Standing On the Shoulders of Giant: Small Membrane Molecules That Associate Laterally With Integrins

A Review Article by Alvin Wong YaoSheng

Supervisor: Asst Prof Tan Suet Mien School of Biological Sciences NANYANG TECHNOLOGICAL UNIVERSITY

Submitted to the School of Biological Sciences in partial fulfilment of BS4210 Final Year Project NANYANG TECHNOLOGICAL UNIVERSITY

April, 2009

DECLARATION

I declare that, in accordance with School requirements this thesis is under 6000 words in length; all presented work was performed within the official project time frame as stated below; all presented work was performed by me, unless otherwise specified; all relevant work experience gained before BS4210 FYP is stated below; the input by my supervisor, or delegated supervisor, into this thesis was limited to reviewing of a single hard copy draft; this thesis is my own work, unless otherwise referenced, as defined by the NTU policy on plagiarism and I have read the NTU Honour Code and Pledge; the included Abstract, Introduction, Results, Discussion and Conclusions sections will be submitted to SafeAssignment no later than 24 hours after hardcopy submission.

BS4210 FYP start date: 10/12/08 BS4210 FYP submission date: 17/04/09 Total number of weeks: 18

Pre-FYP experience - National University Hospital Dept of Lab Medicine student/ lab technologist in Biochemistry, Microbiology and Haematology 1.5 yrs - National University Hospital Dept of Lab Medicine part time lab technologist in Biochemistry STAT lab 0.5yrs

Signature of student:

Alvin Wong

Date: 17/04/09

Integrins are a superfamily of glycoproteins cell adhesion receptors expressed on the cell surface that bind to extracellular matrix ligands, cell- surface ligands, and soluble ligands. They initiate intracellular signal transduction important for maintaining cell physiology thus is required in wide spectrum of functions such as adhesion, proliferation, survival, apoptosis, shape, polarity, motility, haptotaxis, gene expression and differentiation [Luo et al., 2007]. In the immune system, integrins play crucial roles in leukocyte trafficking and migration [Laudanna et al., 2002], immunological synapse formation, costimulation [Dustin, 2001], and phagocytosis [Cain et al., 1987].

Integrins exist as heterodimer consisting of and subunits that are non- covalently bound [Hynes, 2002]. They belong to the class 1 or single pass transmembrane protein [Tan and Law, 2006]. There are 24 known heterodimers in human, form from association between 18 and 8 subunits (Table 1). Integrins and subunits are totally distinct with no detectable homology between them. Sequence identities among subunits are ~ 30% while among subunits with ~45% [Miyazawa et al., 2001].

Integrins Structure

Integrins consist of distinct domains with four domains in subunits containing propeller, thigh, calf- 1 and calf- 2 domains; and eight domains in subunits composing of I- like or I, hybrid, PSI, 4 I- EGF repeats and tail domains (Figure 1). In other nine subunits such as L, 1, 2, M, X, D, E, 10 and 11, an Idomain is inserted in the - propeller. Based on electron microscopy images, all these domains are organised to form a molecule with a large globular head with two stalks [Nemut et al., 1988] (Figure 2 and 3). In support of the proposed shape of integrins, researchers [Hantgan et al., 1990] through crystal structure of V3 showed the head and two stalks. However, the stalks are severely bent, generating a vshaped topology in which the head closely juxtaposed to the membrane-proximal portions of the stalks [Xiong et al., 2001 and 2002]. Based on large numbers of studies, it was established that this bent conformation represent a low- affinity state (Figure 4). Upon ligand binding, it initiates global conformation changes that extend the bent integrin by the switchblade [Luo et al., 2007] or deadbolt [Arnaout et al., 3

2005] model. Even more recent studies link this global conformation changes to intradomain conformation changes associated with ligand binding and affinity modulation.

subunit

The head region is made up of seven segments of ~60 amino acids which are folded into a 7- bladed - propeller domain [Xiong et al., 2001; Xiao et al., 2004]. When an I- domain, approximately 200 amino acids, is present, it is inserted between - sheets 2 and 3 of the - propeller and they become the major ligand binding site. As for nonI domain integrins, the - propeller- I like- domain interface is involved in ligand binding. I-domain plays a central role in ligand recognition and binding. Comparison between non- I- domain 3 and I- domain L subunits showed that the former was unable to bind ICAM- 1 or ICAM- 3 [Leitinger and Hogg, 2000; Yalamanchili et al., 2000]. Binding of the I- domain requires divalent cations which is located on the upper face of I- domain. This is also known as metal ion- dependant adhesion site (MIDAS). Within this site, a metal ion is coordinated by three loops from I- domain, and a glutamate from the ligand. If the metal ion is an Mg2+, serines and threonine form electrostatic bonds to it. Two water molecules also interact with Mg2+ which provides indirect contacts with two aspartates. This lack of direct electrostatic bonds between Mg2+ and aspartates increases the electrophilicity of Mg2+ [Lee et al., 1995a]. If the metal ion is a Mn2+, direct electrostatic bond threonine is severed allowing direct electrostatic bond to aspartate and thus decreases electrophilicity of Mn2+. Lee et al proposed that the Mn2+ bound I- domain may represent the unliganded I- domain (Figure 5).

stalk contains three - sandwich domains. The upper stalk consist of a thigh domain and lower stalk consist of calf- 1 and calf- 2 domains [Xiong et al., 2001]. A small Ca2+ - binding loop located between the thigh and calf- 1 form the genu which serves as the highly flexible pivot point for switchblade extension. Recent studies showed that extension occurs by rearrangement of this thigh/genu interface [Xie et al., 2004].

subunit 4

The head region consists of ~240 amino acids. It is involved directly in ligand binding in integrins lacking I- domain. For I- domain containing integrins, I- like domain function indirectly to regulate ligand binding. In addition to MIDAS, I- like domain contain two other metal ion coordination sites. One of it lies adjacent to MIDAS, thus termed ADMIDAS (ADjacent MIDAS). Its metal ion is coordinated by carbonyl oxygen of the second serine in DXSXS motif, another carbonyl oxygen of methionine at the top of 7 helix, and two aspartate side chains from 1 helix [Xiong et al., 2001]. It was proposed that ADMIDAS function to stabilize the active conformation of integrin. The other metal ion coordination site is ligand- induced metal- binding- site (LIMBS) also located in the I- like domain in close proximity to MIDAS. However recent studies showed that LIMBS is not ligand- associated or induced metal binding site, thus a new nomenclature was proposed as synergistic metal ion binding site (SyMBS) [Zhu et al., 2008].

