2.06 Cell Surface Receptors

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1 2.06 Cell Surface Receptors N opovic and E Wilson, Texas A&M Health Science Center, College of Medicine, College Station, TX, USA ª 2010 Elsevier Ltd. All rights reserved Introduction General Characteristics of Cell Surface Receptors G-rotein Coupled Receptors Overview and General Characteristics athologies Associated with G-rotein Coupled Signaling Enzyme-Linked Receptors Receptor Tyrosine Kinases Transforming Growth Factor- Receptors Ligand-Gated or Receptor-Operated Ion Channels Adhesion Molecules: Mediators of Cell Matrix and Cell Cell Interactions Integrins Cadherins Selectins and Ig CAM Adhesion Molecules Conclusions 90 References 90 Abbreviations BARK -adrenergic ~ receptor kinase BM morphogenetic protein CAM cell adhesion molecule ECM extracellular matrix protein EGF epidermal growth factor FAK focal adhesion kinase FGF fibroblast growth factor GABA -aminobutyric acid A GA GTase activating protein GD guanine nucleotide diphosphate GEF guanine nucleotide exchange factor GCR G-protein coupled receptor GRK G-protein receptor kinase GT guanine nucleotide triphosphate IGF insulin-like growth factor MAK mitogen-activated protein kinase NGF nerve growth factor DGF platelet-derived growth factor I 3-kinase phosphatidyl inositol 3-kinase LC phospholipase RTK receptor tyrosine kinase SH2 src-homology domain 2 SOS son-of-sevenless TGF transforming growth factor VEGF vascular endothelial growth factor Introduction Cells are able to respond to changes in the environment by the interaction of external stimuli with cellassociated receptors which in turn activate signal transduction pathways that serve to regulate the cellular response to the change in environmental conditions. While cells do contain some intracellular receptors (e.g., steroid hormone receptors), the vast majority of the cellular stimuli cannot cross the plasma membrane. This limitation necessitates that, for the cell to interact and respond to changes in external stimuli, there are receptors that are integral plasma membrane proteins and are thus able to interact with stimuli in the extracellular milieu and then initiate intracellular signaling events that regulate changes in cell behavior. This chapter will focus on the membrane spanning receptors and these receptors will be referred to as cell surface receptors for the general class of receptors. Figure 1 illustrates the general relationship between external stimuli, cell surface receptors, intracellular signaling cascades, and altered cellular responses. The external stimuli that initiate these 81

2 82 Receptor Systems External stimuli Cell surface receptors receptor systems is that they all amplify the signal from one receptor ligand interaction to a more longterm signaling event. In this chapter we will focus on four classes of cell surface receptors: the G-protein couple receptors, the tyrosine kinase receptors, other enzyme-linked receptors, and adhesion molecules (Alberts et al. 2007). Contraction / Relaxation Signal transduction Effector Messenger molecules Cell behavior Secretion Growth / Differentiation Metabolism Figure 1 Schematic representing central role of the cell surface receptors in recognizing external stimuli and activation of cellular transduction pathways leading to changes in cellular behavior. Illustrated are external stimuli, cell surface receptors, signal transductions, and their relationship to changes in cellular activities. process are of many and varied types of molecules including hormones, cytokines, neurotransmitters, growth factors, and extracellular matrix molecules and fragments. Interaction of these varied external stimuli leads to both short-term cellular changes including shape changes, secretion, contraction/ relaxation, changes in metabolism, and migration to name a few events, as well as long-term adaptations including changes in gene expression, proliferation, differentiation, and even cell death or apoptosis. The types of molecules that initiate these responses and interact with the cell surface receptors range from ions, small compounds, peptides, and proteins to physical forces such as vibration, pressure, flow, and light. The varied nature of the stimuli that a cell needs to respond to requires a large number and varied types of cell surface receptors that can bind these stimuli and initiate a wide range of signaling pathways that can lead to specific downstream responses. One commonality among the various General Characteristics of Cell Surface Receptors The concept of receptors was first introduced in the context of the mechanisms of action of drugs, and the term was used long before the molecular nature of the various receptors was known. The primary function of physiological receptors is to bind the appropriate ligand on the external surface of the cell and to propagate the regulatory signal in the target cell. Thus, all receptors are