ELSEVIER Biochimica et Biophysica Acta 1242 (1995) 77-98
BB Biochi~ic~a et Biophysica A~ta
Signal transduction by cell adhesion receptors
Carlos Rosales a, Vincent O'Brien a, Loft Kornberg a,b, Rudy Juliano a.*
a Department o f Pharmacology, School o f Medicine, University of North Carolina, Chapel Hill, NC 27599, USA b Research Triangle Institute, P.O. Box 12194, RTP, NC 27709, USA
This review will deal with the emerging field of signal transduction by cell adhesion receptors. Plasma membrane proteins that mediate cell to cell or cell to extracellular matrix (ECM) adhesion have long been of interest because of their importance in embryogenesis [1-3], in the control of hemostasis [4], in the circulation of lymphocytes [5,6], and in the altered invasive and metastatic behavior of malignant cells [7-9]. Several major families of adhesion receptors have been identified and characterized over the last few years: these include the integrins, the cadherins, the selectins, immunoglobulin super-family members, and membrane-associated proteoglycans. Until very recently, most research on these families of molecules has been directed toward elucidation of their structures, identifica- tion of their ligands or counter-receptors, determination of patterns of tissue expression, and evaluation of their role in cell adhesion, morphology and motility. Now, however a
new paradigm is emerging which suggests that cell adhe- sion receptors also have important functions in signal transduction cascades that impinge on the regulation of cell growth and differentiation. The purpose of this review article is to encourage the development of this new paradigm by providing basic information concerning known signal transduction cascades, basic information on cell adhesion molecules, and an overview of what is currently known of adhesion receptor signaling.
2. Families of adhesion receptors
This section will briefly review some of the key fea- tures of the cadherin, immunoglobulin (CAM), selectin, and integrin families of cell adhesion receptors. Since many thousands of publications on cell adhesion receptors have appeared over the last few years [2], only a small number of research articles and reviews will be cited here
Membrane
Catenins
C ll'l I I I I I I
ICAM-2 ICAM-1 VCAM-1 MHC-Class II L P E ctm [32
Cadherins Ig Superfamlly Selectlns
Mac-1
a5 i ~ a l i n
~ x-Actinin
FnR
Integrlns
Fig. 1. Structure of adhesion receptor families. The general structure and representative examples are shown schematically. Cadherins have five extracellular (EC) domains that contain multiple glycosylation sites. The amino-terminal EC1 domain has the adhesive recognition site, while the other domains include putative calcium binding sites. There is a proteolytic cleavage site near the plasma membrane, The cytoplasmic tail interacts with the cytoskeleton via catenin proteins. The immunoglobulin (lg) superfamily contains many members. They all share the immunoglobulin domain, a compact structure of 60-100 amino acids arranged as a 3-stranded beta sheet facing a 4-stranded beta sheet, which is usually stabilized by disulphide bridges. Different members of this family may have extensive or abbreviated cytoplasmic domains. Selectins have an amino-terminal domain which is homologous to calcium-dependent animal lectins, followed by an EGF-type domain, two to nine repeats of a complement regulatory protein domain, and a short cytoplasmic tail. Integrins are heterodimer receptors that contain ot and /3 subunits of approximately 1100 and 750 amino acids, respectively, which are non-covalently associated. Each subunit has a single membrane spanning region, and with the exception of /34, a short cytoplasmic tail. The ot chain extracellular globular domain contains calcium binding sites that are important for ligand binding. Some integrin cytoplasmic domains interact with focal contact proteins such as talin and a-actinin which then bridge to the actin cytoskeleton. Mac-l, complement receptor 3. FnR, fibronectin receptor (t~5/3 1). Molecules are not drawn to scale. For further detail see Refs. [33,38,290-292].
c. Rosales et al. / Biochimica et Biophysica A cta 1242 (1995) 77-98 79
to provide the reader with some key ideas about these molecules. A schematic of the structure of several types of adhesion receptors is presented in Fig.Fig. 1 1.
2.1. The cadherins
The cadherins comprise a family of calcium-dependent adhesion receptors that are usually employed in homotypic cell-cell adhesion [10]. The cadherins can be divided into two subgroups based on their association with cytoskeletal components [11]. The first group includes the N, P, R, B, and E cadherins. These molecules localize in adherence- type junctions, and can form linkages to the actin-contain- ing cytoskeleton. The desmogleins and desmocollins com- prise a group of desmosome-associated cadherins that can form linkages to intermediate filaments [12].
The structure of a typical cadherin consists of an amino-terminal external domain having five tandem re- peats, a single transmembrane segment, and a cytoplasmic carboxy-terminal domain of about 150 amino acids (Fig. 1). The binding functions of the cadherin are localized in the amino-terminal tandem repeat, while the other repeats contain putative calcium binding sites. The cytoplasmic domains of cadherins interact strongly with a group of intracellular proteins known as catenins [13]. Truncation of the cadherin cytoplasmic domain so as to delete catenin binding sites leads to a loss of adhesive function, indicat- ing that catenins modulate the ligand binding activity of cadherins [10]. Since there is considerable homology among their cytoplasmic domains, different cadherins can com- pete for the same pool of catenins [14].
Cadherins play a critical role in the determination of tissue organization. They are specially important in some facets of morphogenesis during development [ 11,15], while in adult organisms, loss of cadherin expression in epithelial tumors is associated with a more invasive and malignant phenotype [ 16,17].
most universally present in nervous tissue [19]. Adhesive interactions mediated by NCAMs are known to be regu- lated by both the abundance of receptor, and its degree of polysialyation [20]. Several other neural cell adhesion molecules belong to the Ig super-family, including L1, MAG, contactin, and Drosophila fasiculins I and II [19], As with the cadherins, the intracellular domains of CAMs may also regulate their adhesive functions [21,22].
CAMs are involved in cell adhesion events in a variety of cell types. Thus, T-lymphocytes express several Ig super-family receptors including CD2, CD4 or CD8, ICAMs 1 and 2, and the T-cell receptor (TCR) itself [5]. These receptors play critical roles in antigen recognition, cytotoxic T cell functions, and lymphocyte recirculation. In contrast to the case of NCAM, which is a homotypic receptor, Ig family proteins involved in the immune system engage in heterotypic interactions. For example, CD2 on T-cells interacts with LFA-3 (another Ig super-family member) expressed on target cells, the TCR interacts with MHC class II proteins (both Ig superfamily), while ICAMs on endothelial cells are recognized by /32 integrins on leukocytes. A number of other Ig family adhesion recep- tors are known. For example, VCAM-1 is a counter-recep- tor for integrin c~4/3 1, that is also involved in leukocyte- endothelial cell interactions [23]. PECAM-1 is an Ig family member that is a homotypic cell-cell adhesion molecule; one of its roles seems to be maintaining tight contacts between adjacent vascular endothelial cells [24]. Carci- noembryonic antigen (CEA) and Deleted in Colon Carci- noma (DCC) are two proteins prominently associated with colon cancer, both of which are Ig family members that seem to have cell adhesive functions [25-28]. Recently a number of transmembrane phosphatases have been identi- fied that belong to the Ig superfamily and seem to be involved in cell adhesion [29,30].
2.3. Selectins
2.2. Immunoglobulin super-family o f adhesion receptors
A diverse array of cell adhesion receptors are included in the immunoglobulin (Ig) super-family (CAMs). Proteins of this family are defined by the presence of one or more copies of the Ig fold, a compact structure of 60-100 amino acids arranged as a 3-stranded beta sheet facing a 4-stranded beta sheet [11]. In most cases the structure of Ig family adhesion receptors includes an amino-terminal extracellu- lar domain, a single transmembrane segment, and a cyto- plasmic tail (Fig. 1).
The 'classic' example of an immunoglobulin super- family adhesion receptor is NCAM, the neural cell adhe- sion receptor [1]. The extracellular domain of NCAM contains five Ig folds; various adhesive functions of NCAM have been associated with different parts of the extracellu- lar domain [18]. NCAM functions as a homotypic, cal- cium-independent adhesion receptor, and seems to be al-
The selectins comprise a recently discovered family of lectin-like adhesion receptors [31]. The structure of a se- lectin includes an amino-terminal domain which is homol- ogous to calcium-dependent animal lectins, followed by an epidermal growth factor (EGF)-type domain, two to nine complement regulatory protein repeats, a transmembrane segment, and a short cytoplasmic tail (Fig. 1). Selectins mediate heterotypic cell interactions through calcium-de- pendent recognition of sialyated glycans. The best defined physiological role for selectins concerns leukocyte adher- ence to endothelial cells and platelets during inflammation [6,32,33]. P-selectin is present in latent form in endothelial cells and platelets and it is rapidly translocated from secretory granules to the cell surface upon cell activation. E-selectin is expressed on endothelial cells in response to inflammatory cytokines. L-selectin is constitutively ex- pressed on leukocytes.
The precise identity of the ligands for the three cur-
80 c. Rosales et al. / Biochimica et Biophysica Acta 1242 (1995) 77-98
rently known selectins is actively being pursued. All three can bind to sialyated, fucosylated lactosaminoglycans, par- ticularly the sialyl Lewis X motif [31]; such motifs can appear on glycolipids as well as on glycoproteins. Re- cently, mucin-like glycoprotein ligands for L-selectin and P-selectin have been cloned [31,34], indicating that glyco- proteins rather than glycolipids are the most significant targets for selectins.
2.4. Integrins
activities to individual integrins. Other reports have impli- cated a cytoplasmic domains in cytoskeletal organization and cell motility [56], in modulation of focal contact formation [52], or in activating integrins ('inside out sig- naling') [57,58]. Some integrins seem to form a complex with focal contact proteins including a-actinin and talin via binding sites in the /3 subunit cytoplasmic domains [59,60]. These interactions could be important in cyto- skeletal organization and may also play a role in signaling processes.