I- like domain is inserted in the hybrid domain. Hybrid domain form the upper part of the stalk. Staining of the hybrid domain by antibodies mapping can only be done when integrin is activated, suggesting an outward swinging during integrin activation [Mould et al., 2003a; Tang et al., 2005]. This outward swinging is linked to the downward shift of the C- terminal 7 helix of the I- domain and seem to have a role in conformational changes involved in both activation and ligand binding [Mould et al., 2003b]. Moreover, the hybrid domain plays another important role in signal transduction from membrane proximal region to head region [Tng et al., 2004].

Hybrid domain is in turn inserted in the plexin- semaphoring- integrin (PSI) domain. The cysteine rich region of PSI domain has shown to maintain integrin in the inactive conformation [Zang et al., 2001].

The reminder of the stalk is built from four integrin epidermal factor- like (IEGF) domains and a tail domain (TD). The I- EGF domain provides link between signals coming from cytoplasmic and transmembrane domains and the head region conformation changes. Experiments showed that antibodies to this region can directly activate ligand binding [Humphries, 2000; Bazzoni and Helmer, 1998; Takagi et al., 1997; Lu et al., 2001]. 5

Following the I- EGF domains is the tail domain. Crystallography showed weak hydrophobic interaction between I- EGF and TD, suggesting flexibility [Arnaout, 2002].

Transmembrane domain

The heterodimer integrin requires a transmembrane (TM) domain to plant itself stably into the cell membrane. However, recent studies showed that the TM domain do not merely serve to link outside and inside domains. Researchers [Li et al., 2003 and 2002] reported that the TM domain may function in mediating clustering of integrins after ligand binding. Moreover, separation of the and TM domain plays a role in regulating the conformation of the extracellular domain. Thus, provide bidirectional signalling across the membrane. Several models have been proposed to explain TM signalling. These models are based on different movements of TM domains such as rotating, tilting, oligomerization between two or more or subunits and piston movement [Williams et al., 1994; Armulik et al., 1999; Adair and Yeager, 2002; Li et al., 2005] (Figure 6).

Cytoplasmic domain

Integrin and subunits cytoplasmic domains are short, no more than 70 amino acids residue. They form linkage with actin- microfilament system [Hynes, 2002]. Their interactions with signalling molecules, cytoskeleton and other proteins play an important role in the transduction of signals from outside as well as inside of the cell [Liu et al., 2000].

In the subunit, its cytoplasmic tail have a conserved GFFKR motif at the membrane proximal region. This motif has shown to maintain integrins in resting state as well as involve in clustering and subunit association. In support of this, Lu and Springer, and Ylanne et al [Ylanne et al., 1995 and 1993] showed that deletion of this motif caused integrin to be constitutively active. Similarly, a sequence LLvixhDRRE (capital letters stands for conserved residue) in subunit when deleted also result in constitutively active integrins. However, deletion nearer to the c6

terminal end blocks activation. In addition, the Asp 723 from this sequence forms a salt bridge with Arg 955 in GFFKR motif in subunit cytoplasmic tail. This association maintain integrins in their low affinity state [Huges et al., 1996].

cytoplasmic tail also contains either one or two NPxY/F motifs. Phosphorylation of tyrosine (Y) in this motif may provide a mode of regulating integrins interaction with other proteins at the cytoplasmic face of the plasma membrane. This allows recruitment of many different proteins, such as talin that binds actin filaments (Figure 7) and thus forms connection to cytoskeleton. Mutation studies to the tyrosine in the first NPxY/F motif to alanine inhibits integrin activation moreover causes structural changes to the motif itself as well as the membrane proximal region [Ulmer et al., 2001]. Liu et al also showed that mutation to NPxY prevent binding signalling and cytoskeletal proteins to tails. Calderwood [Calderwood, 2004] proposed that the membrane distal region of tails regulate activation by interacting with signalling proteins that disrupt the membrane proximal association which is important in maintaining integrins in resting state. Membrane distal region of tails however function to regulate tails conformation and also bind cell- type- specific activator proteins.

Bidirectional signalling

The TM domain of integrins connects the extracellular and intracellular domains. This provides a linkage between the outside and inside of a cell thus allowing bidirectional transmission of mechanochemical signals across plasma membrane [Qin et al., 2004].

Inside- out signalling involves ligation of intracellular signal proteins to the cytoplasmic domain. Before ligand binding, integrin is in bent conformation. This conformation is stabilize by associations between headpiece and lower stalk, between lower and stalk, and between the and TM and cytoplasmic domains. These associations however are not tight. Upon binding an intracellular ligand like talin induces separation of the cytoplasmic and transmembrane domains. This in turn results in separation of the and lower legs. Lower leg separation destabilizes the interface between the lower legs and the headpiece and results in integrin extension 7

(Figure 8) [Eigenthaler et al., 1997; Calderwood et al., 1999]. The extension leading to change in affinity in the ectodomains is through either of the proposed models. In the deadbolt model, an elongated loop of the TD maintains I- like domain in inactive state by preventing shift in the c- terminal 7 helix and the subsequent loop. Inside- out signal will then lead to piston- like movements of the TM regions causing sliding of the extracellular stalks of the and subunits, which disrupts the interaction between the headpiece and the stalk just beyond the membrane [Xiong et al., 2003a and 2003b]. In the switchblade model, dissociation of the and cytoplasmic and transmembrane regions leads to dislocation of I- EGF repeat in the stalk, which causes the head region to extend outwards in a switchblade- like movement [Springer et al., 2007].

