes and subsequent detachmentt of the epidermis from its basal lamina upon physical stress [18, 19]. Similarly, mutationss in the a 7 integrin subunit have been found to result in a mild form of progressive muscularr dystrophy [20, 21]. Alternatively, reduced levels of laminin (a2-chain) in certain muscularr dystrophies result in decreased levels of the a7pl integrin [22-24].

Finally,, integrins play an important role in various aspects of cancer. Integrin expression patternss are altered in metastatic cells, resulting in changes of cell adhesion and migration which oftenn lead to the detachment of cells from the primary tumor, migration and invasion into the target organ.. These metastatic cells gain the capacity to survive without adhesive cues also named anchoragee independent growth. In addition, integrins play a role in the neovascularization of tumors,, that provides the tumor cells with the necessary nutrients for proliferation [25, 26].

Integri nn structure Thee ligand-binding site of integrins is present in the globular head domain formed by the a

andd p-chains, while the rest of the extracellular domain forms the membrane-proximal stalk-like structure.. The small cytoplasmic tails are approximately 30-50 amino acids long and do not contain catalyticc or consensus protein-protein binding motifs. An exception is the cytoplasmic domain of P4,, that contains two pairs of fibronectin-III repeats and is approximately 1000 amino acids long. Thee extracellular domains of several integrin a-subunits (i.e. the collagen binding a 1, a.2, alO, all 1, the E-cadherin binding aE and the leukocyte integrins aD aX, aM, aL) contain a 200 amino acidd insertion, the I domain. Structural analysis of integrin I domains have revealed the presence of aa characteristic metal ion-dependent (Mg2+) adhesion site motif (MIDAS) shown to be critical for ligandd binding [6, 27]. A similar I domain is predicted to be present in the extracellular part of the P-chainss [28], Comparisons of structure have suggested that the seven repeat motifs, present in the extracellularr domain of all the a-subunits, fold into a seven-bladed propeller structure that forms onee globular domain with the ligand-binding site on the upper surface [29]. The optional I domain iss inserted between blades number two and three of this propeller and the divalent cation-binding sitess are located on the lower surface of blades 4-7 [30]. Besides glycosylation of both the a- and p-subunits,, post-translational cleavage of a-subunits, that lack an I domain results in a disulfide-linkedd heavy and light chain. The noncleaved a4-subunit forms an exception to this rule. Post-translationall cleavage has been implicated in the modulation of the affinity of a6 integrins [31].

Integri nn activation and deactivation: inside-out signaling

Thee affinities of integrins for their ligands may vary depending on the cell type in which they aree expressed [32-34]. Leukocyte and platelet integrins require activation induced by agonist (i.e thrombin,, epinephrin) for binding to their ligand. This inside-out signaling has been extensively studiedd in platelets and requires the activity of protein kinase-C (PKC) and Rho-like GTPases.

11 1

ChapterChapter I

Thee current model is that the cytoplasmic domain of the a-subunit inhibits, directly or

indirectly,, the interaction of the (3-subunit with cytoskeletal components. This inhibition is relieved

uponn binding of a ligand to an integrin or by signaling induced by another agonist possibly by

inducingg a conformational change which either splits or slides the a- and p-cytoplasmic tails

relativelyy to each other [35, 36]. Mutation and deletion studies have indicated a crucial role of the

conservedd a (GFFKR) and p (LLxxxihDR) membrane proximal sequences. Changes in integrin

affinityy (interaction of a single integrin with its ligand) are the result of conformational changes in

thee integrins, which can be detected by antibodies that identify activation dependent neo-epitopes.

Certainn cations (e.g. Mn2') and antibodies may activate integrins by stabilizing or inducing their

activee conformation [32]. Integrin-ligand binding is also increased by avidity. The concerted

interactionn of clustered receptors with a ligand can be induced by multiple binding sites in the

ligandd (polyvalent) and/or by cytoskeletal interactions that organize integrin clusters from within

thee cell. For example, in leukocytes it has been found that the integrin subunit p2 first needs to be

releasedd from talin in the cytoskeleton, before it can become clustered and bind ligand.

Subsequentlyy the integrin associates with a-actinin, which leads to a stable interaction with the

cytoskeletonn [37].

Inn adherent cells, integrins are apparently in a constitutive active conformation.