considered to have a ligand-binding domain and an effector domain. The receptors then serve to integrate the signals from the external stimuli to coordinate cellular responses. These properties make the receptor systems excellent targets for drugs and also the sites of action for many chemical toxicants. Three key considerations are often used to classify the nature of the interaction of a stimulus (e.g., drugs, toxicants, and hormone growth factors) with its receptor. First, receptors determine the quantitative relationship between the amount (dose) of the stimulus and the physiologic effect. This relationship is determined by the affinity of the stimulus for the receptor and the total number of receptors present on the cell surface. Second, receptors are responsible for the selectivity of the interaction of a stimulus with the cells service. Selectivity is determined by the molecular nature of the ligand receptor interaction. Finally, receptor interaction and structure lead to the ability to have both agonists (activators) and antagonists (inhibitors), which contributes to their importance in the fields of pharmacology, drug development, and toxicology. The use of radiolabeled ligands allowed for the characterization of receptors before the molecular characteristics of the proteins were known. The use of such experimental systems showed that ligand receptor interactions could be treated like association interactions between two molecules and that an affinity could be determined. These types of studies also showed that there were a limiting number of receptors for the ligand and that there were saturation

3 Cell Surface Receptors 83 kinetics. The concept of multiple receptors for the same ligand was determined by the shape of the binding curves. These processes have been discussed in detail in other texts including Bourne and von Zastrow (2007) and Brunton et al. (2007). We will focus on the description of the primary types of receptors and their role in human pathologies and as targets for toxicants G-rotein Coupled Receptors Overview and General Characteristics The largest family of cell surface receptors is that of the guanine nucleotide binding protein or G-protein coupled receptors (GCR), which are also known as seven-transmembrane receptors because of this common structural feature. Approximately 800 human genes code for receptors in this family, which interact with ligands as diverse as ions, hormones, neurotransmitters, and other sensory stimuli. This family of receptors is the target for approximately 30% of the drugs on the market (Hopkins and Groom 2002). These facts indicate the importance of this class of receptors in both normal physiology and in human disease of the G-protein coupled receptors and as potential targets for toxicants. This family shares common structural features including the characteristic seven-membrane traversing segments, external loops of the membranespanning region and the N-terminal region that form the ligand-binding domain, and the cytosolic loops and C-terminal tail that form regions that are involved in interaction with the guanine nucleotide-binding proteins (G proteins) and regulation by other intercellular proteins (Figure 2) (ierce et al. 2002). This common structure allows for the approximately 800 different receptors to interact with the wide array of extracellular ligands that they bind with both specificity and selectivity and to initiate a number of diverse signaling pathways by interacting with different G proteins that regulate specific signaling pathways. Guanine nucleotide binding proteins or G proteins are heterotrimeric proteins that are composed of,, and subunits. The subunit contains the enzymatic activity and plays the primary role in conveying the signal from the receptor to the effector molecules. The subunits serve to localize the G-protein heterotrimer to the membrane through posttranslational modifications such as myristoylation (Neer 1995), and can also serve as direct activators of signaling events in some cases (e.g., potassium channels) (Ford et al. 1998). The most basic signal transduction pathway of the GCR family is that activation of the receptor by ligand binding results in a conformational change in the receptor. The resultant change in conformation allows for the exchange of guanine nucleotide diphosphate (GD) from the -subunit of the G protein for guanine nucleotide triphosphate (GT). This key step in the process initiates the activation of the -subunit and its release from the subunits. As stated above, both the and subunits are capable of activating effector proteins. Among the G-protein regulated effector systems are adenyl cyclase, phospholipase C-, and numerous ion channels to name just a few. Thus, the binding of the external stimuli results in the amplification of the initial signaling event into the production of many second messenger molecules leading to the change in cellular behavior. This