Outside- in signalling takes place upon inside-out activation. Integrins then binds extracellular ligands that induces conformational changes involving outward swinging of the hybrid domain, separation of the and stalk, and separation of the TM domains which lead to interaction of cytoplasmic tails with cytoplasmic signalling molecules. These molecules consist of enzymes such as c- Src, focal adhesion kinase (FAK) etc, and adaptors such as paxillin etc that assemble within dynamic adhesion structures, including focal adhesions that bind cells to the extracellular matrix and podosomes (small foot- like extensions of plasma membrane) [Shattil et al., 2005].

Integrins modulation

Integrin adhesion is regulated through its affinity as well as avidity. Affinity refers to the strength of a single integrin binding its ligand and is increased by conformation changes. Avidity refers to the overall strength of cellular adhesive interactions resulted from the combination of both the affinity of individual receptor- ligand bonds and the total number of bonds formed also known as valency [Kim et al., 2004] . Experiments shown that changes in both affinity and avidity are important in integrinligand binding [Hogg et al., 2000]. Affinity and avidity regulation may occur sequentially, and could be driven by independent pathways [Giagulli et al., 2004].

Affinity regulation involves conformational changes at the head region upon ligand binding which is then propagated down the stalk, the TM, and finally reaching the cytoplasmic tails. In I- domain containing integrins, on binding to ligand causes downward shift of the I- domain 7- helix in the C- terminal direction in which a conserved Glu [Lee et al., 1995b] on the 7- helix acts as an intrinsic ligand that binds to the I-like domain MIDAS [Alonso et al., 2002]. This in turn reshapes the 6- 7 loop, and activates the I- domain MIDAS (Figure 9). In the case of non Idomain integrins, a small ligand is required to initiate ligation of bigger ligands. This small ligand will bind initially to the interface between I- like domain and - propeller which in turn causes the downward shift of the c- terminal helix and thus shifting the I- like domain away from - propeller resulting in exposing the top of - propeller which allow binding of larger ligands [Mould et al., 1997].

Avidity regulation involves strength control through three ways including (1) changes in number of integrins expressed. Bainton et al. showed that there are several integrins stored in intracellular pools that can be expressed on cell surface quickly upon ligation. Moreover, cells can selectively mobilize particular intracellular integrins type pools so as to generate differential responses [Sengelov et al., 1993]. (2) Changes in cell shape can affect adhesion by increasing or decreasing the contact area [Stewart et al., 1996]. (3) Integrins clustering can increase adhesion efficiency even when receptors remain in their low affinity state [Yoshikazu et al., 2007].

Another mode of integrins modulation is lately gaining acceptance and increasingly studied is: how the type of adhesion and signalling following integrins ligation may be modulated by lateral association with other membrane molecules. First of such component is Integrin- associated transmembrane protein (IAP). It functions as receptor for cell- binding domain of throbospondin [Lindberg et al., 1996], and can regulate vitronectin binding to V3 [Lindberg et al., 1993; Gao et al., 1996] and activate IIb3 through thrombospodin binding [Chung et al., 1997]. They may also interact with 21 in vascular smooth muscle cells [Wang et al., 1998]. Second are growth factor and insulin receptors. Co-immunoprecipitation showed that these molecules coprecipitates with V3 upon treatment of cells with platelet derived growth factor (PDGF) or insulin [Schneller et al., 1997a and 1997b]. This may suggest that growth factor and integrin signalling pathway intersect. Other 9

components that have been coprecipitated are urokinase plasminogen activator receptor with 1, 2 and 3 [Wei et al., 1997], CD98 with 31 [Fenezik et al., 1997], CD36 with IIb3, CD46 with 31, and EMMPRIN/basigin/CD147 with 31 and 61 [Chang et al., 1997]. In addition, dystrophin complex may associate with 1 through and sarcoglycan [Yoshida et al., 1998]. Finally, the association between 41 with transmembrane chondroitin sulphate proteoglycan is implicated in integrinmediated melanoma cell adhesion.

This review paper will elaborate more on some of these components that interact laterally with integrins and provide modulatory function. Since the discovery of complement receptor- 3 (CR3/M2)- FcIIIB interaction, the number of integrinassociated partner proteins has grown dramatically in the past decade to about 20 (Table 2). They can be classified into two major classes being transmembrane (~7 members) and glycosylphosphatidylinositol (GPI)- linked (~5 members). The rest are receptors, transporters, adherence proteins and channels.

Transmembrane Partner Proteins

This class constitute the largest number of integrin- associated partner proteins which included FcRIIA, voltage- gated potassium channels, CD98, TM4SF and IAP. Their interaction with integrin brought about modification of integrin properties as well as involvement in transmembrane signalling via kinases and membrane channels. In this category, we will focus on TM4SF also known as tetraspans.

Tetraspans is a large family of transmembrane proteins which have two extracellular loops, four TM domains, and a short N- and C- terminal cytoplasmic tails (Figure 10) [Maecker et al., 1997; Hemler et al., 1996 and 1998]. Their TM domains are the highly conserved area, particularly their sequence of hydrophobic residue and polar amino acids. Cytoplasmic tails within members also contain two to three conserved charged residues in the 4- amino acid cytoplasmic loop. Its extracellular domains are mostly different between members with exception of the PXSC motif, three cysteine repeats and glycosylation sites.