Phosphorylationn of integrin cytoplasmic domains [38, 39] and proteolytic cleavage [40-42] have

beenn suggested to affect integrin-ligand binding. However, there is no consensus as yet on the

generall mechanism that regulates integrin-mediated cell adhesion. Cell migration is a process that

requiress the precise regulation of integrin-mediated adhesion/release [43]. The repeated cycle of the

assemblyy and disassembly of adhesive complexes (focal adhesions) at the cells1 front, where the

polymerizationn of actin drives the protrusion of the cell membrane, cytoskeletal contraction and

detachmentt of the rear results in a net displacement of the cell. It has been found in migrating

fibroblastt that a pool of integrin surface molecules is internalized and recycled to the cells posterior

[44-46].. Alternatively, ligand-bound integrins in the rear of the cell are left as footprints on the

underlyingg substrate [46, 47].

Splicee variants of integrins

Alternativee mRNA splicing leads to additional complexity of the integrin family [48].

Variantss of both the extracellular and cytoplasmic domains have been reported. Alternative

extracellularr domains may account for different ligand binding affinities or variations in the state of

activation,, while variants of the cytoplasmic domain may modulate integrin activity, cytoskeletal

associationss and/or signaling events.

Extracellularr variants of Drosophila aPS2 differ in their affinities for D-laminin [49].

However,, no differences in laminin binding affinity of the extracellular domain variants of a6 (XI

orr X1X2) have been reported [50, 51]. Activation and migration on different laminin isoforms of

thee variants of the a7 extracellular domain (XI , X2) was found to be regulated in a cell type

specificc manner [52, 53]. Similarly, a single amino acid polymorphism in the extracellular domain

off P3 leads to a different state of platelet activation [54]. The biological relevance of two secreted,

truncatedd products of the extracellular domains of p3 and al lb remains to be determined [55. 56].

Variantss of the cytoplasmic domain have been found of the three closely related laminin

receptorr a-chains: i.e. a3A, B; a6A, B; a7A. B, and C. Their sequences differ after the membrane

proximall GFFKR motif, which is crucial for correct heterodimerization and the regulation of

12 2


bindingg activity of the integrins. Splicing of the mRNA of these laminin receptors is regulated in a developmentallyy and tissue specific manner and is conserved in men and mice. The homology betweenn the A and B cytoplasmic variants of different a subunits is higher than that between the variantss of the same a subunit [48]. During embryogenesis the first variants expressed are the a3B, a6BB and a7B cytoplasmic variants, which later in development are replaced by the A variants of a subunitss in many tissues. The function of this developmental regulated switching of variants is currentlyy unknown. Notwithstanding the observation that ct6A and cc6B show differences with respectt to phosphorylation and cell migration in vitro, no specific differences have been detected withh respect to their ligand specificity and/or activation [57]. Similarly, no differences have yet beenn found for the variants of the cytoplasmic domain of a3 and a7 with respect to regulation, migrationn and ligand binding. Although the lack of cc6 expression results in embryonic defects and deathh around birth due to severe blistering of the skin [58, 59], genetically modified mice that expresss only the cc6B integrin variant have a normal phenotype [60]. The only effect detected in thesee mice is a reduction in the number of lymphocytes and a decreased migration of lymphocytes onn fibronectin in vitro. Thus the switching from a6B to a6A appears not to be crucial for embryonicc development [60].

Cytoplasmicc variants of several integrin p-subunits have been described. The best studied are thee five cytoplasmic variants of the pi subunit, the most abundantly expressed integrin subfamily: pii A, B, Cl, C2 and D (this thesis, [48]). Antibody inhibition studies and studies with mice in which thee gene encoding pi has been disrupted by homologous recombination have demonstrated the criticall role of pi-integrins in development, cell differentiation, migration and the assembly of the extracellularr matrix proteins [13]. piA is present in all tissues, except cardiac and skeletal muscle, whichh instead express the plD variant (this thesis). The BIB and piC variants are minor forms and aree present in man, but not in the mouse [61]. Both variants behave as inactive integrins, which is probablyy due to their failure to become localized at focal adhesions. Expression of piB or piC variantt in cells decreases the ability of cells to adhere and to migrate on extracellular matrix components.. Similarly, expression of plB or piC inhibits DNA synthesis and cell proliferation, whereass pi A does not inflict such inhibition [62-68].