series of events is illustrated in Figure 2 (Bourne 1997). There are multiple genes encoding each of the G-protein subunits, which contribute to the diversity of the overall signaling system. Currently, there are at least 16 known subunits, 5 distinct subunits, and 11 subunits in mammals (Luttrell 2006). Table 1 shows some of the more common G proteins, some of the interacting receptors, and the associated signaling molecules. The expression patterns of the various subunit types vary widely. Some of the subunits, such as G s, are expressed ubiquitously and couple multiple receptors to the stimulation of the same effector systems (e.g., adenyl cyclase and cyclic adenyl monophosphate (cam)). Other G subunits, such as transducin, which couples the GCR rhodopsin to cyclic GM phosphodiesterase are restricted only to specific cell types, in this case the retinal epithelial cells. Thus, mutations in specific G proteins or their receptors may have more widespread physiologic effects or may be more restricted depending on the expression pattern of the various components of the system (Luttrell 2006). The key points in determining how long the signaling pathway will remain active upon stimulation include how long the stimulus is present, the activation status of the receptor (to be discussed in more detail later), the length of time that the G protein is in the GT bound or active form, thus maintaining the activated status of the effector system, and the relative halflife or the second messenger molecules. A number of other regulatory molecules modulate these various points. For example, guanine nucleotide exchange

4 84 Receptor Systems Hormone 1 G α G β G γ Effector 4 GD Hormone 2 Effector Effector G α G β G γ G α G β G γ GT GD Hormone 3 GD GT Effector G β G α G γ 5 GT Receptor kinase Second messenger Arrestin Figure 2 Schematic of G-protein coupled receptor activation. (1) Inactive GCR-G-protein-effector complex poised to interact with agonist. (2) Agonist binding to receptor induces conformational change in receptor and initiates exchange of GD for GT. (3) G subunit dissociates from subunits and interacts with and activates the effector system leading to increased second messenger production. (4) GT hydrolysis leads to inactivation of signaling process and reassociation of the subunits. (5) Activation of receptor kinases (GRKs) leads to phosphorylation of the receptor and arrestin binding. A process that results in desensitization of receptor and possible targeting for lysosomal degradation. factors (GEFs) regulate the exchange of GD for GT upon activation. In the GCR pathway, the activated receptor is one of the most prominent GEF. GTase activating proteins (GAs) serve to speed up the catalytic rate of GT hydrolysis to GD (Siderovski and Willard 2005). Misregulation of any of these sites can cause pathologies (Brunton et al. 2007). Constitutive activation of certain GCRs due to subtle mutations in receptor structure has been shown to give rise to disease such as retinitis pigmentosa, precocious puberty, and malignant hyperthyroidism (Spiegel and Weinstein 2004). It was observed that many agonists became ineffective after prolonged exposure of the cells or tissues to them. This process is termed refractoriness, tachyphylaxis, or desensitization. This mechanism of action has been of considerable interest because of the profound limitation on the efficacy of some drugs and on the duration of action of certain hormones or stimuli. This phenomenon has been observed with multiple signal transduction systems, but has been most studied with the adrenergic system (Hoffman and Taylor 2001). Multiple points of regulation of receptor responsiveness have been identified including the receptors, G proteins, adenyl cyclase, and the phosphodiesterase that hydrolyzes cam. How long the receptor is desensitized and the type of desensitization is determined by which of the components is modified. Heterologous

5 Cell Surface Receptors 85 Dimerization LCγ LCγ TK TK TK TK TK l3k TK GRB2 SOS GA Src Ras Figure 3 Schematic of receptor tyrosine kinase (RTK) activation and signaling. Signaling through RTKs is initiated by binding of ligand, which leads to dimerization of the receptors. Dimerizations result in autophosphorylations of the receptors. The phosphorylated receptor then serves as a scaffold that organizes a complex signaling machine. A key pathway that is activated by RTKs is the ras-mediated activation of mitogen-activated protein kinase (MAK) leading to increased transcription of immediate early genes. Table 1 Examples of prominent G-protein coupled receptors G-rotein type Examples of receptors Major second messenger Gs -adrenergic receptor Increased cam Endothelin Receptor II Glucagon Gi 2-adrenergic Decreased cam 5-hydroxytryptamine (1A)(5-HT(1A)) Acetylcholine (muscarinic) Gq Endothelin receptor I Diacylglycerol and I3 Histamine (H1) receptor Leading to increased intracellular calcium Angiotensin