10

Some tetraspans members like CD9, CD81 are ubiquitously expressed while others are limited to specific cell types. They can interact with large number of cell surface components as well as among themselves. One such component is integrins (Table 3). Experiments through deletion studies showed that their interaction takes place in the extracellular domain of the - subunit [Berditchevski et al., 1997a; Yauch et al., 1998] with hydrophobic interaction playing certain roles [Berditchevski et al., 1997a], and possibly with contribution by - subunit because interaction is seen in 61 but not in 64 [Indig et al., 1997; Yauch et al., 1998]. An in depth look into CD151- 31 association mechanism points to the sequence between residue Leu149 and Glu213 in the second half of the large extracellular loop (LECL) being critical in maintaining stability in this association [Indig et al., 1997]. In addition, mutation of the cysteine repeats within the LECL of the conserved CCG and PxxC motifs prevent the direct association of CD151 and 31. Thus led to mapping the tetraspans- binding site on the extracellular domain of - subunit. In support of this, Yauch et al. [Yauch et al., 1998] substitute the cytoplasmic and TM domains of 3 with corresponding regions from 5 and demonstrated that it does not affect association with tetraspans. Contrary to this observation, Mannion et al. reported that mutation of the two metal- binding sites on 4 prevent stable association with its tetraspans. However similar mutation on 3 did not affect association with CD151. This suggests that the interacting interfaces between different integrins and tetraspans differ. Recently, Ono et al. found that interaction between CD82 with 31 and 51 depends on glycoslation status of both integrins and tetraspans. As different cell have different degree of CD82 glycosylation, it may provide a tissue- specific control mechanism to regulate their assembly.

The purpose of integrins- tetraspans association is difficult to determine. As yet, there is no evidence indicating that this interaction influence integrins conformation or ligand binding having an influence on stabilizing integrin- tetraspan complex. The roles of tetraspans in integrin- mediated cell adhesion seem controversial. Lately, this is modified to post- adhesion strengthening. However, its involvement in cell in cell motility is well studied. Cell migration is a complicated phenomenon controlled by highly regulated processes both at the rear and leading edge of the cell. Both migratory and stationary cells have integrin- tetraspans complexes found clustered in 11

the leading edge of cells as well as current and previous [Berditchevski et al., 1995, 1996 and 1997a; Indig et al., 1997; Tachibana et al., 1997] intercellular contact sites [Yanez et al., 1998]. However, they are not found in focal contacts [Berditchevski et al., 1995, 1996, 1997a and 1999; Indig et al., 1997; Tachibana et al., 1997]. Cotransfection of CHO with CD9 supported this by showing IIb3- CD9 complex concentrated at the leading edge but not at focal adhesion even though IIb3 is present there [Indig et al., 1997]. In addition, the CD9 transfected CHO did not alter focal adhesion formation [Indig et al., 1997]. In contrast, transfections with CD9 in HT1080 cells alter actin cytoskeleton organisation [Berditchevski et al., 1999]. On top of that, recent experiments showed that association between tetraspans and actin cytoskeleton is weak [Berditchevski et al., 1999]. Thus led to the suggestion that ligand- tetraspans binding is an alternate signalling pathway resulting in the weak cell- matrix- cytoskeleton association suitable for lamellipodial extension and retraction. All studies on tetraspans- integrin association points to its more important functional role in migration than adhesion [Yanez et al., 1998; Hemler et al., 1996; Skubitz et al., 1996]. This is supported by a study using co immunoprecipitation and antibody [Yauch et al., 1998; Yanez et al., 1998]. This study unlike its previous predecessor showed that most part of 31 integrin associate with CD151 (another member of the tetraspans family) with no additional cell surface protein being coprecipitated. Co- immunoprecipitation under stringent conditions further suggests stable specific interaction. After showing their strong association, antibody study using anti- CD151 or anti- 31 showed significant reduction in neutrophil migration. In addition, anti- CD151 (but not anti- CD9) or anti- 31 (but not 61) specifically inhibited neurite outgrowth without affecting adhesion [Yanez et al., 1998].

Interaction between tetraspans and integrins may provide indirect association of integrins that do not possess intrinsic enzymatic activity with enzyme or other signalling molecules. Experiments showed that conventional protein kinase C (cPKC) isotypes can associate with CD81 and CD151 after activation [Hemler et al., 1998]. cPKC is known to regulate actin cytoskeleton, and would be a suitable candidate to effect the post adhesion strengthening. In this instance, tetraspans three c- terminal residues at the cytoplasmic tails can recognise type one or three PDZ domains which can form link to cytoskeleton. The intracellular adaptor that forms the linkage and possesses the PDZ domain is yet to be identified. And mutation on the three c12

terminal residue blocks this strengthening [Lammerding et al., 2003]. In a separate study, it was shown that phosphatidylinositol 4- kinase (PI4K), which is a key enzyme in synthesizing C4- phosphoinositides, interact constitutively with multicomponent complex of CD63, CD81 and 31 [Berditchevski et al., 1997a and 1997b] and also with CD151- 31 complex [Yauch et al., 1998]. This enzyme interacts with tetraspans rather than 31. In support of this, treatment of CD63- CD81- 31 complex with antibodies against either tetraspans blocks tyrosine phosphorylation of FAK while treatment with anti- integrin did not. However this doesnt hold for CD9 expressing HT1080 cell line. Its FAK phosphorylation differs from wild types and also dependant on the adhesion substrate used [Berditchevski et al., 1999]. In this case, anti- CD63/CD82/CD151 mixed with collagen substrate can lead to FAK phosphorylation. In contrast, when those antibodies are used alone, FAK phosphorylation level lowered even lesser than those in suspended cells. Therefore, signalling through tetraspans seems very complex.

Biosynthetic labelling experiments may suggest another role for tetraspans- integrin association. This role involves maturation and trafficking of integrins themselves. The study reported that CD151 associate with pre- 3 subunit even before 1 subunit joins the complex [Berditchevski et al., 2001]. Moreover, CD151 mutants that sequestered in the endoplasmic reticulum retain their ability to bind 31. In addition, immunoelectron microscopy and immunofluorescence studies showed abundant tetraspans in various types of intracellular vesicles [Escola et al., 1998; Hamamoto et al., 1994]. All these data point to a probable role for tetraspans in sorting and/or turnover of integrins. Bonifacino et al. [Bonifacino et al., 1999] proposed that tetraspans c- terminal tyrosine- based sorting motif (Tyr- X- X- ) can recruit clathrin adapter proteins to integrin complexes and thus direct them along different trafficking routes.