Thee plD isoform is very homologous to pi A. Both variants share the first 24 amino acids of theirr cytoplasmic domains and the two NPXY focal contact localization sequences (cyto-2 and -3 domains)) in the C-terminus are also conserved. In non-muscle cells, transfected piD is localized in focall contacts and activates focal adhesion kinase (FAK), MAP-Kinase and RhoA, similarly to pi A ([69,, 70, 71], this thesis). However, Baudoin et al. [72] have shown that piA and piD are not functionallyy equivalent in embryonic development. The replacement of piA by piD results in abnormall migration of neuroepithelial cells and embryonic lethality in mice, despite the finding that reciprocall replacement in striated muscle of piD by piA does not lead to severe abnormalities in vivovivo [72].

Variantss of the cytoplasmic domain of p3 (A-C) and p4 (A-E) have also been reported. The P3BB variant is similar to BIB; it lacks the NPXY motifs, which results in the defective localization inn focal adhesions and impaired phosphorylation of FAK. In addition expression of p3C in cells resultss in reduced adhesion and decreased cell survival [73]. The p4A-subunit is the most abundant P44 variant, while the function and characteristics of the other minor p4 variants remain to be elucidated. .

13 3

ChapterChapter 1

Studiess using mutations, truncations or domain swaps of integrin cytoplasmic domains have indicatedd that the cytoplasmic tails of the integrin p subunits (i.e. the conserved NPXY/F motifs are required)) contain sufficient information for localizing integrins into preexisting focal contacts, presumablyy by binding to cytoskeletal components enriched at those sites [74-77]. Furthermore, expressionn of (3 cytoplasmic domains, as chimeric proteins containing the extracellular and transmembranee domains of the interleukin-2 receptor (IL2R) at high levels, results in a dominant negativee effect on focal contact formation and integrin-mediated signaling. This is probably due to thee capturing of cytoplasmic focal contact components by the cytoplasmic domain. In addition, studiess in the fruitfly have demonstrated that the cytoplasmic domain of integrin (3-subunits is sufficientt for correct localization [78].

Outside-inn signaling

Adherentt cells in cell culture form specialized structures, focal adhesions or focal contacts at sitess where there is close contact between the plasma membrane and the underlying extracellular matrixx and at which integrins, signaling and cytoskeletal molecules are colocalized (see Fig. 1). A hierarchyy in the recruitment of focal adhesion components is observed upon integrin aggregation whenn the integrin is bound to ligand (receptor occupancy) [79, 80]. Furthermore there are differencess in the constituents and the activation state of the different focal contacts even within the samee cell [81-85]. The adaptor protein tensin and the tyrosine kinase FAK (focal adhesion kinase) aree the first proteins to be recruited after integrins cluster. Ligand binding to the clustered integrins leadss to the recruitment of cytoskeletal components such as, vinculin, talin and a-actinin. Furthermore,, ligand binding and tension induces phosphorylation events at focal adhesions by memberss of the Src protein-kinase family including: cSrc, Csk, Fyn, which leads to the recruitment andd activation of downstream signaling molecules e.g. activation of the Rho-like GTPases (this thesis),, phospholipase-Cy (PLCy), and the ERK and INK signaling cascades [86, 87]. Ultimately, thiss integrin signaling cascade leads to cytoskeletal rearrangements (this thesis), anchorage dependentt completion of the cell cycle and integrin-mediated gene transcription [88]. The Rho familyy of GTPases play an important role in cytoskeletal rearrangements [89]. These proteins act as molecularr switches, which are active when they are bound to GTP and inactive when bound to GDP.. The GTP loading of Rho-like GTPases is regulated by GEFs (guanidine exchange factors), GAPss (GTPase-activating proteins) and GDIs (guanidine dissociating factors). As a key regulator of actomyosin-basedd contractility, RhoA plays a central role in the formation of both stress fibers and focall adhesions [90].

Thee formation of signaling complexes, which are modified and strengthened by multiple interactionss between its components, is a recurring theme in integrin-mediated signaling. These stabilizingg and modifying interactions make it difficult to define the regulation and crucial primary interactionss in integrin-mediated signaling [91]. Hemidesmosomes form a separate class of cell-matrixx adhesive structures found in the epidermis. Hemidesmosomes are linked to the intermediate filamentt system which involves the interaction of the cytoplasmic domain of p4 of the oc6p4 integrinn with associated proteins (plectin and BP230), this in contrast to the actin linkage in the otherr integrin-cytoskeletal associations [18.92].