The integrins are a family of cell-surface glycoproteins that act as receptors for extracellular matrix proteins, or for membrane bound counter-receptors on other cells. Each integrin is a heterodimer that contains an a and a /3 subunit with each subunit having a large extracellular domain, a single membrane spanning region, and in most cases (other than /34), a short cytoplasmic domain [35,36] (Fig. 1). The integrin receptor family of vertebrates in- cludes at least 15 distinct a subunits and 8 or more /3 subunits which can associate to form at least 21 distinct integrins [11,37,38]. The c~//3 pairings specify the ligand binding abilities of the integrin heterodimers. Some inte- grins, such as t~5/31, the 'classic' fibronectin receptor, bind to a single ECM protein [39]. More commonly, an individual integrin will recognize several distinct extracel- lular matrix proteins [40,41]. Cells often display multiple integrins capable of interacting with a particular ECM protein [11,37], thus integrin expression is often apparently redundant. Some integrin ot subunits undergo alternative splicing in a tissue-type specific and developmentally regu- lated manner [42]; this suggests discrete intracellular func- tions for individual integrins.
The relationships between integrin structure and the various functions of integrins are an active area of investi- gation. The ligand binding regions of both /3 1 and /3 3 subunits have been localized [43-45], while in a subunits, occupancy of the metal ion domains has been shown to be critical for ligand binding [46]. However, the most detailed information currently available concerns the cytoplasmic domains of integrins. Truncation of the /31 or /3 2 cyto- plasmic domains reduces integrin affinity for its ligands and impairs /3 1 recruitment to focal contacts [47-49]. Further, the /3 1 cytoplasmic domain alone, even expressed as part of a fusion product with another membrane protein, seems sufficient for focal contact localization and at least some aspects of signal transduction [50,51]. The evaluation of the role of c~ chain cytoplasmic domains has been more complex. One hypothesis is that the a cytoplasmic domain modulates the function of the /3 cytoplasmic domain, preventing recruitment to focal contacts unless the integrin is occupied by its ligand [52,53]. Cytoplasmic truncation of different a subunits has been reported to cause either activation (al Ib) [54] or inactivation (a4) [55] of ligand binding. These reports clearly show that different ct chains possess different intrinsic properties that confer diverse
3. Signaling pathways
Recently, it has become clear that adhesion receptors can initiate biochemical signaling cascades within the cell. This raises the possibility that adhesion receptors may impinge upon signaling paths previously defined in con- nection with other types of receptors. We will now provide a brief and simplified description of some of the major signal transduction pathways currently known for the pur- pose of comparing these pathways to the less well under- stood pathways of adhesion receptor signaling.
3.1. Receptor tyrosine kinase pathway
This pathway begins with a cell-surface receptor that is a tyrosine kinase (RTK) (e.g. receptors for epidermal growth factor, nerve growth factor, and platelet-derived growth factor) [61] or that is associated with such a kinase (e.g. T-cell receptor or interleukin-6 receptor) [62,63] and continues all the way to the nucleus. The proto-oncogene protein Ras is central to this pathway therefore this signal- ing cascade is also known as the Ras pathway. Ras signal- ing pathways seem to be present in all eukaryotic organ- isms from yeast to humans [64].
The receptor tyrosine kinase pathway starts with the binding of a growth factor to its tyrosine kinase receptor. This ligand interaction induces the receptor to cluster in the plane of the membrane resulting in activation of the receptor's kinase domain and autophosphorylation of spe- cific tyrosine residues [61]. Adaptor proteins, such as Grb2/Sem5, bind to a Ras-activator protein (mSosl) [65- 68] bringing it to the receptor to form a stable complex [67,69,70]. Activation of Ras is induced by the exchange of GDP for GTP. On the other hand, inactivation is mediated by GTPase activating proteins (GAPs), that stim- ulate the intrinsic GTPase activity of Ras. The balance between Sos 1-type stimulators and GAPs determines the level of activity of Ras in a particular cell [71]. Once activated, Ras has effects on cytoplasmic serine/threonine kinase cascades [72], that involve Raf-1 [73], MEK (also named MAP kinase kinase) [74-77], and mitogen-activated protein (MAP) kinases (also named ERKs) [78], and possi- bly also ribosomal $6 kinases (Rsk) [79]. MAP kinases can migrate from the cytosol into the nucleus after growth
C. Rosales et al. / B iochimica et Biophysica Acta 1242 (1995) 77-98 81
RTK
@ ® 1
Nucleus 1
DNA ~ Gene =' Expression SRE
Fig. 2. Signal transduction pathway of tyrosine kinase receptors. RTK, receptor tyrosine kinase; Grb2, connector protein with SH2 and SH3 domains; Sos, Ras activator protein; Ras, the protein product of c-ras; GAP, GTPase activating protein; Raf-1, a kinase, the protein product of c-raf; MEK, MAP kinase kinase; MAPK, mitogen-activated protein ser/threo kinases, also known as ERKs; Elk-l, a component of the transcription factor complex that recognizes the serum-responsive ele- ment; SRE, serum-responsive element motif in DNA.
factor stimulation [80] finally delivering the signal into the nucleus by phosphorylating transcription factors [81-83] (Fig.Fig. 2 2).
The elucidation of this pathway has provided an attrac- tive model for signal transduction from plasma membrane receptor tyrosine kinases all the way to nuclear transcrip- tion factors. However, it is by no means complete; for example, it does not explain specificity. Different growth factors, all signaling through receptor tyrosine kinases, induce very different responses in cells [84]. Other levels of control must exist to account for this diversity in cellular responses. Signals intersecting or diverging at various points in this pathway, or activation of other pathways by a particular receptor would result in specific responses [85]. In T-lymphocytes, for example, Ras is activated by the unique hematopoietic-specific guanine nucleotide exchange protein Vav after T-cell receptor stim- ulation [86] (Fig.Fig. 3 3). Crosstalk between major signal transduction pathways is also possible. Recent evidence indicates that the cAMP signaling pathway is intercon- nected to the tyrosine kinase receptor pathway [87-89] (Fig. 3). These reports demonstrate that increases in intra- cellular cAMP concentrations inhibit transmission of growth stimulatory signals through the Ras pathway [64]. Raf-1 is also able to deliver a signal directly to the nucleus via activation of the nuclear factor NF-KB by phosphoryla- tion of its inhibitor I K B [90], thus bypassing the MAP kinase route. A further indication that the Ras pathway is connected to other signaling pathways is suggested by the
fact that Raf-1 can be directly activated by protein kinase C (PKC) [91]. There is also evidence for a pathway from G proteins to MEK that bypasses both Ras and Raf [92] (Fig. 3).
One of the key concepts to emerge from recent studies of the Ras pathway is the idea of the signal transduction complex. Transmission of the signal depends not only on changes in the activity of the protein components of the signaling path, but also on the physical association of those components mediated primarily by SH2 and SH3 domains [61,65,67,69,70,93-96]. Thus ideas about compartmental- ization and organization figure prominently in our under- standing of signaling mediated by mitogens and acting through the RTK/Ras pathway. As we will see below, intracellular organization also seems to be an important aspect of signaling mediated by adhesion receptors.
3.2. G-protein pathway
Many classes of hormones initiate a signal transduction pathway by interacting with receptors that couple to GTP- binding proteins (G proteins). Ligand-receptor interaction
Set R RTK Membrane NTKR
Nucleus
I I
DNA ~ ~ Gene Expression
Fig. 3. Signals intersect at various points in signal transdnction pathways. The minus sign on top of the arrow from cyclic AMP indicates inhibition of the ras pathway at a point between Ras and Raf-1. RTK, receptor tyrosine kinase; NTKR, non-tyrosine kinase receptor; Ser R, G-protein- coupled serpentine receptor; Grb2, connector protein with SH2 and SH3 domains; Sos, Ras activator protein; Ras, the protein product of c-ras; GAP, GTPase activating protein; Src-like, kinase with structural homol- ogy to Src coupled to a non-tyrosine kinase receptor; Vav, a hemato- poietic-specific GTP exchange protein; Raf-l, the protein product of c-raf; MEKK, MEK kinase; MEK, MAP kinase kinase; MAPK, mitogen-activated protein ser/threo kinases, also known as ERKs; cAMP, cyclic AMP; NF-kB/IkB, the NF-kB transcription factor and its cytoplas- mic inhibitor protein; Elk-l, a component of the transcription factor complex that recognizes the serum-response element.
82 C. Rosales et al. / Biochimica et Biophysica Acta 1242 (1995) 77-98
activates the G-proteins which subsequently interact with effector enzymes to generate messengers such as cyclic AMP (cAMP), or 1,2-diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP 3) [97-101]. G proteins are hetero- trimers formed by a , /3 and 7 subunits [102,103]. The /3 and 7 subunits function as a dimer and control the func- tion of the t~ subunit. The c~ subunit binds GTP but has an intrinsic GTPase activity that spontaneously hydrolyzes
GTP to GDP; in an unstimulated cell most G proteins have GDP bound.
All the receptors that interact with G proteins have a common structure. They are called serpentine receptors having seven hydrophobic transmembrane c~ helixes, a large cytoplasmic loop between ~ helixes 5 and 6, and a large hydrophilic tail at the C-terminus [104]. When a sepentine receptor binds its ligand, the complex interacts
Fig. 4. cAMP-dependent signal transduction by G-protein-coupled receptors. Upon ligation of a hormone to a serpentine receptor (Ser R), adenylyl cyclase is activated via the G-protein a subunit. Cyclic AMP (cAMP), in turn, activates cAMP-dependent protein kinase by dissociating its catalytic domain (C) from the regulatory subunits (R). cAMP-dependent protein kinase then phosphorylates several target proteins in the cell.