Emerging Transmembrane Partner Proteins

Recently, there is growing number of transmembrane partner proteins that are found to interact in- cis with integrins. One such is EMMPRIN, which is expressed on leukocytes, has found to associate with 31and 61 [Berditchevski et al., 1997b]. They are about 50KDa consisting of two immunoglobulin family domains. It was 13

shown to function in regulating matrix metalloproteinase production [Petty et al., 2002].

Another emerging partner protein is platelet derived growth factor beta (PDGF ) receptor. Interaction between integrin and PDGF receptor can lead to tyrosine phosphorylation of PDGF receptor even in absence of PDGF [Sundberg et al., 1996].

One other partner protein is L- selectin. Cross- linking of L- selectin on neutrophil surfaces causes change in cell shape, microfilament organisation, and 2 integrin adhesion. RET experiments [Simon et al., 1999] showed 2 integrin and L- selectin in close proximity and suggest that it function in strengthening adhesion during cell- cell interaction.

An even more recent discovery is junctional adhesion molecules (JAM). The JAM family consist of members made up of two extracellular Ig domains and a short cytoplasmic tail ending with a type II PDZ- binding motif. JAM- L interacts in- cis with integrin, very late antigen- 4 (VLA- 4/ 41), on resting T- lymphocytes and monocytes to maintain themselves in monomeric form. However upon VLA- 4 activation, the VLA- 4- JAM- L complex dissociate leading to dimerization of JAML. This provide a regulatory mechanism in which JAM- L and VLA- 4 functions are co-ordinately regulated, enabling JAM- L to increase integrin- dependant adhesion of leukocytes to endothelial cells [Luissint et al., 2008].

Glycosylphosphatidylinositol (GPI)- linked Partner Proteins

This class of membrane proteins include FcRIIIB, CD14 and uPAR associate with the lipid bilayer via a single lipid molecule [Petty et al., 1999]. The absence of TM and cytoplasmic domains, without other contributing factors, makes it unable to mediate transmembrane signals. However, its association with integrin give them the ability to mediate transmembrane signalling. Hence it was proposed that GPI- linked proteins function as rapidly diffusing membrane scouts that relay (in this case)

14

inflammatory signals to integrins [Petty et al., 1996]. In this class, we will be focusing on urokinase- type plasminogen activator receptor also known as uPAR.

uPAR is a highly glycosylated 55KDa GPI- linked membrane receptor for urokinasetype plasminogen activator (uPA). It is also a receptor for vitronectin [Wei et al., 1994] and recently included high molecular weight kininogen (HKa) as a third ligand [Colman et al., 1997]. uPAR consist of three domains. uPA binds domain 1 while its other two ligands bind regions of domain 2 and 3 [Preissner et al., 2000; Colman et al., 1997]. Upon uPA binding, it catalyzes the conversion of plasminogen to plasmin, a protease. Plasmin can also activate other molecules in the proteolytic cascade that can digest extracellular proteins. Such function is important in cells like activated monocytes, T- cells, neutrophils etc. Interaction between uPAR and integrin CR3 lies in the lectin- like sites. It binds one or more carbohydrate moieties of the uPA/uPAR complex. This is supported by experiments using specific saccharides to block the lectin- like sites [Bohuslav et al., 1995] and prevented their association. On top of that, recent reports showed that protein- protein interaction between them may also be important [Wei et al., 1996; Simon et al., 2000]. Simon et al. mapped an uPAR binding site on the 4th blade of the - propeller [Simon et al., 2000]. By using a peptide mimicking this region, he was able to block uPAR- CR3 association as well as adhesion to vitronectin and fibrinogen. Bohuslav et al. [Bohuslav et al., 1995] modified immunoprecipiton protocol that preserves uPAR- CR3 interaction thus showed uPAR coprecipitate with CR3. This interaction has shown to induce cytoplasmic signals such as tyrosin phosphorylation and upregulating calcium level. On the other hand, such observation is not seen in neutrophils treated with anti- CR3 or neutrophils from patients suffering from leukocyte adhesion deficiency (LAD) [Cao et al., 1995]. In neutrophils, uPAR and integrins association is very dynamic to allow migration. During migration, neutrophils changes from spherical to elongated shape. In the spherical neutrophils, CR3 and uPAR is associated. When elongated, CR3 is localize in the uropod while uPAR in the lamellipodium. This attaching/detaching reaction between CR3 and uPAR is rapid and reversible [Kindzelskii et al., 1996a and 1997]. uPAR in the lamellipodium will associate with another integrin, complement receptor- 4 (CR4/X2). Based on degradation of Bodipy (boron-dipyrromethene)- BSA in the extracellular environment suggest connection between spaitial patterns of pericellular proteolysis and of uPAR 15

expression [Kindzelskii et al., 1996a]. Moreover, pericellular proteolysis oscillate in time with the same frequency as CR4- uPAR oscillation [Kindzelskii et al., 1998], further suggesting coordinated behaviour of uPAs proteolytic action which is probably mediated by tempo of cellular signalling machinery [Petty et al., 2000 and 2001]. Such oscillation is important in the repeat detaching/attaching cycles involved in cell migration.

uPAR- integrin interaction involvement in signalling can be classified into caveolindependent or caveolin- independent. Caveolin is a 21KDa membrane- associated protein that concentrates in membrane domains called caveolae. The hydrophobic domain of caveolin is expressed in the inner surface of the plasma membrane where they inter- interact forming caveolin scaffolding of caveolae. At the cytoplasmic side, it can interact with signal transduction molecules like Src kinase and G proteins [Okamoto et al., 1998]. In support of the caveolin- dependant mechanism, Wei et al. [Wei et al., 1999] demonstrated in kidney 293 cell line that uPAR- integrin signalling is affected by the number of caveolin expressed. Removal of these caveolin enhances fibronectin binding and prevents vitronectin binding and uPAR- integrin association. In the case of human neutrophils that express uPAR- CR3 and uPAR- CR4 interactions [Kindzelskii et al., 1996b and 1997] yet doesnt express caveolin; furthermore, lots of neutrophils activities like chemotaxis, oxidase priming etc required mediation by uPA, suggesting that a caveolin- independent signalling mechanism must exist.