14 4


Figur ee 1. Schematic representation of a focal adhesion and an adherens-junction Transmembranee integrins bind to the extracellular matrix and associate within the cell to a complex of cytoskeletal proteinss that link them to actin filaments. These focal adhesions form also the basis for the assembly of signaling complexess of which several components are indicated. The right sight of the figure shows an adherens-junction. The homophilicc adhesion receptors, cadherins are linked via (3-catenin and a-catenin to the actin cytoskeleton. Abreviations:: a = integrin a-subunit; P = integrin p-subunit; a-act = o>actinin; ce-cat = a-catenin; (3-cat = p-catenin; Cass = pl30Cas; ECM = extracellular matrix; FAK= focal adhesion kinase ; GF = growth factor; GR= growth factor receptor;; ILK = integrin linked kinase; pl20 = pl20 catenin; pax = paxiilin; vine = vinculin; zyx= zyxin.

Crosss talk with integrins

Integrinss have been shown to affect the expression and activity of other integrins and different typess of adhesion molecules (see also chapter 5). Occupancy of integrins by ligand has been shown too activate ligand binding of other integrins in a positive feed back loop [93], Conversely, inhibition off one integrin by another is known as trans-dominant inactivation [53, 94-96]. Integrin-mediated signalingg or potential changes in integrin-cytoskeletal interactions may account for these effects. Integrin-mediatedd adhesion can also modify cadherin-based intercellular adhesions [97]. A role of integrinss in the down-regulation of cadherin activity during neural crest cell migration and epifhelium-mesenchymee transitions (EMT) has been implicated, suggesting the existence of an oppositee effect of cadherin and integrin-mediated adhesion. EMT occurs during specific stages of

15 5

ChapterChapter 1

embryonicc development but also under certain pathological conditions as in the loss of epithelial polarityy in breast tumor cells [98, 99].

Syndecanss are transmembrane heparan sulfate proteoglycans whose external glycosaminoglycann chains bind ligands in the extracellular matrix. Syndecan-4 promotes integrin-mediatedd focal contact formation, probably by signaling through its cytoplasmic association with PKCandPIP22 [100].

Cytoplasmicc proteins binding to integrins

Ass mentioned above, the small cytoplasmic domains of integrins have no intrinsic catalytic capacityy or protein-protein interaction motifs. Nevertheless, recent studies using yeast two-hybrid interactionn assays and biochemical assays have identified an increasing number of integrin binding partnerss (see Table II and references therein, reviewed in [33, 34, 101-103]). These proteins can be groupedd based on their proposed functions in three categories: 1) cytoskeletal proteins which anchor thee integrins to the cytoskeleton and could serve as docking sites for signaling molecules and a-actinin,, talin and filamin belong to this subgroup; 2) potential signaling molecules form the largest groupp and include several kinases, such as ILK, FAK, and proteins of unknown function and 3) calciumm binding and accessory molecules which probably play a role in integrin activation and the cyclingg or correct folding of integrins, respectively. The biological relevance of most of these interactionss remains to be determined.

Transmembranee proteins interacting with integrins

AA different class of integrin associating molecules consists of several transmembrane and membranee associated proteins, which act as co-receptors or scaffolding molecules (Table III ) [104]. Memberss of the tetraspan protein (TM4) family or tetraspanins [105], bind directly to the extracellularr domain of several integrin a-subunits. Their interaction with integrins affects cell migrationn and surface expression levels of integrins, and they are thought to cluster integrins and otherr molecules into higher order complexes. Co-immunoprecipitation of integrins with several receptorr kinases such as the EGF (epidermal growth factor)-receptor tyrosine kinase and PDGF (platelett derived growth factor)-receptor tyrosine kinase has also been reported. These assumed complexess could facilitate efficient crosstalk between adhesion and growth factor induced signaling.. It has been established that both the signaling pathways induced by soluble signaling factorss and integrin-ECM interactions converge at the level of FAK [106], and that integrin-mediatedd adhesion reduces the threshold of growth factor-mediated signaling. This cooperation of growthh factor-induced and adhesion-dependent signaling is the basis for anchorage-dependent growth. .