SerR Membrane
PIP2
G-Protein
od [. Ca .2
Endoplasmic Reticulurn
Active , Inactive Protein Protein
Fig. 5. InositoMipid signal pathway. Binding of a ligand to its serpentine receptor (Ser R) triggers activation of a G protein that, in turn, activates phospholipase C (PLC). The enzyme cleaves phosphatidylinositol 4,5-bisphosphate (PIP 2) to inositol 1,4,5-trisphosphate (IP 3) and 1,2 diacylglycerol (DAG). IP 3 diffuses in the cytosol to the endoplasmic reticulum, where it interacts with its specific receptor (IP3R), releasing Ca 2÷ from the lumen of the endoplasmic reticulum. Ca 2÷ binds in the cytosol to calmodulin and the complex Ca2+/calmodulin then activates several proteins in the cell. DAG remains in the membrane, where together with Ca 2+ and phosphatidyl serine, it activates protein kinase C (PKC), Activated PKC, in turn, phosphorylates several cellular proteins to modify their activities.
c. Rosales et al./ Biochimica et Biophysica Acre 1242 (1995) 77-98 83
with the ce subunit of the G protein causing it to displace GDP and bind GTP. During activation, the /33' dimer dissociates and the o~ subunit is freed to directly activate adenylyl cyclase or other targets [105]. The activity of the effector enzyme is terminated rapidly because GTP is quickly hydrolyzed to GDP, causing the reassociation of a and /33, subunits resulting in the inactivation of the G protein and subsequently the enzyme.
One common pathway of G protein-mediated signaling involves activation of adenylyl cyclases. The properties of the different types of adenylyl cyclases result from their different susceptibility to regulation by Ca ÷2, the /33, subunits of G proteins, and protein kinases [106-109]. The next step in the signal transduction pathway is often the activation, by cAMP, of specific enzymes; in most cases the cAMP-dependent enzymes are protein kinases [110] (Fig.Fig. 4 4).
Other molecules can also function as G protein-linked second messengers. A rise in the cytosolic concentration of Ca 2+ ions induces many cellular responses (for review see [1 1 l, 1 12]). Some serpentine receptors are coupled via a Gq protein to one of several phospholipase C enzymes [111,113]. Activation of phospholipase C (PLC) by the receptor-ligand complex on the surface of the cell results in hydrolysis of the plasma membrane phospholipid, phos- phatidylinositol 4,5-bisphosphate (PIP2), generating two signaling molecules, IP 3 and DAG [1 13] (Fig.Fig. 5 5). IP 3 binds to specific receptors of intracellular membrane com- partments [114,115], inducing the release of Ca 2+ into the cytosol [112]. Ca 2+ then binds to calmodulin, a ubiquitous small protein in eukaryotic cells [116]. Finally, calcium- calmodulin complexes activate several enzymes (mainly protein kinases) involved in different functions within the cell [ 117] (Fig. 5).
DAG, the other product of PIP 2 hydrolysis by PLC, is an activator of some PKC isoforms. DAG-responsive PKCs are found in the cytosol in an inactive form. Ca 2- induces PKC translocation to the plasma membrane where it can be activated by DAG. PKCs are important enzymes that couple the receptor signal with the functional machinery of the cell by activating or deactivating a number of proteins through phosphorylation of serine/threonine residues [118] (Fig. 5).
3.3. Other pathways
Recently there has been a rapid development of knowl- edge about other signal transduction pathways. While we cannot provide an extensive review, the reader should be aware of two additional pathways: (a) the JAK-STAT pathway; (b) the sphingolipid pathway. The Janus kinases (JAKs) are a family of cytoplasmic tyrosine kinases that couple to cytokine receptors such as those for interferons and some interleukins. Upon activation, the JAK kinases can trigger the Ras signaling pathway, but in addition, they can also directly tyrosine phosphorylate and activate cyto-
plasmic proteins (termed STATs) that then migrate to the nucleus and participate in transcription of specific genes [119-122]. The sphingolipid signaling pathway can be activated by a variety of agents, with the prototype being the inflammatory cytokine TNFa. Engagement of the TNF receptor activates a sphingomyelinase that cleaves sphingomyelin and releases ceramide. Subsequently, ce- ramide activates both protein kinases and protein phospha- tases as part of a complex and as yet poorly understood signaling pathway. Ultimately these changes lead to phos- phorylation and activation of transcription factors includ- ing c-jun and NF-KB. The sphingolipid signaling pathway has been implicated in negative regulation of cell growth and in apoptosis [ 123-125].
3.4. Regulation o f signaling by the cytoskeleton
A connection between cytoskeletal reorganization and biochemical pathways of signal transduction has been dis- cussed extensively but many details remain to be deter- mined [126-129]. It seems reasonable that appropriate cell shape and cytoskeletal organization might influence the functions and biosynthetic capabilities of the cell [ 130,131 ]. One interesting concept of signaling by cell adhesion receptors is a mechanochemical one; the integrins or other receptors would essentially play the role of a physical link between the extracellular matrix and the cytoskeleton, while the organization and mechanical tension of the cyto- skeleton would directly influence events in the cytoplasm and nucleus. This 'tensegrity' concept has been elegantly developed by Ingber and his colleagues [132]. However, there may also be very immediate linkages between cyto- skeletal organization and well known second messenger pathways. For example, in fibroblastic cells, relaxation of stress on the cytoskeleton can trigger a marked increase in cAMP levels [133]. Thus biochemical events rather than structural ones may be the key mediators in cytoskeletal effects on signal transduction.
Evidence is emerging supporting the idea that there are bi-directional connections between signaling pathways and the organization of the cytoskeleton. For example, the Ras-related small GTP-binding proteins Rho and Rac are known to be involved in controlling actin polymerization [134-136]; it is also clear that growth factor receptors are coupled via Ras to Rho and Rac, and thus influence cytoskeletal assembly [137]. Recently, it has been shown that Rho activates phosphatidylinositol 3-kinase in prepara- tions of platelets [138], and also phosphatidylinositol 5- kinase in mammalian cells [139], suggesting a mechanism by which Rho could regulate cytoskeletal reorganization. Activation of the phosphatidylinositol 5-kinase also affects the availability of substrates (PIP 2) involved in growth factor signaling [ 139].
A number of other reports indicate that the cytoskeleton can be influenced by several signal transduction pathways. For example, proteins involved in actin cross linking have
84 C. Rosales et al. / Biochimica et Biophysica Acta 1242 (1995) 77-98
been identified as substrates for protein kinase C [ 126,140]. Conversely, cytoskeletal proteins can regulate signal trans- duction by several different mechanisms. For example, actin filaments modulate enzymatic activities through di- rect interaction with phosphatases and kinases [141,142]. L-plastin, an actin bundling protein, has been implicated in the IPa-independent mechanism of Ca 2+ release from intracellular stores after Fc receptor ligation in leukocytes [143,144]. The cytoskeletal protein ankyrin has been found to block the IPa-dependent release of Ca z+ by direct competition with IP 3 for the IP 3 receptor [145,146]. All these reports indicate that, in some cases, the cytoskeleton is clearly involved in the process of signal transduction.
Proteins are organized spatially in the cell via interac- tions with the cytoskeleton, as exemplified by the forma- tion of focal contacts after integrin interaction with extra- cellular matrix proteins [127]. The actin cytoskeleton may be a site where signals are organized by bringing together molecular complexes. This idea is supported by a number of observations concerning signaling events after disrup- tion of the actin cytoskeleton by cytochalasin D treatment: this includes inhibition of Ca 2+ release by Fc receptor binding of immune complexes in neutrophils [144], reduc- tion of focal adhesion-associated kinase (pp125 FAK) phos- phorylation [147], and inhibition of MAP kinase activation after engagement of integrins in fibroblasts [148]. Further, recent fluorescence microscopy observations in cells treated with beads coated with anti-integrin antibodies show that numerous signal transduction proteins, as well as cyto- skeletal proteins, accumulate at sites of integrin clustering [149]. Thus, as in the case of formation of signal transduc- tion complexes involving interactions of SH2/SH3 do- mains, the cytoskeleton may provide a framework for physically linking cell surface receptors with components involved in signal transduction pathways.
4. Signal transduction by adhesion receptors
4.1. Integrin signaling and pp125 FAK
Evidence from several laboratories suggests that inte- grins are capable of transmitting biochemical signals from the extracellular matrix to the cell interior [37,150]. Our laboratory was the first to demonstrate that ligation of integrins in carcinoma cells leads to enhanced tyrosine phosphorylation of a complex of proteins of about 130 kDa [151]. Similar observations emerged from other labo- ratories studying fibroblasts [152] or platelets [153]. A 125 kDa focal adhesion-associated kinase (pp125 FAR) was originally identified as being one of several proteins that are highly phosphorylated in src-transformed chicken em- bryo fibroblasts [154]. Subsequent cloning of the cDNA for this protein showed that it is a novel tyrosine kinase which can localize to focal adhesions [155]; full-length mouse [156] and human ppl25 FAK [157] versions have also
been cloned. Interestingly, this kinase is structurally dis- tinct in that it shows no homology to other known kinases except in its catalytic domain, nor does it contain SH2 or SH3 domains [155,156,158].