The importance of uPAR- 2 integrin interaction is demonstrated in vivo in uPARnegative knockout mice. These mice have lowered 2- integrin- dependant emigration of leukocytes into inflamed peritoneum in contrast to wild type [May et al., 1998]. In addition, recruitment of neutrophils to lung upon infection with P. aeroginosa is significantly reduced in uPAR- negative mice [Gyetko et al., 2000]. Wild type mice can also have their neutrophil recruitment blocked using anti- CD11b. Waltz et al. [Waltz et al., 2000] also demonstrated that neutrophils recruitment is also blocked by using a hybrid protein consisting of amino terminal fragment of uPA and human Fc domain which blocks uPA binding site on uPAR. Therefore, 2- integrin dependant cell trafficking during inflammatory reactions are linked to partner protein uPAR. Recently, Tang et al. [Tang et al., 2008], demonstrated using reporter monoclonal 16

antibodies and fluorescence resonance energy transfer analysis the conformational changes in M2 upon uPAR interaction (Figure 11 and 12). This observation is in line with uPAR and 51 interaction as reported by Wei et al. [Wei et al., 2005]. Tang reported uPAR interact with M2 in its bent confirmation. This in turn causes hybrid domain movement leading to the reorientation and separation of the TM. It is proposed that this TM separation maybe one of the event in outside- in signalling of uPAR- M2 interaction [Tang et al., 2008].

Emerging GPI- Linked Partner Proteins

The number of emerging GPI- linked partner proteins grow over the decade. GPI- 80 is one such candidate. GPI- 80 is human adherence- related protein that interacts with CR3 integrin [Suzuki et al., 1999]. 2 integrin dependant adhesion of neutrtophils, transmigration of endothelial barriers and cytoskeletal reorganization have shown to be inhibited with anti- GPI- 80 mAb [Suzuki et al., 1999; Suzuki et al., 1997]. Recent studies by Huang et al [unpublished] showed close proximity between CR3 and GPI80 on neutrophil surfaces.

CD66b has also shown to interact with CR3. CR3 contributes to changes in adhesion during CD66 ligation [Ruchaud-Sparagano et al., 1997]. Studies showed physical interaction between them on neutrophil surfaces. Ottonello et al reported that this association can be blocked by D- mannose [Ottonello et al., 1999]. It was suggested that their interaction lead to certain cytolytic phenomena since cytolysis is also inhibited by D- mannose but enhanced by mAb VIM- 12, which mimic GPI- linked protein association with CR3.

Future Prospect: Medical Significances

Through the decades of studies in integrin- partner proteins has allow further understanding into the phenomena of cell adherence. However, there have yet made a major contribution in clinical medicine. In the case of GPI- linked- integrins complexes related diseases, studies have proven that a medical condition, pyoderma gangrenosum (PG) a disease that causes tissue to become necrotic, causing deep 17

ulcers that usually occur on the legs (Figure 13) , is found to be due to deficiency in integrin- GPI- linked protein interactions [Wollina, 2005 (unpublished), Shaya et al., 1998]. There are two possible classes of treatment currently under study. They are antagonistic high affinity carbohydrates and inhibitory peptides [Simon et al., 2000]. An example would be the interaction between 41 and transmembrane chondroitin sulfate proteoglycan is required to mediate melanoma cell adhesion. A sequence in the 4 integrin subunit bind directly to chondroitin sulfate glycosaminoglycan but when used as an antagonist, can block 41- mediated melanoma cell adhesion [Iida et al., 1998]. Another example in potential clinical application is in creating a peptide that mimic uPAR. This peptide mimetic will compete with uPAR to bind the W4 region of the - propeller of integrin CR3 and block cell adhesion to fibronogen, vitronectin, and cytokine- stimulated endothelial cells. Specific saccharides that block the lectin- like site on CR3 also prevent interaction with uPAR [Bohuslav et al., 1995]. Thus both peptide mimetic and saccharides can block uPAR- CR3 interactions and cell adherence. However, these potential mechanisms are not mutually exclusive. Regardless of the mechanisms involved, specifically designed peptides and saccharides screened on basis of the uPAR- CR3 interaction could lead to novel drugs to control metastasis as well as inflammation. Metastasis involves changes in cell adhesion and motility. This process consist of many cell- surface molecules involved n cell- ECM or cell- cell interactions, these include selectins, immunoglobulin like receptors, cadherins, integrins and proteoglycans. These molecules act in complex to hold cells in place or in other instance to sustain cell movement. One such complex is the integrin- tetraspan. Reports showed they play critical roles in tumour development, invasion or metastasis [Hemler et al., 1996; Keely et al., 1998 and Boudreau et al., 1998]. Data from different studies which are also mentioned under the transmembrane partner proteins section of this paper suggest that tetraspans might interrupt integrin movements in cell motility. This is further supported by the association of integrin- tetraspan complexes with PI 4- kinase [Berditchevski et al., 1997a and 1997b], which produce phosphatidylinositol- 4, 5- bisphosphate (4, 5PIP2), a regulator of cytoskeleton architecture. Anti- tetraspan antibodies like anti- 3 integrin, induce PI 3- kinase- dependent production of matrix metalloproteinase 2 (MMP- 2) in breast carcinoma cells [Sugiura et al., 1999] indicating that tetraspans are involved in the control of a matrix proteinase that allows malignant cells invasion of adjacent tissues through destruction of the ECM. Such mechanistic insight could 18

lead to new treatments for the prevention of metastasis. Furthermore understanding of integrin- tetraspan function could improve methods for prognosis prediction or even treatment of malignant tumours.

Integrin partner proteins, though small in size compared to integrins, have big roles to play in basic cell functions. As they are small in size, researchers have overlooked them only until the past two decades. However, their mechanistic functions have yet been fully understood. As research in finding more integrin partner proteins get more intensive as well as our understanding of cell adherence broaden, it should also be possible to utilize these fundamental insights in cell function into practical clinical treatments in metastasis and inflammation. Through this, drugs that have new targets can replace less efficient drugs already in market for cancer and auto/inflammatory diseases and hopefully for other integrin- partner proteins related disorders.