16 6


Tablee II Cytoplasmicc proteins that associate with integrin cytoplasmic domains

Protein n Integrinn Assay Remarks References s

CytoskeietalCytoskeietal proteins Tall in Filaminn A. B F-actin n a-Actinin n Myosin n Skelemin n Plectin/HDl l p27BBP/eIF6 6 BP230 0

P;aIIb b P1.2,3,7 7 a l .2 2 pi.2 2 P3 3 Pl.3 3 P4 4 p4 4 P4 4

P P P.Y2H H P P P P P P Y2H H P.Y2H H Y2H H Y2H H

cytoskeietall protein, localized in focal adhesions actinn cross-linking cytoskeietall protein actinn cross-linking F-actinn contraction, binds upon P3 phosphorylation cytoskeietal// M-band protein bindss intermediate filaments bindss nuclear matrix and intermediate filaments bindss intermediate filaments

[147-15] ] [150,, 152, [154,, 155] [156-158] ] [159,, 160] [161] ] [162,, 163] [164] ] [19,, 165]

153] ]

AdaptorAdaptor and signaling proteins ICAP-11 pi Y2H Rackk I p 1.2,5 Y2H

Wait-1 1 M1BP P Cytohesin-1/3 3 Mss4 4 IRS1 1 Paxillin n

She e

Grb b

ProteinProtein kinases ppl255 FAK

P7:cc4.E E PIA.D D

P2 2 a3 3 avP3 3 a4 ;p i ,3 3

P3,4 4

p3 3

P1,2,3 3

Y2H H Y2H H Y2H H Y2H H P P P P

P P

P P

p59ILK K Pl,3 3 Y2H H

Chaperone/CatciumChaperone/Catcium binding Calnexin n BiP P Calreticulin n CIB B Melusin n

P l ; a6 6 ullbp3 3 a a al lb b PIA.D D

P P P P P P Y2H H Y2H H

phosphorylatedd upon binding, cell migration T 7xWDD repeats, in vivo binding is phosphorylation dependent,, binds PKC and Src, 5xx WD repeats downregulatedd during muscle differentiation PHH and Sec7 domains with GEF activity, cell adhesion T GEFF activity for Rab proteins containss PH domain, binds insulin receptor containss SH2, S1I3 binding motifs, and L1M domains bindss FAK, localized in focal adhesion containss PTB and SH2 domain, binds Grb2; Trk, EGF and PDGFF receptors containss SH2, SH3 domains, binds upon p3 phosphorylation

Tyrr kinase, localized in focal adhesions, phosphorylatedd upon integrin and growth factor engagement bindss Src, paxillin, Grb, pl30Cas. etc. Ser// Thr kinase contains ankyrin repeats.binds PINCH, activatedd by P13-kinase. regulates GSK-3 and PKB/AK.T, rolee in anchorage-independent growth,(cell adhesion I)

chaperone e chaperone e integrin-dependentt Ca2* influx and adhesion Ca2** -dependent binding bindss in absence of cations, striated muscle specific

[166,, 167]

[168] ] [169] ] [170] ] [171-173] ] [174] ] [175] ]

[176-178] ]

[179-181] ] [182] ]

[178.. 183]

[184-186] ]

[187] ] [188] ] [189,, 190] [191.192] ] [193] ]

Nuclear/cytopiasmicNuclear/cytopiasmic proleins and transcription (co)faclors P3-endonexinn P3 Y2H binds cyclin A. affinity and adhesion of aIIbP3 t

(155 P p3-endonexin-like, adhesion Ï. cell migration T «« or p Y2H contains 4'/2 LIM domains, heart specific, localizes in

focall contacts and nucleus P22 Y2H binds Jun. shuttles from plasma membrane to nucleus upon

ligandd binding BIN11 u3 Y2H binds Myc, tumor supressor

TAP-20 0 FIIL2 2

JAB1 1

[194,, 195, 196; [197] ] [198] ]

[199] ] [174] ]

PP = biochemical protein-protein interaction assay: Y2H = yeast two-hybrid; SH2 = Src-homology domain 2: SH3 = Src-homologyy domain-3: PTB = phosphotyrosine binding domain; PH = pleckstrin-homology; GEF = guanidine exchangee factor