Our laboratory [159] and others [147,153,156,160-162] have shown that pp125 FAK is phosphorylated on tyrosine in response to integrin-clustering or cell adhesion. The increase in tyrosine phosphorylation is associated with an increase in pp125 FAK autophosphorylation and activity towards exogenous substrates in kinase assays in vitro [153,160]. ppl25 ~gK is also phosphorylated on tyrosine in response to integrin activation in platelets [153,161], and upon exposure of fibroblasts to bombesin, vasopressin, PDGF, endothelin, or PKC activators [37,150- 152,154,158,163-166]. Experiments with fibroblasts that were transfected with a kinase-inactivated version of pp 1 2 5 FAK , indicate that autophosphorylation is the primary basis of pp125 FAR tyrosine phosphorylation and that Y397 is the major autophosphorylated site [167]. It is not known how the FAK phosphorylation signal is generated from the cell exterior, but it seems to require the /31 subunit of integrins [51,152]. Experiments with agents that disrupt the actin-cytoskeleton suggest that at least partial integrity of the actin-cytoskeleton is needed for ppl25 FAR phospho- rylation [147]. At this point it is not clear whether ppl25 FAK interacts directly with integrins, or whether other events (possibly activation of other kinases) may precede ppl25 ~AK activation [161] (Fig.Fig. 6 6).
Several lines of indirect evidence suggest that ppl25 FAR is involved in assembly of the cytoskeleton: (1) phospho- tyrosine staining seems to be more intense in new, more peripheral focal adhesions than in the central, older focal adhesions [147]; (2) maximal pp125 FAR phosphorylation occurs coincidently with cell adhesion, but prior to cell spreading [159]; (3) treatment of fibroblasts with the tyro- sine kinase inhibitor, herbimycin A interfers with cell adhesion-induced ppl25 FAR phosphorylation and inhibits formation of focal contacts and stress fibers [147]; (4) the focal adhesion associated proteins, paxillin and tensin, are substrates (in vitro) for pp125 FAK and their phosphoryla- tion increases upon cell adhesion [168]. Thus, current evidence suggests that ppl25 FAK is somehow involved in cytoskeleton assembly. Paradoxically, ppl25 FAR may also participate in the focal adhesion disorganization observed in src-transformed fibroblasts. There is several-fold more phosphotyrosine associated with pp125 FAR in src-trans- formed fibroblasts than in normal fibroblasts and the activ- ity of ppl25 FAK increases concurrently [160]. Tyrosine phosphorylated pp125 FAK can stably associate with src via the src-SH2 domain [158,167]. It was suggested that the physical association of pp125 FAR and src may lead to the recruitment of src to focal adhesions, causing a hyperphos- phorylation of focal adhesion-associated proteins and dis- organization of focal adhesion structures [167]. An interac- tion between ppl25 FAK and the fyn tyrosine kinase, an- other src family member, has also been reported [169].
C. Rosales et al. / Biochimica et Biophysica Acta 1242 (1995) 77-98 85
M ~ Integrin
1 Nucleus 1
D~CO000CC< ] Fig. 6. A comparison of signaling pathways mediated by tyrosine kinase receptors versus integrins. The left hand side of the figure shows the tentative pathways of integrin-mediated signal transduction. The right- hand side shows the better established pathway for RTK signal transduc- tion. Arrows indicate connections which are reasonably well established, while question marks indicate more hypothetical connections. Additional arrowheads along a path indicate the existence of multiple steps rather than a direct interaction. RTK, receptor tyrosine kinase; pp125 yAK, the integrin-regulated 125 kDa focal adhesion kinase; Syk, an SH2 containing cytoplasmic tyrosine kinase; Grb2, connector protein with SH2 and SH3 domains; Sos, Ras activator protein; Ras, the protein kinase product of c-ras; Rat-I, the protein product of c-raf; MEK, MAP kinase kinase; MAPK, mitogen-activated protein ser/threo kinases, also known as ERKs; NF-KB/IKB, the NF-kB transcription factor and its cytoplasmic inhibitor protein; X, a hypothetical linker protein that may permit inte- grins to interact with pp125 FaK or with Syk. The pathway involving Syk and NF-KB has been demonstrated only in myeloid cells.
The association between pp125 vAK and src family members leads to increased phosphorylation of pp125 FAK which can create binding sites for other proteins with SH2 domains. Thus, recent work has documented association between pp125 FAK and PI-3-kinase [170] and also Grb2 [171]. These observations suggest that the integrin- pp125 rAK interaction can lead to the formation of com- plexes that include some of the same proteins that are recruited to signal transduction complexes formed by acti- vation of receptor tyrosine kinases. Whether similar or distinct downstream events are activated via integrins and via RTKs remains to be determined.
Although it is postulated that pp125 FAK is involved in focal adhesion formation, the biological function of pp125 FAr~ remains a mystery, pp125 FAr~ is highly con- served between the mouse, chicken, and human versions suggesting some fundamental role for the kinase [155- 157,172]. When pp125 FAK was overexpressed in fibrob- lasts, there were no changes in total protein phosphoryla- tion or cell morphology [167]. However, these cells were not reported to have been assayed for changes in cell adhesion and motility. These experiments did, however.
localize the focal adhesion targeting (FAT) sequence of pp125 FA~ to the C-terminal portion of pp125 Far~, which is also known as p41 ~NK (FAK-related non-kinase), and which can be expressed autonomously in vivo via an alternatively processed mRNA [158]. One would expect p41 vRNK to be a dominant negative regulator of pp125 FAK by excluding ppl25 yAK from focal adhesions. However, chicken fibroblasts which overexpress p41 ~NK show no changes in cell morphology or growth [167]; once again these cells were not assayed for changes in cell adhesion or motility. Interestingly, pp125 vA~ seems to be overex- pressed in human metastatic tumor tissue [173]. It is not known if this contributes to metastasis; however, it sug- gests a possible role for pp125 FAK in cell motility [174].
Evidence is accumulating that protein tyrosine phospha- tases are also involved with the regulation of cell adhesion. When fibroblasts are dissociated from the substratum, there is a rapid loss of protein-associated phophotyrosine [152,175] and an increase in tyrosine phosphatase activity in cell extracts in vitro [175]. Data from our laboratory has shown that when cells are dissociated from the substratum using trypsin or mechanical disruption, pp 125 FAK rapidly losses phosphotyrosine (Kornberg and Juliano, unpub- lished data). These data strongly suggest that cell detach- ment activates a tyrosine phosphatase; perhaps this phos- phatase regulates pp125 FAK.
4.2. Other integrin signaling events
Although there has been a great deal of interest in the integrin/pp125 FAK connection, it seems clear that other tyrosine kinases exist that are responsive to integrin stimu- lation. For example, in T-lymphocytes ligation of /32 integrins stimulates tyrosine phosphorylation and activa- tion of phospholipase Cy [176]. In monocytes /31 integrin ligation induces tyrosine phosphorylation and consequent activation of immediate-early genes [177]. In B cells cross- linking of c~4/31 by either mAb or natural ligands induces the tyrosine phosphorylation of a 110 kDa protein [178]. In these cases there is no evidence for involvement of ppI25FAK; rather other integrin-responsive kinases are likely involved. Recent observations indicate that the inte- grin-responsive tyrosine kinase in monocytic cells is Syk, a cytoplasmic tyrosine kinase previously known to be in- volved in signaling mediated by Fc receptors and B-cell receptor [ 179,180]; Lin et al., submitted.
A number of integrin-triggered signaling processes have been observed in various cells, where the relationship to integrin-mediated tyrosine phosphorylation is currently un- clear. Integrin clustering or integrin-mediated adhesion has been reported to activate the Na+ /H + antiporter [181]; induce Ca 2+ transients [182-184]; activate calcineurin [185]; play a role in leukotriene production [186]; affect Ca 2+ activated proteases and the distribution of P1-3-kinase [187,188]; modulate a K ÷ channel via pertussis toxin sensitive G proteins [189]; activate phospholipase A2
86 c. Rosales et al. / Biochimica et Biophysica Acta 1242 (1995) 77-98
(PLA2) [ 190]; and affect cAMP levels [191]. These reports suggest that different integrins may utilize several distict biochemical pathways to deliver signals to the cell interior; this diversity may, in part, be responsible for the speci- ficity in the responses to various integrins.
The role of calcium in integrin signaling events is particularly interesting. Cell spreading on fibronectin or vitronectin leads to a rise in intracellular calcium that arises from calcium influx through voltage independent channels [184]. This calcium influx is regulated by a 50 kDa transmembrane integrin-associated protein flAP) with channel-like structure previously identified as a protein that binds to the /3 3 integrin subunit [192-194]. Block of calcium influx by anti-IAP antibodies does not affect integrin stimulated changes in intracellular pH, nor does it affect pp125 FAK activation. These observations suggest that calcium transients are a relatively late event in integrin signaling and are either independent of, or downstream from pp125 FAK. This would be consistent with observa- tions on bombesin activation of pp125 FAK which suggest that calcium is not involved [166].
Cytoplasmic proteins that bind to integrins may provide additional signaling pathways not yet fully understood. A 190 kDa protein that binds to the /3 3 integrin in PDGF stimulated cells has been described [195], although its role is unclear. In insulin-stimulated cells the insulin receptor substrate IRS-1 has been reported to bind to /3 3 integrins [196]. These observations suggest a cooperative signaling interaction between growth factor receptors and integrins, as do the observations of Chong, et. al. [139]. In another set of interesting observations, a protein of 60 kDa that has homologies to the calcium binding protein calreticulin has been reported to bind to the KXGFFKR motif found in the cytoplasmic domains of integrin a chains; a similar motif is found in steroid hormone receptors leading to the inter- esting but controversial hypothesis that integrin ligation may affect these nuclear receptors [197].