19

 subunits
1

Corresponding subunits
1* 10* 11* 2*

Ligands
Laminin, collagen Laminin, collagen Collagen Laminin, collagen, thrombospondin, E-cadherin, tenascin Laminin, thrombospondin, uPAR Thrombospondin, MAdCAM-1, VCAM-1, fibronectin, osteopontin, ADAM, ICAM-4 Fibronectin, osteopontin, fibrillin, thrombospondin, ADAM, COMP, L1 Laminin, thrombospondin, ADAM, Cyr61 Laminin Tenascin, fibronectin, osteopontin, vitronectin, LAPTGF-, nephronectin Tenascin, VCAM-1, osteopontin, uPAR, plasmin, angiostatin, ADAM, VEGF-C, VEGF-D ICAM, ICAM-4 ICAM, iC3b, factor X, fibrinogen, ICAM-4, heparin ICAM, iC3b, fibrinogen, ICAM4, heparin, collagen ICAM, VCAM-1, fibrinogen, fibronectin, vitronectin, Cyr61, plasminogen Fibrinogen, thrombospondin, , fibronectin, vitronectin, vWF, Cyr61, ICAM-4, L1, CD40 ligand Fibrinogen, vitronectin, vWF, thrombospondin, fibrillin, tenascin, PECAM-1, fibronectin, osteopontin, BSP, MFG-E8, ADAM-15, COMP, Cyr61, ICAM-4, MMP, FGF-2, uPA, uPAR, L1, angiostatin, plasmin, cardiotoxin, LAP-TGF, Del-1 Laminin Osteopontin, BSP, vitronectin, CCN3, LAP-TGF- LAP-TGF-, fibronectin, osteopontin, ADAM E-cadherin MAdCAM-1, VCAM-1, fibronectin, osteopontin LAP-TGF-

3 4 (VLA- 4)

5 (Fibronectin receptor)

6 (Laminin receptor) 7 8

L* (LFA- 1) M* (Mac- 1/CR3) X* (CR4) D*

IIb

V (Vitronectin receptor)

4 5 6 7

6 V V E* 4 V

Table 1. The 24 integrin heterodimers. The subunits with I domains are asterisked. Names in bracket are synonyms of the respective heterodimers.

20

Figure 1. Organization of domains within the primary structures. Some subunits contain an I domain inserted in the position denoted by the dashed lines. Cysteines and disulfides are shown as lines below the stick figures. (Picture and text adapted from Springer et al., 2002)

21

Figure 2. Schematic of the course of the and subunit polypeptide chains through domains from the N to C termini. Subunit on the left is and right is polypeptide chains. Generally, subunit consist of four domains; - propeller, thigh, calf- 1 and calf- 2. In nine other subunits, an I- domain or I may be present. subunit consist of 8 domains; I- like or I, hybrid, PSI, 4 I- EGF repeats and tail domains (Picture adapted from Springer et al., 2007)

22

Figure 3. Structure of the extracellular segment of V3. (A) Ribbon drawing of crystallized V3 [shown in blue (V) and red (3)]. (B) Model of an extended extracellular domain of V3. (Picture adapted from Xiong et al., 2001.)

23

Figure 4. The structure of V3 in the bent conformation. The V and 3 subunits are colored in green and red, respectively. (Picture adapted from Springer et al., 2007)

24

Figure 5. Structural rearrangement of the M I domain MIDAS. (a) Structure of the closed M I domain MIDAS. (b) Structure of the open I domain MIDAS. Glu314 from a neighbouring M I domain coordinates with the MIDAS magnesium. Purple and green spheres are Mn2+ and Mg2+ ions, respectively, and red spheres are coordinating water- molecule oxygens. [From PDB ID codes 1JLM and 1IDO]

25

Figure 6. Proposed model for separation of TM domain. The inactive integrin is characterise by inter- subunit interaction at the TM as well as at the membrane proximal region. The interaction packs the TM helices together in a long tilted fashion. When this packaging is separated, it gives rise to active integrin with high affinity for its ligand. (Picture adapted from Hong, 2007)

26

Figure 7. Different pathways by which integrins can link to the actin cytoskeleton. Integrin link actin through four pathways; ILK, - actinin, talin and filamin. In the case of ILK, - actinin and talin, associated proteins like - parvin, parvin, vinculin and Arp2/3 are required. Together these form connection between integrin and actin cytoskeleton. (Picture adapted from Miners Lect 2008)

27

Figure 8. Biogenesis of focal adhesions. (a) Many integrins that are not bound to the extracellular matrix (ECM) are present on the cell surface in an inactive conformation, which is characterized by bent extracellular domains that mask the ECM- binding pocket. This conformation is stabilized by interactions between integrin transmembrane domains, membrane- proximal extracellular domains and a salt bridge between the cytoplasmic domains. (b) When talin is recruited to the plasma membrane and activated in association with phosphatidylinositol phosphate kinase type- I (PIPKI), it binds to the cytoplasmic tail of integrins. This interaction separates the cytoplasmic domains and induces the integrins to adopt the primed conformation. (c) The integrin extracellular domains extend and unmask the ligand- binding site, allowing the integrin to bind specific ECM molecules. The separated integrin cytoplasmic domains and talin form a platform for the recruitment of other focal-adhesion proteins. Integrin- linked kinase (ILK), and isoforms of particularly interesting Cys- His- rich protein (PINCH) and parvin form the IPP complex in the cytoplasm, and this complex is recruited to focal adhesions through interactions with other factors, such as paxillin. Other proteins such as vinculin and focal adhesion kinase (FAK) are recruited to the nascent focal complex in a sequential manner. The maturation of focal adhesions involves clustering of active, ligandbound integrins and the assembly of a multiprotein complex that is capable of linking integrins to the actin cytoskeleton and communicating with signalling pathways. (Picture and text adapted from Legate et al., 2006)