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ChapterChapter 1

Tablee II I Transmembranee proteins

Protein n

Tetraspanins Tetraspanins CD9;; CD37; CD53; CD63;CD81;CD82 2 CD151/PETA; ; NAG2;; CO-029

IgIg superfamily proteins CD147/EMMPRIN N CD47/IAP P CD46/MCP P CD36 6

Growth-factorGrowth-factor receptors PDGFF receptor-p VEGFF receptor-2 ErbB2 2

GPI-linkedGPI-linked receptors uPAR(CD87) ) FcyRIIIB(CD16B) ) CD14,, LPS, LBP

thatt interact with integrins

Integrin n

(a3pii andaópi, occasionallyy allb, a2pi, a4pl ,a5pl ,aLp2,, a6p4)

a3pl ,a6pl l a2Bl,avB3,aIIbB3 3 a3pi i <xlIbB3,a3pi,a6Bl l

avp3 3 avp3 3 a6B4 4

PU2,, 3 aMp2,, aDp2 aMp2 2

TypeType II transmembrane proteins CD98 8 BP180 0

plA A a6pi;a6p4 4

References s

[34,, 101, 104]

[200] ] [201,202] ] [203] ] [204,, 205]

[206] ] [206] ] [207,, 208]

[209-211] ] [212] ] [212] ]

[213,214] ] [163,215] ]

OtherOther proteins Caveolinn [31,2 [211,216]

Adaptedd from [34, 101, 104]

Integrin ss in myogenesis Thee process of myogenesis includes migration of myogenic precursor cells from somites to

peripherall areas, proliferation, differentiation and subsequent withdrawal from the cell cycle, and finallyy fusion to form mature contractile myotubes. This skeletal muscle differentiation pathway is orchestratedd by myogenic basic helix-loop-helix transcription factors, including myogenin, Myf-5 andd MyoD [107]. These transcription factors regulate the sequential expression of muscle specific contractilee and cytoskeletal proteins that build the unique cytoskeletal structure of the sarcomere, whichh form the contractile building blocks of cardiac and skeletal muscle [108] (see Fig. 2.). The molecularr architecture of a sarcomere facilitates the mechanical sliding of actin filaments along myosin.. The polar actin filaments are mechanically linked to actin arrays of successive sarcomeres att the Z-line. The Z-line is connected at focal contact-like structures, costameres (greek for rib-like structures),, to the lateral muscle fibers [109, 110]. Skeletal muscles contain adhesive structures that resemblee in their protein composition the focal adhesions which are studied in cell culture; i.e.

18 8


myotendinouss junctions (MTJ, muscle-tendon attachment sites). Integrins are also present in neuromuscularr junctions (NMJ, axon-muscle attachment site). In contrast to the fused skeletal musclee fibers, cardiac muscle consists of mononucleated cardiomyocytes, which are connected to eachh other at intercalated discs. The intercalated disc consists of three classes of junctional complexes:: i.e. the adherens junctions (integrin-ECM), desmosomes (cadherin-based) and gap junctions,, the latter play a role in the electric coupling of heart cells.

myoblastss myotube

costamere e

Sarcomere e

intercalatedd disc

cardiacc muscle

Figur ee 2. Schematic represen-

tationn of a skeletal and cardiac

musclee cell

(A)) Mononucleated myoblasts fuse to formm multinucleated myofibers which differentiatee into muscle fibers. Differentiatedd muscle cells have their cytoskeletonn organized in contractile units,, the sarcomeres, that facilitate the slidingg of myosin (thick bars) along actinn filamins (horizontal black bars). Thee actin filaments are linked to the sarcolemmaa by Z-lines (Z) at integrin richh lateral attachments sites (costameres).. Specialized cell-tendon contacts,, myotendinous junction (MTJ) andd neuromuscular junction (NMJ) are indicated.. (M = M-line).

(B)) Mononucleated cardiac cells are linkedd together by intercalated discs, celll adhesion structures containing, integrin-basedd adherens-junctions, cadherin-basedd desmosomes and gap-junctions.. The costameres are localized laterally. .

Variouss families of adhesion molecules have been implicated in the development and biologicall function of skeletal and cardiac muscle. It has been demonstrated that inhibition of cell adhesionn by antibodies directed against N-CAM [111,112], N-cadherin [113, 114], ADAM 12 [115] orr pi [116, 117] disturbes the differentiation and/or fusion of myoblasts into myotubes in vitro. However,, most null mutations of adhesion molecules in mice appeared to have no effects on myoblastt fusion, probably due to compensation by other such molecules [11, 118-120].