4.3. Down-s t ream events in integrin signaling
An important issue for integrin signaling is what hap- pens after activation of pp125 yAK or other integrin-respon- sive tyrosine kinases. Identification of downstream events should help in understanding the overall process of inte- grin signal transduction. Recently some insights into downstream events have been obtained by several groups. Our laboratory has shown that integrin mediated cell adhe- sion can activate MAP kinases [148]. When confluent 3T3 fibroblasts are harvested and placed in suspension cuture in the absence of mitogens, the levels of activity of the p42 and p44 forms of MAP kinase are quite low. When the cells are given the opportunity to adhere to a substratum coated with integrin ligands such as fibronectin or RGD peptides, there is a dramatic elevation of MAP kinase activity. Attachment to substrata coated with non-specific adhesive molecules such as poly-D-lysine fails to activate
the kinase, suggesting that this activation is an integrin-de- pendent phenomenon. In rat fibroblasts, a translocation of MAP kinases from cytoplasm to nucleus occurs in parallel with the activation of the enzymes. Interestingly, the drug cytochalasin D, which disrupts actin microfilaments, com- pletely blocks integrin-mediated MAP kinase activation, showing that the cytoskeleton is involved in this process. The initial time course of MAP kinase activation during integrin-mediated adhesion is similar to the kinetics of pp125 FA~: activation. In addition, tyrosine kinase ihhibitors that block pp125 FAK phosphorylation also block MAP kinase activation. This suggests that pp125 FAK activation may be upstream of MAP kinase activation, but the con- nection is by no means firmly established.
MAP kinases play a critical role in mitogenic signal transduction since they are directly implicated in linking cytoplasmic signaling cascades to control of transcription in the nucleus [92,198,199]. An early nuclear event in- duced by growth factors is activation of the c-fos proto- oncogene. Expression of c-fos is regulated by a transcrip- tion complex (p67SRV/p62rCF) that interacts with the so- called serum-response element (SRE) in the c-fos promoter region. It has been shown that phosphorylation of the p62 TcF (Elk-l) component is directly mediated by MAP kinases and that this alters transcriptional activation [200,201]. Thus c-fos expression, an early event in cell cycle traverse, is immediately tied to activation of MAP kinases. This suggests that integrin activation of MAP kinase could play a direct role in the regulation the cell cycle.
Another group has shown that pp125 yAK can interact with Grb2, the SH2/SH3 domain protein that is part of the Ras signal transduction pathway [171]. Further, there is evidence in lymphoid cells that treatment with anti-c~ 2/31 antibodies can increase the GTP loading of Ras [202]. These observations suggest a close parallelism between integrin signaling and RTK signaling. In both cases initial ligand binding events activate a tyrosine kinase, and Grb2 can bind to the autophosphorylated kinase. In the integrin cascade, we know that MAP kinase is activated, but it is far from clear whether the intermediate events involving Ras and Raf are similar to those of the RTK cascade (Fig. 6). There are several alternate possibilities for integrin activation of MAP kinases. As one example, it is clear that integrin-mediated adhesion can increase PKC activity [203]. In yeast, a signaling pathway leading from PKC c~ to MAP kinase has been delineated [204]; this pathway involves a yeast homolog of MEKK, a kinase that activates MEK, and which is often coupled to G-protein signaling cascades [92]. If a similar pathway existed in mammals, it would offer the possibility that integrins could by-pass Ras and Raf in activating MAP kinases. Recent observations have shown that both PKC c~ [205] and G proteins [206] can be found in focal adhesion sites where they might be able to interact with integrins.
In non-transformed fibroblasts, both soluble mitogenic
c. Rosales et al. / Biochimica et Biophysica Acta 1242 (1995) 77-98 87
factors and anchorage to the substratum are required for cell cycle traverse and cell division. Recent evidence shows that anchorage dependence is a G1 event and that cells progressing past G1 no longer require anchorage [207]. One direct effect of anchorage to a substratum is to regulate the levels of cyclin A in NRK fibroblasts [208]. It is tempting to speculate that anchorage dependence of cell growth involves integrin mediated signal transduction. As discussed above, integrin ligation has been shown to trig- ger cytoplasmic tyrosine kinases such as pp125 FAK, as well as activation of MAP kinases known to play a critical role in mitogenic regulation. However, the signals provided by anchorage must involve more than the activation of com- ponents of the RTK/Ras mitogenic pathway; otherwise anchorage signals could be substituted by high levels of soluble mitogens acting through this pathway, and such is not the case. Thus at this point it seems unlikely that the signals provided by substratum anchorage and the signals provided through RTKs are the same, though it is probable that they at least overlap. One obvious possibility is that integrin ligation promotes cytoskeletal assembly, which in turn allows the formation of signal transduction complexes that cannot be formed in non-anchored cells. At this point, however, the possible relationship between integrin signal- ing and anchorage dependence of cell cycle traverse re- mains ill-defined. It will be necessary to work out further details of the integrin signaling cascade and determine how it may impinge on regulation of cyclins and cyclin-depen- dent kinases.
4.4. Signal transduction by CAMs and cadherins
There is relatively little currently known about the signal transduction mechanisms of cell-cell adhesion re- ceptors including CAMs, cadherins, and selectins. Nonetheless, some information is beginning to emerge suggesting that signals arising from these molecules may also be important regulators of cell growth, differentiation, and behavior. As in the case of the integrins, it seems likely that the signaling events initiated by cell-cell adhe- sion receptors may impinge on previously known signal transduction pathways such as those triggered through receptor tyrosine kinases or through serpentine receptors and G proteins.
CAMS
Recently it has become clear that CAMs can couple to signal transduction processes in cells of central nervous system (CNS) origin. Thus, stimulation of the neural cell adhesion molecules NCAM and LI have been shown to infuence second messenger systems, particularly intra- cellular [Ca 2÷ ] levels, but also intracellular pH, and cAMP levels [209-211]. The type and magnitude of the response differed in various types of neuronal cells; in most cases either antibodies to L1 and NCAM, or the purified molecules themselves, were able to stimulate responses
[21l]. The calcium transients observed subsequent to L1 stimulation have been attributed to an influx of calcium through an L-type channel [209],
The mechanisms involved in NCAM mediated calcium signaling in neuronal cells have been worked out in some detail by using rat cerebellar neurons or PC12 pheochro- mocytoma cells as model systems [212]. These studies involved NCAM-transfected mouse fibroblasts, which were used as a substrate to promote neurite outgrowth by neu- ronal cells [213]. Subsequent studies indicated that for both NCAM and N-cadherin, neurite outgrowth required a sig- naling process that involved G-proteins and N-and L-type calcium channels [214]. Thus treatment with pertussis toxin, which blocks G proteins of the Gi subtype, or treatment with a combination of N- and L-type calcium channel blockers, could completely inhibit neurite outgrowth, The signaling process initiated by NCAM or N-cadherin could be substituted for by opening the calcium channels by other means. For example, a calcium channel agonist promoted neurite outgrowth of PC12 cells on control mouse fibroblasts, as well as enhancing neurite outgrowth on NCAM transfected fibroblasts [215]. Recent experi- ments with tyrosine kinase inhibitors have suggested that tyrosine kinases are active in the signaling process leading to neurite extension; one kinase seems to be upstream of the activation of calcium channels [216]. Evidence using neuronal cells from 'knock out' mice suggests that the tyrosine kinase fyn may be involved in signals by NCAM [217], while the kinase src may be involved in signaling by L1 [218]. Finally, a role for arachidonate metabolites in this process has been established; the arachidonate metabo- lites may act as a second messenger in activating the calcium channels [219]. Thus the model that emerges from these studies is that neural CAMs (LI, NCAM) or N- cadherin, can act via tyrosine kinases, a G-protein, and arachiodonate metabolites, to open L-or N-type calcium channels, and trigger an influx of calcium; it is the increase in [Ca 2÷ ] level that ultimately controls the neurite exten- sion process (Fig.Fig. 7 7). (However, see Note Added in Proof.)
Interestingly, other CAMs in addition to NCAM can trigger neurite extension; for example 3T3 cells transfected with the cDNA for the Deleted in Colon Carcinoma CAM [220], can support neurite outgrowth of PC12 cells. Fi- nally, other cell surface proteins including Thyl [221] and the beta subunit of the Na+/K+-ATPase [222] have also been implicated as signaling molecules in neurite out- growth. It is interesting to note that NCAM signaling in neuronal cells seems to incorporate elements of both tyro- sine kinase mediated and heterotrimeric G protein medi- ated signal transduction.
A much more general role for Ig superfamily members in signal transduction is suggested by the structure of certain families of tyrosine kinases and tyrosine phospha- tases. A number of transmembrane tyrosine phosphatases have been identified that have extracellular domains sug-
88 C. Rosales et al. / Biochimica et Biophysica Acta 1242 (1995) 77-98
• N-CAM Ca+2
PC12 Cell }'( / Plamna ~ / L-Type Calcium
Activation of Neurlte Extension
Fig. 7. Various second messengers are involved in the signal transduction pathway that leads to neurite growth, in PC12 cells signals from G proteins and a tyrosine kinase (Fyn?) have been reported to play a role in the activation of neurite outgrowth via an L-type calcium channel. Arachidonic acid metabolites are thought to be responsible for the opening of the channel. The increase in cytoplasmic Ca 2+ concentration is the direct stimulus for neurite extension. Question marks on arrows indicate that the relationship between the various second messengers involved is not clear at the present time. See Note Added in Proof for another version of this model.
gestive of adhesive functions. The prototype is CD45, which plays an important role in T-cell regulation [223]. A number of transmembrane phosphatases are known that have Ig motifs in their extracellular domains [30]; at least one of these phosphatases has been demonstrated to have cell adhesive function [29]. Furthermore, a transmembrane tyrosine kinase (Dtrk) with Ig family external domain motifs has been identified in Drosophila [224]; this protein can promote homophilic, calcium independent, cell-cell adhesion and the adhesion process stimulates kinase activ- ity. CAMs coupled directly to kinase and phosphatase domains would seem ideally conformed to play a vital role in adhesion-dependent signal transduction events, but the nature of that role has yet to be defined.