receptor I 5-HT(1C) Go Unknown neurotransmitters in brain unknown Transducin Rhodopsin cgm hosphodiesterase Decreased cgm desensitization occurs when activation of one receptor pathway causes diminished responsiveness of a number of receptor pathways that use the same downstream signaling components. For example, stimulation of the -adrenergic pathway can result in desensitization of other receptor systems that utilize cam as a second messenger system (Hoffman and Taylor 2001). Homologous desensitization occurs when the activation of the same signaling systems causes desensitization of the same receptor-activated system. One of the best-studied examples of homologous desensitization is agonist-stimulated phosphorylation of the ~ -adrenergic receptor resulting in decreased sensitivity to further agonist stimulation. -Adrenergic receptor kinase (BARK) was shown to phosphorylate the receptor only when the receptor was occupied by the agonist. Subsequently, other receptor kinase family members were identified that phosphorylate a wide

6 86 Receptor Systems Table 2 Representative integrin heterodimers and their major binding partners Heterodimer Binding proteins Recognition sequence 1 1 Laminin, collagen 2 2 Collagen DGEA 3 1 Fibronectin, laminin, collagen RGD 4 1 Fibronectin, VCAM, osteopontin RGD, EILDV 5 1 Fibronectin RGD 6 1 Laminin, merosin 7 1 Laminin v 1 Fibronectin, vitronectin RGD v 3 Vitronectin, fibronectin, von Willebrand factor, RGD fibrinogen, denatured collagen, osteopontin v 5 RGD IIb 3 Fibrinogen, fibronectin, von Willebrand factor RGD array of G-protein coupled receptors. The general family of kinases is referred to as G-protein receptor kinases (GRKs). Because these kinases only phosphorylate the agonist-occupied receptors, they provide a mechanism for achieving homologous agonist-specific desensitization. For complete desensitization of the receptor signaling pathway binding of an arrestin protein is required. The phosphorylated residues on the cytoplasmic tail of the receptor form the binding site for arrestin, which in turn serves to attenuate signaling. The arrestin-bound receptor may then be targeted for further processing and internalization, which can lead to either longer-term downregulation and/or activation of other signaling pathways (DeWire et al. 2007). Better understanding of these processes will be important for understanding the mechanism of action for drugs and for potential targets of toxicants athologies Associated with G-rotein Coupled Signaling Mutations in the receptors, G protein, or other regulatory proteins can lead to either loss of function or gain of function of the various pathways. Loss of function mutations may result in agonist resistance and hence mimic hormone or agonist deficiency. Gain of function mutations result in constitutive, agonist-independent signaling and would mimic an excess of the hormone or agonist. The phenotype of the mutations can vary depending on the range of expression of the receptors and whether the mutation is somatic or germ line. Additionally, mutations in these pathways can alter cell proliferation and contribute to sensory defects. G proteins are key targets for bacterial toxins such as cholera toxin and pertussis toxins. Both of these toxins act to stimulate cam production, but by different mechanisms. Cholera toxin AD-ribosylates the G subunit rendering it constitutively active and leads to massive increases in cam production. In contrast, pertussis toxin acts on G i to inhibit the inhibition of adenyl cyclase. This pathway also results in an increase in cam production. A number of other bacterial toxin targets are related to signal transduction pathways related to G proteins (assador and Iglewski 1994). In conclusion, the GCR family of receptors and the associated regulatory proteins regulate a number of key pathways. These proteins serve as key targets for bacterial toxins, pharmacological targets, and sites of numerous pathologies when mutated Enzyme-Linked Receptors As noted above, the GCRs do not contain any enzymatic function themselves; the G proteins serve to link the receptor to the enzymes or effector systems that regulate production of the second messenger molecules in response to binding of the stimulus to the extracellular domain of the receptor. In contrast, enzyme-linked receptors contain catalytic function within the receptor itself that is regulated in response to agonist binding to the receptor. Examples of this type of receptor include the serine/threonine kinase receptors of which the transforming growth factor (TGF)- receptor is an example, the guanylyl cyclase receptors including the atrial natriuretic peptide receptors, ligand-gate ion channels such as the acetyl choline receptor, and the most prominent member of this class of receptors, the tyrosine kinase receptors. For the purpose of this chapter, we will focus primarily on the tyrosine kinase receptors (Alberts et al. 2007).