Studiess in the fruitfly , Drosophila and the roundworm, Caenorhibitis elegans [121, 122] havee indicated a role of integrins in the maintenance of muscle integrity. In Drosophila mutants, lackingg the pPS integrin subunit, the architecture of the sarcomere is disrupted and the myotendinouss junctions become detached after muscle contraction, despite normal myoblast differentiationn and fusion [123]. Alterations of the sarcomeres have also been observed in cardiomyocytess and skeletal muscle cells, obtained after in vitro differentiation of embryonic stem cells,, lacking pi [124, 125].

19 9

ChapterChapter 1

Fetall myoblasts express a5p i, a6|M and avfl3 integrins. which are down regulated during

myogenesiss [126-129]. These integrins have been suggested to play a role in myoblast migration

andd myofibrilogenesis. The relative ratios in expression of these integrins was found to influence

myogenesiss in vitro [130, 131]. However, the absence of the a6 integrin subunit does not lead to

detectablee cardiac or muscular defects [58], although ot5 deficiency, as analyzed in chimeric mice,

causess muscular dystrophy [132]. A transient expression of a4 has been implicated in the secondary

wavee of myoblast fusion [133]. However, loss of a4 in experiments with a4 null chimeric mice and

inn in vitro myogenesis studies of a4-deficient ES cells, showed no obvious muscular defects [134].

Duringg myogenesis expression of the a7B[31A integrin is first induced. Subsequently. a7Bp iA is

replacedd by the oc7BplD and a7Ap iD integrins, which are the major integrin variants in adult

musclee (this thesis, [135]). Expression of the collagen receptors ctlOpl and ocl ipi in adult skeletal

musclee tissue has also been reported [129, 136-138], as has the induced expression of the integrin

oc9pll [139, 140]. Notably, expression of the ocipi and a3pi integrins in myoblasts has only been

detectedd in in vitro myogenesis studies [141, 142].

Cardiacc cells express different integrins during heart development. Integrins a l p l , a3Api

andd a6Api are expressed at low levels at the onset of cardiogenesis [143]. Subsequently, the

expressionn of a i pi and ce3Api decreases, while a6Api remain present [144, 145]. In addition, the

integrinn oc7Bpi is induced in heart postnatally where it remains the only variant of a7 expressed in

adultt heart [146].

Scopee of this thesis

Inn this thesis studies on the expression and the role of a splice variant of the cytoplasmic tail

off the pi integrin subunit are presented. In chapter 2 the cloning of the integrin p iD cytoplasmic

variantt is described, which is specific for skeletal and cardiac muscle. Chapter 3 investigates the

expressionn pattern of p lD integrins in adult muscles and during mouse embryogenesis. It is shown

thatt the expression of p lD is specific for adult skeletal and cardiac muscle cells, where it is

localizedd at muscle specific cell-matrix junctions; the costameres, neuromuscular- and

myotendinouss junctions in skeletal muscle, and the intercalated disks and costameres in cardiac

muscle.. Furthermore, the expression of the ubiquitous pi A is switched towards p iD in muscle

tissuess during mouse embryonic development. In chapter 4 the effects of the expression of either of

thee splice variants pi A or pi D in, a pi-deficient epithelial cell line, GE11 are outlined. Expression

off either of the pi-splice variants induced the disruption of intercellular adhesions and cell

scattering.. The functional relationship between integrins, cadherins and Rho-like GTPases was

furtherr analyzed. In chapter 5 the filamin protein family of actin cross-linking proteins is reviewed.

Inn chapter 6 we show that a novel splice variant of filamin-B interacts with the cytoplasmic

domainss of both p iA and plD. Cell tranfection studies with Green fluorescent protein-tagged full

lengthh filamin-B variants indicated that filamin-B isoforms have different cellular localization and

differentt effects on myogenesis. In chapter 7 a summary of and a discussion on the role pi integrin

variantss in myogenesis and signaling is presented.

20 0


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23 3

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UvA-DARE (Digital Academic Repository) Beta-1 integrin ... · Besides their role in the maintenance of tissue structure and polarity, cell adhesion ... laminins,, collagens, fibronectin)