28

Figure 9. Communication between I and I domains. Springer et al. proposed that - Glu310 acts as an intrinsic ligand that binds to the I- domain MIDAS and, thus, axially displaces the I- domain 7- helix in the C- terminal direction, reshaping the 6- 7 loop, therefore activating the I- domain MIDAS. (Picture adapted from Springer et al., 2007)

29

1 partners uPAR (3, 5, 6, V) TM4SF (3, 4, 6) IAP (2)

Reference * * * Berditchevski, F., Chang, S., Bodorova, J., and Hemler, M. E. (1997) J. Biol.Chem. 272, 2917429180 * Fenczik, C. A., Sethi, T., Ramos, J. W., Hughes, P. E., and Ginsberg, M. H.(1997) Nature 390, 8185 * Yoshida, T., Pan, Y., Hanada, H., Iwata, Y., and Shigekawa, M. (1998) J. Biol.Chem. 273, 15831590 Iida, J., Meijne, A. M. L., Oegema, T. R., Yednock, T. A., Kovach, N. L., Furcht,L. T., and McCarthy, J. B. (1998) J. Biol. Chem. 273, 59555961 Luissint, A. C., Pierre G. Lutz , David A. Calderwood , Pierre-Olivier Couraud, and Sandrine Bourdoulous: JAM-L mediated leukocyte adhesion to endothelial cells is regulated in cis by 41 integrin activation. J Cell Bio 2008: 183: 1159- 1173

CD147 (3, 6) Voltage-gated K+ channel type 1.3

CD98 (3) CD46 (3)

dystrophin complex

Transmembrane chondroitin sulfate proteoglycan

JAM- L 2 Partners

CD66b

Ruchaud-Sparagano MH, Stocks SC, Turley H, Dransfield I: Activation of neutrophil function via CD66: differential effects upon beta 2 integrin mediated adhesion. Br J Haematol 1997;98:612620. Suzuki K,Watanabe T, Sakurai S, et al.:A novel glycosylphophatidyl inositol-anchored protein on human leukocytes: a possible role for regulation of neutrophil adherence and migration. J Immunl 1999;162:42774284. Simon SI, Cherapnov V, Nadra I, Waddell TK, Seo SM, Wang Q, Doerschuk CM, Downey GP: Signaling functions of L-selectin in neutrophils: alterations in the cytoskeleton and colocalization with CD18. J Immunol 1999; 163:28912901. * * * * * *

GPI 80

L- selectin 3 Partners uPAR (V) TM4SF (IIb) IAP (V, IIb) PDGF receptor (V) Insulin receptor (V) CD36 (IIb)

Table 2. Summary of some laterally associated proteins with their partner integrins. Items in brackets denotes associated chain. * represent reference from Todd et al. ,1996, 1997; Woods et al. ,2000; Porter et al. ,1998; Hemler et al. ,1998.

30

Table 3. Summary of all known integrin- tetraspans complex (Table adapted from Berditchevski et al. , 2001)

31

Figure 10. Structural features of tetraspanins. Tetraspanins contain four transmembrane domains, a short extracellular loop (EC1), a very short intracellular loop (typically 4 amino acids), and a longer extracellular loop (EC2), flanked by relatively short N- terminal and C- terminal cytoplasmic tails (of 8- 21 amino acids, with a few exceptions). The EC2 is subdivided into a constant region (yellow, containing - helices A, B and E), and a variable region (blue), containing various protein- protein interaction sites. Whereas most protein- protein interactions sites and monoclonal antibody epitopes map to EC2, little is known about the structure or function of EC1. All tetraspanin contain a CCG motif after the B helix, and two other conserved cysteines (yellow), which are arranged to form two intramolecular disulphide bonds (red lines). Many tetraspanins contain two additional cysteines (green), which form another intramolecular disulphide bond (dotted red line). In exceptional cases, there might be eight cysteine (and four disulphide bonds) or seven cysteines (which form three intramolecular, and one intermolecular, disulphide bond) within EC2 (not shown). Transmembrane domains 1, 3 and 4 typically contain polar amino acids (Asn, Glu, Gln; red ovals) of unknown function. Nearly all tetraspanins also contain membrane proximal cysteine that undergoes palmitoylation. In addition to those highlighted here, at least 18 other amino acids (which are in EC2, the transmembrane domains and the Intracellular loop) are also highly conserved (6595% amino acid identity) among all known tetraspanins. (Picture and text adapted from Hemler et al., 2005)

32

Figure 11. An illustration of the domain organization in M2 and uPAR. The model of M2 was built using Modeler8v1 and PyMOL (W. L. DeLano 2002) using these structure coordinates. The bent M2 was generated using V3 coordinates 1L5G as template. The M I domain 1BHO was included. The structures of 2 plexin- semaphorin- integrin (PSI), hybrid, integrin epidermal growth factor 1 (EGF1), - 2, and - 3 were from 2P26 and 2P28. uPAR coordinates were from 2FD6. The M2 model only serves as an illustration in the absence of a complete structure of an I domain- containing integrin. The detail position of the I domain in an intact integrin has not been determined. (Picture and text adapted from Tang et al., 2008)

33

Figure 12. Hypothetical model of M2 conformational changes induced by uPAR. Lateral interaction of M2 with uPAR induces hybrid domain movement in an overall bent conformer, consequently triggering the separation of the TMs. (Picture adapted from Tang et al., 2008)

34

Figure 13. Ulcer manifestation in pyoderma gangrenosum (PG). An uncommon ulcerative cutaneous condition which may occur in association with systemic disease like inflammatory bowel disease (IBD). PG usually starts suddenly and often at the site of a minor injury as a small pustule, red bump or blood blister. The size and depth of the ulcerations vary greatly, and they are often extremely painful. The small opening will eventually led to chronic wound in which the skin breakdown resulting in ulcer that deepen and widen rapidly. (Picture adapted from Hazel et al., 2002)

35

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