Cadherins. As described above, the signaling processes involved in neurite extension can be triggered by N- cadherin as well as by CAMs. In addition to those events, other examples have emerged where cadherins seem to play an important role in signal transduction. Evidence for this role came initially from two quite distinct directions; from developmental studies, and from investigations of epithelial carcinogenesis. As discussed in section 2.1, catenins are intracellular proteins that bind to and modu- late the functions of cadherins. Sequencing of beta catenin
led to the finding that it is a close homolog of the Drosophila segment polarity gene armadillo [225], thus suggesting that cadherin-catenin complexes may transduce information between cells during development. This con- cept is reinforced by the observation that fat, a Drosophila tumor suppressor gene encodes a cadherin analog [226]. Studies on the effect of tyrosine kinase oncogenes on epithelial cell morphology and organization led to the concept that the catenein-cadherin complex is one target for such oncogenes [19,227]. Tyrosine phosphorylation of catenins leads to the disruption of the strong cell-cell contacts that are characteristic of normal epithelial cells. This correlates well with previous observations that carci- noma cells with low cadherin expression displayed a strongly invasive phenotype [16], thus suggesting that functional cadherin/catenin complexes are essential for maintenance of normal epithelia.
A key observation relating cadherins/catenins to ep- ithelial growth control came with the identification of the APC gene, which is altered in individuals with adenoma- tous familial polyposis (who are thus disposed to colon cancer), as well as in some sporadic colon tumors. The APC protein product has been shown to bind to catenins, especially beta catenin [228,229], thus potentially modulat- ing cadher in /ca ten in complexes. Alterations in APC/catenin complexes may be involved in growth regu- latory signaling. In any case, the presence of cadherins, catenins, and APC in appropriate functional ratios seems necessary for the maintainence of a normal epithelial phenotype. Finally, suggestions of additional signaling roles for cadherin-like molecules comes with the identification of the ret proto oncogene, which has a cadherin-like extracellular domain coupled to an intracellular tyrosine kinase domain [230].
5. Regulation of gene expression by cell adhesion recep- tors
5.1. Early studies on matrix effects on gene induction
There are a large number of observations in the litera- ture indicating that adhesive interactions with extracellular matrix can affect cell differentiation and gene expression
Table 1 Examples of gene expression regulated by extracellular matrix
Cell type ECM interaction Genes induced Ref.
fibroblast adhesion fibroblast fibronectin melanoma receptor ot v/33 breast epithelium basement membrane monocyte fibronectin
laminin collagen
c-fos, pro o~-1 collagen metalloprotease type IV collagenase /3-casein immediate early genes (c-los, c-jun, IL-1, IL-8, TNF)
[231,232] [233] [2341 [238-240] [241,243]
C. Rosales et al. / Biochimica et Biophysica Acta 1242 (1995) 77-98 89
(Table 1). Attachment of fibroblasts to a substratum has been reported to rapidly increase expression of c-fos mes- sage and later to induce pro-alphal collagen message [231,232]. The cross-linking of the ot5fll integrin with antibody, or by cell adhesion to fibronectin fragments, induces the expression of metalloprotease genes in fibrob- lastic cells [233], while stimulation of the a v/3 3 integrin in melanoma cells induces type IV collagenase [234]. Specific integrin engagement in myoblasts seems essential for myogenic differentiation [235]. While different types of extracellular matrix can selectively stimulate the expres- sion of liver-specific genes in cultured hepatic ceils [236,237]. One of the best examples of the role of matrix attachment in regulation of gene expression occurs in breast epithelial cells, where the ability to express milk proteins in response to hormones is contingent on the presence of an appropriate extracellular matrix [238-240].
5.2. ln tegrin-mediated gene induction in monocy t ic cells
A good example of integrin mediated gene induction occurs in monocytes [241,242]. This system has allowed new insights into the relationships between matrix pro- teins, integrins, protein kinases, transcription factors, and gene regulation [150]. When peripheral blood monocytes are plated onto tissue culture plastic or on to substrata coated with extracellular matrix ligands such as fi- bronectin, collagen, or laminin, there is a rapid and pro- found induction of a number of so-called immediate-early (IE) genes including transcription factors such as c-fos, c-jun, I~cB, and MAD-6/A20, as well as cytokines such as IL-1/3, IL-8 and TNFc~ [241,243]. There is some selec- tivity of gene expression since plating the cells onto different ECM proteins caused preferential expression of some of the IE genes [242]. Cross linking of /31, but not /3 2, integrins with antibodies was sufficient to trigger the induction of multiple IE genes, indicating a critical role for /31 integrins in this response [244]. Many of the genes induced by integrin ligation have NF-KB motifs in their upstream regulatory regions [150]. Preliminary data using electrophoretic mobility shift assays (Haskill, et. al., un- published data) or NF-K B reporter constructs (Rosales and Juliano, unpublished data) has provided direct evidence that the NF-KB transcription factor is activated subsequent to integrin ligation. This sets the stage for exploring the pathway between the initial engagement of integrins and the specific downstream event of NF-KB activation.
Recently. it has become clear that integrin-mediated tyrosine phosphorylation is critically involved in IE gene induction in monocytes [177]. Thus when monocytes are plated on to ECM proteins, or when /31 integrins are cross-linked by antibody, there is a dramatic increase in protein tyrosine phosphorylation prior to IE gene expres- sion. Blockage of tyrosine phosphorylation with the in- hibitors genestein or herbimycin A also abolished IE mes- sage expression, indicating that the two events are causally
Integrln Oluster
r e r l e
v, /,
Nucleus
DNA ~ ~ Transcription
NF-kB IE Gene site
Fig. 8. Activation of immediate early genes by integrin receptors in monocytes. Clustering of integrins at the plasma membrane of monocytic cells results in activation of a tyrosine kinase and eventually of NF-KB, causing the induction of immediate early genes which contain NF-KB responsive elements in their 5'-regulatory sequences. MAPK kinase also seems to be activated upon integrin engagement but its relationship with Syk and NF-kB remains unknown. Multiple arrow heads indicate that several intermediate steps are involved in the signaling pathway. Syk, a cytoplasmic tyrosine kinase; MAPK, mitogen-activated protein ser/threo kinases; NF-K B/I K B, the NF-kB transcription factor and its cytoplasmic inhibitor protein; IE Gene, an immediate early gene, such as c-fos, c-jun, IL- 1 fl, IL-8, TNFa.
linked. Interestingly, pp125 FAK does not play a role in this process, since this kinase is absent from monocytic cells [177,245]. Preliminary evidence suggests that the integrin- responsive tyrosine kinase in monocytic cells may be Syk (Lin et al., submitted). Ligation of /31 integrins in mono- cytic cells also causes activation of the p42 form of MAP kinase (Lin and Juliano, unpublished data), but it is not yet clear whether this has anything to do with integrin-media- ted IE gene induction (Fig.Fig. 8 8). Thus some of the connections between integrin ligation and gene induction are beginning to be worked out, but much remains to be done.
The discussion above emphasizes the widespread in- volvement of integrins in regulation of gene expression and cell differentiation. However, a recent study of the role of /31 integrins in the differentiation of F9 embryonal carcinoma cells places a cautionary note on this interpreta- tion [246]. Sequential homologous recombination was used to knock out all three copies of the fl 1 integrin gene in polyploid F9 cells. F9 cells can be induced to differentiate to either parietal or visceral endoderm, processes that lead to changes in both cell morphology and the expression of specific genes. Ablation of /31 integrins markedly altered the process of morphological change, but tissue specific gene expression was not affected. Thus in this system, key gene induction responses are not coupled to integrin medi- ated processes. It may be that certain sets of gene induc- tion events require signals mediated by cell adhesion re- ceptors such as integrins, while other induction events need only signals provided by soluble growth or differenti-
90 C. Rosales et al. / Biochimica et Biophysica Acta 1242 (1995) 77-98
ation factors. The role of integrins in regulating gene expression in differentiated cells, possibly in collaboration with soluble growth and differentiation factors, should prove to be an interesting and productive area of research.
5.3. Gene induction mediated by CAMs and selectins
In contrast to the situation with integrins, there is a relative paucity of information on gene induction mediated by cell-cell adhesion molecules such as cadherins, se- lectins, and Ig family CAMs. However, this is clearly an area of great interest, and some work is beginning to emerge. One report examined the role of NCAM in regu- lating metalloproteinase expression in glial cells [247]. Tranfection of a rat glioma cell line with a human NCAM isoform that contains a transmembrane domain resulted in down-regulation of 92kDa gelatinase and interstitial colla- genase expression. By contrast transfection of an NCAM isoform having a glycosyl-phosphatidyl inositol anchor, but lacking a transmembrane domain, failed to affect met- alloproteinase levels. This indicates that the transmem- brane domain of NCAM plays a role in modulating gene expression in these cells, and in particular, regulates pro- teases that can degrade the extracellular matrix. In another study, CEA was transfected into rat myoblasts [248]. These cells can ordinarily be induced to undertake a program of myogenic differentiation, including induction of the tran- scriptional regulator myogenin, and expression of muscle actin and myosin. Ectopic expression of CEA disrupted the normal program of myogenic differentiation, and main- tained the cells in a proliferative state. Transfection with CEA mutants deficient in cell adhesion failed to block myogenic differentiation, indicating that homotypic CAM interactions are necessary for the effect.