7 Cell Surface Receptors Receptor Tyrosine Kinases The receptor tyrosine kinases (RTKs) are a large superfamily of receptors that function as the receptors for a wide array of growth factors, including epidermal growth factor (EGF), nerve growth factor (NGF), platelet-derived growth factor (DGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), insulin and the insulin-like growth factors (IGF), and the ephrins and angiopoietins. Most of the RTKs function to regulate complex functions such as proliferation or differentiation, and as such the signal transduction pathways often terminate in the regulation of transcription and gene expression (Alberts et al. 2007). The RTKs are characterized by a single transmembrane spanning region. The extracellular region contains the growth factor or ligand-binding region and the intracellular portion of the receptor contains the tyrosine kinase enzymic activity. For most family members, agonist binding initiates the signaling pathway by bringing two receptors together to form a dimer. The formation of the dimers allows for the tyrosine kinase on one half of the dimer to phosphorylate tyrosine residues on the other dimer. This process has been referred to as autophosphorylation or transphosphorylation. The phosphorylated tyrosine residues act as docking sites for enzymes and other components of the signaling machinery. The proteins that bind to the phosphorylated tyrosines usually contain a consensus amino acid sequence that has been termed src-homology domain 2 (SH2). Among the proteins that bind to activated RTKs are tyrosine kinases (e.g., members of the src family), tyrosine phosphatases, adapter proteins (e.g., grb), phospholipase, (LC ), and phosphatidyl inositol 3-kinase (I 3-kinases). Thus, the activation of individual RTKs can serve as initiation of multiple downstream signaling pathways (Alberts et al. 2007). The resulting signaling events can regulate both cytosolic and nuclear events. The activation of LC results in the generation of the second messenger molecules diacylglycerol and inositol tris-phosphate. These molecules regulate the activity of protein kinase C and the release of intracellular calcium stores, respectively. The activation of these pathways, like the LC pathway regulated by the GCRs, serves to alter contractile and cytoskeletal properties of the cells in addition to the regulation of transcriptional events. Activation of I 3-kinase is key to the regulation of the Akt signaling pathway but RTKs. Akt is a key regulatory pathway governing cell survival in addition to other regulatory events. The key pathway regulating cell proliferation and growth is the activation of the mitogen-activated protein kinase (MAK) pathway. The activation of this pathway is initiated by the recruitment of the adaptor protein Grb2 to the phosphorylated tyrosines on the cytosolic tail of the receptor. This adapter protein then recruits the GEF, son-of-sevenless (SOS), which in turn serves to activate the small GTase, ras. Ras functions to activate the raf protein kinase, which in turn activates the MAK, cascade. The activation of MAK results in translocation to the nucleus where it phosphorylates key transcription factors that regulate the transcription of immediate early genes that go on to regulate the expression of proteins that initiate cell growth (Simon 2000). As key regulators of cell growth and differentiation, each step in the signaling pathway (i.e., growth factor, receptor, and signaling), when mutated, can result in cancer, and thus may serve as key sites for toxicant-induced injury. For example, v-sis is a virally encoded oncogene that is homologous to DGF and competes with DGF for the binding to its receptor. Mutation of downstream regulatory proteins such as ras results in unregulated cell growth and cancer (erona 2006). However, the focus of this particular chapter will be on the receptors themselves. Aberrant protein tyrosine kinase activity of the RTK family is linked to the development and progression of human cancers. The erbb family of receptors was first implicated in cancer when the avian erythroblastosis tumor virus was found to encode an altered form of the human EGF receptor also known as erbb1. Mutations in this pathway are also found in many breast cancers and specific mutations are used to determine the overall prognosis of the cancer and also to determine the appropriate treatment plan (Rowinsky 2003). Defects in insulin receptor signaling are associated with increased cell proliferation and altered signaling processes associated with diabetes. These are only a few of the numerous mutations in RTKassociated signaling that have been shown to be primary defects in the initiation of cancer or that function tumor promoters (orter and Vailancourt 1998). In addition to their role in cancer, RTKs play critical roles in the regulation of many other physiologic processes, and breakdown in the normal processes leads to other disease processes. The VEGF receptors play important roles in regulating

8 88 Receptor Systems angiogenesis or the growth of new blood vessels (Breen 2007). This process is important in tumor growth and in metastasis of cancer cells. lateletderived growth factor and its receptors are important in development of atherosclerosis and other vascular disorders (Boucher and Gotthardt 2004). In addition, the insulin receptor and insulin insensitivity are key regulators of diabetes and related vascular problems (Nigro et al. 2006). Like the GCRs, there are precise mechanisms for shutting down signaling through the receptors and these processes are tightly regulated. A major deactivation pathway, receptor downregulation, involves ligand-induced endocytosis of the RTK and subsequent degradation in lysosomes. A complex molecular machinery that uses the small protein ubiquitin as a key regulator assures proper endocytosis and degradation of RTKs. Data have been presented above that overactivation of RTK signaling pathways is strongly associated with carcinogenesis. Newer data also suggest that inappropriate receptor downregulation can also result in oncogenesis (Bache et al. 2004; Kirisits et al. 2007) Transforming Growth Factor- Receptors The TGF- family includes the TGF- 1,2, and 3, the bone morphogenetic proteins (BMs), inhibins, and activins. These growth factors play important roles in development and tissue morphogenesis, and in adults play important roles in normal tissue remodeling and adaptation. Additionally, these pathways have been linked to specific pathologies. The TGFs are important in fibrosis of various tissues including lung, kidney, and liver are important in the pathogenesis of asthma and renal failure. The receptors for these growth factors are quite complex and are composed of multiple subunits. The generalized structure of these receptors includes interaction between type I and type II receptors. Both types of receptors are transmembrane proteins and contain a serine/threonine kinase domain. The type 1 receptor contains a conserved glycine/serine rich sequence binding of TGF-, which results in the stable association of two receptor subunits of each type and phosphorylation of gly/ser region on the type I receptor by the type II receptor. This sequence of events results in activation of the type I receptor with subsequent autophosphorylation and the phosphorylation of the small mothers against decapentaplegic (SMAD) proteins. SMADs are considered ligand-induced transcriptional regulators of TGF- signaling. SMADs activate transcription through DNA binding and organization of nucleoprotein complex. Signaling mediated through these receptors are key to renal and pulmonary fibrosis, cardiovascular disease, and other pathologies (Feng and Derynck 2005) Ligand-Gated or Receptor- Operated Ion Channels Ligand-gated or receptor-operated ion channels are a special classification of activity-associated receptors. In this case, instead of activating an enzyme activity upon ligand binding, ligand binding regulates the activity of the ion channel. Thus, the relationship is similar to the enzyme-linked receptors and will be discussed in this section. Several examples of ligandgated ion channels include receptors for several neurotransmitters nicotinic cholinergic receptor, -aminobutyric acid A (GABA), and receptors for glutamate, aspartate, and glycine. The general organization of these receptors is that they are composed of multisubunits; each subunit spans the plasma membrane multiple times. Association of the subunits forms the pore or channel and changes in conformation of the subunits upon ligand binding regulates opening and closing of the channels. Depending on the specific receptor, ligand binding may occur only on a subunit that appears once in the overall structure (e.g., sulfonylurea receptor) or multiple ligand binding subunits may be present (e.g., the nicotinic acetylcholine receptor). Ligand-gated channels may also be regulated by protein phosphorylation of the channel subunits subsequent to signaling through other receptors (Sheng and ak 2000) Adhesion Molecules: Mediators of Cell Matrix and Cell Cell Interactions We will review briefly the various types of adhesion receptors in this section. We will present a brief overview of the integrin family of adhesion molecules, the selectins, cadherins, and the Ig cell adhesion molecules (CAMs). These receptors have been reviewed in detail previously (Juliano 2002). These receptor molecules play important roles in a number of basic processes including cell proliferation, migration, development, and tissue remodeling in adults. All of these receptor types involved fairly

9 Cell Surface Receptors 89 large extracellular domains that allow for interaction with extracellular matrix proteins (ECM), and/or adhesion molecules of adjacent cells, single transmembrane domains, and short cytoplasmic tails that are involved in organizing the actin cytoskeleton and recruiting signaling molecules. Early studies of these molecules focused on their roles in adhesion. In addition to this role, it is now clear that these receptors play major roles in cell signaling and regulation of key cellular events Integrins Integrins are specialized cell surface receptors that interact primarily with ECM proteins such as fibronectin, collagen, and laminin. Some specialized integrins interact with cell surface receptors on other cells. Integrins are heterodimers composed of an and a subunit. Currently 18 distinct subunits and 8 -subunits have been identified in vertebrate organisms. The distinct interaction of specific combinations forms the specificity of interaction with ECM proteins. Upon interaction with ligand the integrins undergo conformational changes that result in reorganization of the cytoskeleton and organization of complex protein signaling complexes within the focal adhesion sites (see Figure 4). The affinity of an integrin for its ECM ligand is modulated by both intracellular signaling processes and by ions in the extracellular milieu (Ginsberg et al. 2005). Engagement of integrins by their specific ECM ligand results in direct activation of signaling processes. The observation that cell adhesion and resulting integrin clustering lead to increased tyrosine phosphorylation was a seminal event in beginning to understand the regulation of signaling processes by integrin receptors. One of the first molecules to be discovered was focal adhesion kinase (FAK), a non- RTK that is now known to become activated by