A report concerning a role for selectins in gene expres- sion has appeared recently [249]. Sulfatides have been established as ligands for L-selectin. When neutrophils were treated with sulfatides, a transient increase in cytoso- lic calcium was observed; this was followed by increased expression of messages for TNFa and IL-8. Antibodies to L-selectin also were able to induce calcium transients but effects on gene expression were not examined. Thus initial evidence suggests that signals transmitted via selectins (possibly calcium transients) can affect gene regulation in neutrophils. Based on the studies cited here, it seems clear that signals transmitted by Ig family CAMs or by selectins can have a major impact on regulation of gene expression in some types of cells.
6. Signaling by cell adhesion receptors: a role in cancer
Tumor formation is a result of attenuation or the cir- cumvention of mechanisms that control both the prolifera- tion of cells and their spatial positioning within the body.
[250,251]. While a large body of work has sought to evaluate the role of adhesion receptors in the invasive and metastatic behavior of tumor cells [8,252], relatively little attention has been given to the role of these molecules in regulating tumor cell proliferation. Much of the focus instead has centered on studying signaling pathways of soluble growth promoters and growth inhibitors. This has proved to be a fruitful area as many proto-oncogenes and tumor suppressors have been shown to function in signal- ing pathways [253]. A consensus view of cancer is that mutational activation of dominant oncogenes or inactiva- tion of tumor suppressors leads to irreversible activation of proliferative pathways leading to uncontrolled growth. However, this is an incomplete view of cancer since normal cells, in addition to soluble factors, also require attachment to a substratum for proliferation (anchorage-de- pendent growth). Tumor cells lose this requirement for anchorage, are less adhesive and often display a less well organized cytoskeleton than their normal counterparts [9,251,254]. Overexpression of adhesion receptors or focal contact proteins involved in ECM-cell adhesion restores cytoskeletal organization and reduces tumorigenicity [255- 257]. Also, cell adhesion receptors have been identified as tumor suppressor genes, most notably in colon carcinomas [258]. These insights, coupled with the emergence of cell adhesion receptors as signaling receptors [150,259] has prompted fresh interest in the role of cell adhesion receptor molecules in regulating growth of normal and malignant cells.
6.1. lntegrins
Changes in integrin subunit expression in transformed cells in vitro and tumor cells in vivo suggests that integrins may be important in tumor progression. A summary of these studies is provided in a recent review from our laboratory [254].
The main conclusions from this large body of work are that:
1. In carcinomas (which are the major human neo- plasms) integrin expression is disperse and discontinuous, in contrast to normal epithelial cells where these molecules are usually displayed in a polarized fashion, confined to the basal and basolateral membranes where they interact with the underlying basement membrane. Such disorgani- zation of integrin receptors correlates with disorganization of the basement membrane itself -a hallmark of most carcinomas. However, it is not clear if changes in integrin expression are the cause of the observed basement mem- brane disruption or if this disruption prevents the accumu- lation of integrins within the plane of the basolateral membrane.
2. Quantitative changes in integrin expression levels occur which may be specific to particular tumor types.
3. Increased expression of a v/3 3 on melanomas corre- lates with an increased metastatic potential.
c. Rosales et aL / Biochimica et Biophysica Acta 1242 (1995) 77-98 91
4. There is a loss, or significant reduction in the expres- sion of the a5/31 fibronectin receptor in both transformed and tumor cells.
These studies must be interpreted with caution and their relevance to tumor biology can be questioned for at least two reasons, one is that tumor cell lines do not necessarily accurately represent tumor cells that grow in vivo, and two, expression levels on tumor cells may not correlate with functionality since a particular adhesion receptor may be overexpressed but be non-functional, perhaps requiring activation [58].
An effective way to understand the role of integrins in tumor behavior is to alter the expression of selected inte- grins in tumor cell lines and evaluate effects of altered expression on tumor cell proliferation and invasive and metastatic capabilities. Chinese hamster ovary (CHO) cells selected for reduced expression of the a5/31 integrin show enhanced tumorigenicity in nude mice upon subcutaneous injection [260]. Overexpression of this integrin in CHO cells resulted in reduced proliferation and increased an- chorage dependence for growth in vitro and reduced or abolished tumor formation in nude mice [261]. Our labora- tory has shown that a human colonic carcinoma cell line (HT29) transfected with a cDNA for the c~5 integrin subunit shows reduced tumorigenicty in nude mice com- pared to a mock transfectant [262]. Overall, the effect of experimental manipulation of the levels of cell-surface expression of c~5/31 in malignant cells on their growth in vitro and tumorigenicity in vivo correlates well with obser- vations describing the levels of surface expression of this integrin in transformed cells and tumors.
The concept that c~5/3 1 integrin expression has a nega- tive regulatory effect on tumor growth seems to conflict with other observations indicating that fibronectin or pep- tides containing the RGD sequence have a stimulatory effect on cell growth. In melanoma cell lines, the 120 kDa cell-binding (RGD-containing) fragment is a potent mito- gen, but only for cells expressing c~5/31 integrin [263]. In subclones of an erythroleukemia cell line expressing differ- ent levels of ce5/31, RGD peptides were shown to be mitogenic, to promote anchorage-independent growth and to stimulate cyclin A/cdc2 kinase activity and hyperphos- phorylation of the retinoblastoma protein [264]. These conflicting data can be reconciled if c~5/3 1 integrin pro- vides a signal that results in inhibition of cell growth, be it promotion of growth arrest or programmed cell death (PCD), under conditions when the receptor is unliganded, and that the negative signal is abolished and/or a positive signal generated when the integrin binds its ligand [254].
In contrast to a5/3 1, loss of cell-surface expression of c~v/33 integrin leads to reduced tumorigenicity in nude mice, while restoration of this integrin by transfection of the c~ v subunit cDNA restores tumorigenicity [265]. These observations are consistent with previous studies which linked an increased malignancy of melanoma cells to enhanced c~v/33 expression [266,267]. Also, the c~vb6
integrin, a fibronectin receptor that recognizes the RGD motif, has been shown to enhance the proliferative capac- ity of SW480 colon carcinoma cells both in vitro in 3-dimensional collagen gels and in vivo in nude mice. This activity could be attributed to the presence of an 11 amino acid region at the cytoplamic carboxyl terminus of the /36 chain [268].
How do integrin-induced signals account for the ob- served alterations in tumor cell behavior? As discussed above, integrin signals appear to influence cellular prolifer- ation and differentiation as well as cell survival and so may directly or indirectly (via promoting growth arrest, terminal differentiation and/or programmed cell death) influence the growth of a tumor in vivo. A new role for integrin receptors in the regulation of PCD in both normal cells and tumor cell lines has recently emerged. Normal epithelial or endothelial cells maintained in suspension undergo apoptosis [269,270], a form of PCD defined by characteristic morphological and biochemical features [271]. Cell attachment via binding to dishes coated with fibronectin or anti-/31 integrin antibodies, but not other cell-surface molecules, can suppress apoptosis in 2-dimen- sional cultures [269]. Other workers have shown that, for normal endothelial cells, occupancy of integrin receptors by their ligand is not sufficient to suppress apoptosis, instead cell spreading must occur to ensure survival [272]. A recent report has demonstrated that mammary epithelial cells will undergo apoptosis if their attachment to an exogenous ECM is disrupted by anti-/31 integrin antibod- ies or if their ECM is degraded as a result of overexpres- sion of stromelysin- 1, a proteinase. Interestingly, cell adhe- sion to type- 1 collagen, fibronectin or tissue culture-treated plastic was not sufficient to block apoptosis, even though the cells adhered strongly and were well spread on the substratum [273].
Integrins also appear to regulate apoptosis in tumor cells. A recent report demonstrated that apoptosis could be triggered by inhibition of intercellular contact [274]. These workers used a colon carcinoma cell line, LIM 1863, which will grow in vitro as a spheroid (or 'organoid') comprised of differentiated cells (goblet and columnar cells) around a central lumen. When transferred to a medium depleted of calcium ions the organoid disaggre- gated to a single cell suspension. Reformation of the organoid took place when the single cells were placed in medium containing calcium ions, and this reformation could be blocked using an antibody to the integrin a v subunit. Cells blocked from forming organoid structures underwent apoptosis. LIM 1863 cells express ce v in asso- ciation with both /35 and /36 but not /33, but the identity of the integrin(s) mediating the apoptotic effect remains to be determined [274].
Two reports, from the same group, implicate a v/33 integrin as a direct regulator of tumor cell apoptosis in vitro and an indirect regulator of tumor survival in vivo. In the first study, human melanoma cells, lacking av/33,
92 C. Rosales et al. / Biochimica et Biophysica Acta 1242 (1995) 77- 98
undergo apoptosis in 3-dimensional collagen gels in the absence of growth factors but are protected from apoptosis by heterologous expression of t~ v/3 3 [275]. The second report demonstrated that integrin antagonists, such as cyclic RGD peptide or inhibitory antibody, can inhibit tumor-in- duced angiogenesis of developing chick chorioallantoic membrane by promoting selective apoptosis of endothelial cells which comprise the angiogenic blood vessels. This in turn leads to a rapid regression of xenografted human M21-L melanoma tumor fragments [276].
Apoptosis occurs frequently in tumors and it has been claimed that this mechanism of cell elimination is respon- sible for the continuous cell loss observed in most tumors [277]. Abnormalities in the triggering of apoptosis may promote cancer development by allowing for the accumu- lation of dividing cells and by obstructing the elimination of cells with genetic lesions having a potential to promote malignancy. With the demonstration that integrins are important in triggering apoptosis in anchorage-dependent cells, it can be argued that the loss of certain integrins from tumor cells, in particular t~5/3 l, may promote tumor development by eliminating a major trigger for PCD, i.e. the presence of unliganded integrin on cells detached from the ECM (Fig.Fig. 9 9).
Tyrosine phosphorylation of cellular proteins appears to be an important signaling mechanism in PCD triggered via integrins, since transformation of MDCK cells with v-src conferred partial resistance to apoptosis [270], while treat- ment of endothelial cells with orthovanadate, an inhibitor of protein tyrosine phosphatases, can promote cell survival in the absence of ECM attachments [269]. These observa- tions suggest one mechanism by which oncogenes encod- ing tyrosine kinases may promote anchorage-independent growth and contribute to tumorigenicity. As a counter- point, mutations which inactivate a putative key protein tyrosine phosphatase which is regulated by cell adhesion
[175], may also confer resistance to apoptosis and therefore promote tumor growth.
6.2. Cadherins
Decreased expression or functional inactivation of cad- herins in carcinomas is associated with a more malignant phenotype, while restoration of cadherin expression by gene transfer can render transformed cells less invasive (reviewed in [278,279]). Thus cadherins may function as tumor suppressors, indeed the Drosophila tumor suppres- sor gene, fat, encodes a cadherin-like cell adhesion molecule [226]. As mentioned in section 4.4, two reports demonstrate that tyrosine phosphorylation regulates cad- herin/catenin interaction within epithelial cells: transfor- mation of epithelial cells with a src oncogene leads to disruption of cadherin-mediated cell-cell adhesion, loss of differentiation and adoption of an invasive phenotype [19,227]. Cadherin levels are not altered in these cells, however both catenins and cadherins are phosphorylated on tyrosine residues and are apparently targets for the oncogene.
Evidence for the involvement of cadherins in regulating cell proliferation has come from a study identifying an interaction between catenins and the protein product of the tumor suppressor gene APC (see section 4.4). Mutation of the APC gene is one of the earliest, if not the initiating event, in colon cancer [280] and individuals inheriting a mutated APC gene are predisposed to colonic neoplasms and develop thousands of hyperplasic polyps some of which progress to cancer. Furthermore, the mouse onco- protein, wnt-1 has been shown to influence cell prolifera- tion and regulate cell-cell adhesion apparently by stabiliz- ing cadherin//3-catenin complexes [281]. Thus evidence is emerging which connects oncoproteins and tumor suppres- sors to cadherin/catenin complexes and suggests that such
A. Normal or Trmslormed Cells Atlmched to ECM
ECM
Supress~on of PCD
B. Normal Cells Detached from ECM C. Tram~nwd Cells Oetached from ECM
Er~o~lenous r-,Integnn Ectoo0c Expression ol
~ ~-~------. .~ ~.~ Integrin
/ ~ ' ~ LOSS of Tyro~ne / ~ "Tumor
Fig. 9. Integrin signaling and the control of programmed cell death (PCD) in normal and transformed cells. (A) In normal or transformed cells binding of integrins to extracellular matrix (ECM) ligands results in the generation of an intracellular signal that contributes to the suppression of programmed cell death (PCD). (B) When normal cells are detached from the ECM, un-liganded integrin(s) deliver an intracellular signal that promotes PCD. (C) In transformed cells the loss of certain endogenous integrins may lead to tumor development by ablating the normal signals that trigger PCD, while the presence of an ectopically expressed and un-liganded integrin (e.g. ~5/31) may restore the signal that triggers PCD. The presence of oncogenic tyrosine kinases or the loss of tumor suppressor proteins may contribute to tumorigenesis by inhibiting PCD (as indicated by an arrow labeled with a negative sign). In each case a role for p125 FAK is implied, although no evidence is available, at present, to suggest that this kinase is involved in signaling pathways that regulate PCD.
C. Rosales et aL / Biochimica et Biophysica Acta 1242 (1995) 77-98 93
complexes may regulate cell proliferation. Another possi- bility is that cadherins may be involved in regulating programmed cell death [282] or may regulate transmission of the signal that instructs cells to stop dividing once they have formed an epithelial sheet. Aberrations in the in- tegrity and organization of cadherin complexes, e.g. loss of APC function, may abolish or diminish cadherin function and result in hyperplasia as cells escape control signals that serve to regulate their growth and spatial positioning within the epithelium.
6.3, CD44
Although proteoglycans were only briefly mentioned in section 2 of the review, the critical role of one such molecule in cancer merits some discussion. Receptors for the glycosaminoglycan hyaluronic acid (HA) are involved in homotypic and heterotypic cell adhesion. The HA recep- tor CD44, exists in a variety of forms within the same cell, as a result of alternative splicing and other post-transla- tional modifications [283]. This molecule has been shown to play a positive role in cell transformation and metasta- sis. Oncogenic transformation with src or ras induced overexpression of CD44 in rat intestinal epithelial cells [284], while non-metastatic tumor cells adopt an invasive phenotype after gene transfections with cDNA for one of the CD44 variants [285]. Overexpression of various forms of CD44, in particular those expressing the polypeptide sequence encoded by the v6 exon, appear to be particularly prevalent in both colon and breast carcinoma [285].
Evidence has emerged that CD44 proteins can interact with the actin cytoskeleton, apparently mediated through association with members of the ERM (ezrin, radixin, moesin) family of adaptor proteins [286]. Also, an ERM- like protein has been shown to be the product of the tumor suppressor encoded by the neurofibromatosis type 2 gene [287]. This represents a somewhat analogous situation to cadherins/catenins and the APC gene product, and impli- cates the CD44/ERM complexes in the regulation of cell growth.
6.4. lmmunoglobul in superfamily
The tumor suppressor gene DCC, first identified by its loss in colon tumors, encodes a protein with significant homology to members of the immunoglobulin superfamily. Like other NCAM homologues it can stimulate neurite outgrowth in PCI2 cells when ectopically expressed in mouse fibroblasts. DCC expression is lost in late adenomas i.e. at a pre-invasive stage of colon cancer suggesting that it may function to limit epithelial cell proliferation. DCC expression can also influence the response of PC12 cells to differentiate and growth-arrest when treated with nerve growth factor (NGF); there was inhibition of NGF-media- ted morphological differentiation upon transfection of PC 12 cells with an antisense DCC construct and a reversion of
the neuronal phenotype by addition of antisense oligo- nucleotides to the culture medium [288]. Taken together with the demonstration that DCC promotes neural cell differentiation, it has been proposed that DCC induces differentiation and controls cell proliferation [258].
In many colonic epithelial malignancies carcinoembry- onic antigen CEA is overexpressed 10-100 fold, and the cellular distribution of the molecule is altered which may result in the disruption of cell-cell adhesion with subse- quent loss of tissue architecture [289]. As mentioned in section 5.3 it has recently emerged that CEA may influ- ence the differentiation and, therefore, the proliferative capacity of cells. Upon gene transfection, it has been shown to inhibit differentiation of myoblasts [248]. Ectopic expression of CEA can therefore disrupt the orchestrated program of gene expression required for differentiation and therefore must exert a profound effect on cellular signaling pathways involved in controlling differentiation. The intercellular adhesion function of CEA is critical for this effect; mutants unable to promote homotypic interac- tions also failed to block myogenesis [248]. Perhaps CEA functions in a similar manner in colon cancer, locking colon epithelial cells at a proliferative stage in their differ- entiation or actively promoting proliferation while sup- pressing differentiation. In comparing CEA and DCC it is notable that these two Ig superfamily members have seem- ingly opposed roles in the control of cell growth and differentiation. Whether this is due to these two molecules being coupled to different signaling pathways remains to be determined.
7. Summary
Over the last few years, it has become clear that cell adhesion receptors function in signal transduction pro- cesses leading to the regulation of cell growth and differ- entiation. Signal transduction by both integrins and CAMs has been shown to involve activation of tyrosine kinases, while CAM signaling in neural cells involves G proteins as well. In the case of integrins, some of the downstream signaling events intersect with the Ras pathway, particu- larly the activation of MAP kinases. In fibroblasts, integrin mediated anchorage to the substratum regulates cell cycle traverse, while in epithelial cells, loss of anchorage can trigger programmed cell death. In many cell types, but particularly monocytic cells, integrin ligation has a pro- found impact on gene expression. Preliminary evidence also implicates CAMs and selectins in gene regulation. A consistent theme in signal transduction mediated by adhe- sion receptors concerns the role of the cytoskeleton. Inte- grin mediated signaling processes are interrupted by cyto- skeletal disassembly. Identification of the APC and neu- rofibromatosis type 2 tumor suppressors suggest that cyto- skeletal complexes also play a key role in signaling by cadherins and CD44, respectively. Thus, signaling by cell
94 C. Rosales et al. / Biochimica et Biophysica Acta 1242 (1995) 77-98
adhesion receptors may involve aspects that impinge on previously known signaling pathways including the RTK/Ras pathway and serpentine receptor/G protein pathways. However, novel aspects of signal transduction involving cytoskeletal assemblies may also be critical.
8. Note Added in Proof
While this review was in press, a very exciting model has emerged that may better explain how adhesion molecules may initiate signaling for neurite growth. The three CAMs that have been associated with neurite exten- sion, N-CAM, LI, and N-Cadherin, each contain a His- Ala-Val motif within a 20 amino acid domain that has been named the 'CAM homology domain'. This motif was also found in the extraceUular domain of the fibroblast growth factor receptor (FGF-R), suggesting a possible direct interaction between FGF-R and adhesion receptors. Antibodies directed to the FGF-R CAM homology domain were able to inhibit the stimulation of neurite growth caused by N-CAM, L 1, or N-Cadherin [293]. Moreover, a soluble L1-Fc chimera, was able to stimulate neurite out- growth, even though it did not act as an adhesive support for neurons [294]. This suggests that adhesion receptors may interact directly with RTKs to initiate signaling cas- cades. It also suggests that the tyrosine kinase most imme- diately relevant to neurite extension may be FGF-R rather than a src family kinase such as fyn; however, the exact role of src-related kinases remains to be defined.
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