the integrin matrix interactions. FAK can also serve as a scaffolding protein and upon activation and autophosphorylation can interact with other proteins including c-src, I 3-kinase, paxillin, and talin. In addition to the complex assembly of tyrosine kinase related signaling players that ultimately lead to the activation of MA kinase and hence contribute to cell cycle regulation, activation of integrin signaling cascades plays an important role in activating small GTases such as rho, rac, and cdc 38. These proteins are critical in regulating the organization of both actin stress fibers and cortical actin around the perimeter of the cell. Thus, upon integrin activation a FAK α integrin α actinin Vinculin ECM β integrin Actin Src Talin Figure 4 Schematic of integrin and relationship to extracellular matrix and cytoskeleton organization. Integrins are heterodimers composed of an and subunit. The external domains form the binding site for interaction with extracellular matrix proteins (ECMs). The short cytoplasmic tail interacts with actin-binding proteins and a variety of signaling molecules including FAK and c-src. Integrins can mediate both outside in signaling and inside out signaling. number of other signaling and actin-binding proteins become associated with the complex leading to the formation of the complex structure of the focal adhesion kinase. These processes have been reviewed in more detail elsewhere (Romer et al. 2006). The complex nature of the integrin ligand specificity has been extensively studied because of the relationship between integrin binding and a number of crucial cellular events. The identification of the peptide sequence in fibronectin that interacted with 5 1 integrin was an important step in understanding the interaction of specific integrins with the ECM. The sequence Arg-Gly-Asp (RGD) was found to be the core sequence involved in interacting with a number of integrin heterodimers with surrounding sequences adding to the specificity. eptides have been used to decipher the role of specific integrins in cellular responses. It has been harder to identify the exact amino acid that bind integrins. Sequence in more complex ECM proteins such as laminin have been harder to identify the precise sequences involved in binding to specific integrins; however, this is a promising area of research as an arsenal of different peptide mimetics

10 90 Receptor Systems could then be used to differentially antagonize specific integrins or as scaffoldings for tissue engineering (reviewed in Takagi (2007); Temming et al. (2005)). In addition to modulation of intracellular signaling by binding of matrix to the integrin (outside in signaling), integrin with the extracellular matrix can be modified by intracellular signaling processes (inside out signaling). It is now clear that focal adhesion/integrin signaling processes play very important roles in regulating complex cellular events. In particular, it became clear that regulation of cyclic progress requires key signals from the integrins. In particular, specific integrins seem to be key for complex signaling through specific RTKs (Ginsberg et al. 2005). Construction of mouse models in which the expression of specific integrins is null has aided in our understanding of the complex relationship between specific integrins in development and their contribution to specific disease (Bouvard et al. 2001). The use of mouse models and individual deletions of integrin subunits has allowed for the determination of the contribution of individual integrins to specific processes. For example, deletion of the 1 subunit results in embryonic lethality because of its extensive involvement in many processes and its interaction with numerous subunits (Fassler and Meyer 1995; Werner et al. 2000). Other deletions result in more localized defects, such as the deletion of the 7 subunit leads primarily to a muscular dystrophy phenotype ref Cadherins The cadherins are calcium-dependent homotypic cell cell receptors. The classic cadherin family members include N,, R, B, and E cadherins, each of which contains 100 common amino acid repeats in their large extracellular domain. Cadherins localize to specific regions in the plasma membrane known as adherens junctions and interact with like cadherins on adjacent cells. These cell cell interactions are important in forming cell barriers and polarization of cells. On the cytosolic tails, the cadherins interact with proteins termed catenins (, ) that link the cadherins to the actin cytoskeleton. In this regard, the cadherins are similar to the integrins, in that upon binding they serve to regulate the organization of the actin cytoskeleton and to activate signaling cascades (Juliano 2002). Modulation of cadherin receptors is known to occur in various diseases including both toxicant- and aging-induced renal failure Selectins and Ig CAM Adhesion Molecules Compared to the integrins and cadherins, relatively little is known about the signaling processes mediated by members of the Ig CAM and selectin cell cell adhesion molecules. The Ig CAM family members are characterized by large Ig-type repeats in the extracellular domain with a single transmembrane domain and a short cytoplasmic tail. It is known that members of this family are involved in neural development and in immune cell interaction with endothelial and other tissue cells. These molecules play key roles in mediating the immune response and targeting leukocytes to the tissues. In this role, the expression, activation, and signaling are key to the regulation of inflammatory responses (Juliano 2002). The selectins (L-, E-, and -) also have large extracellular domains that contain lectin-like domains, EGF-like domains, and complement regulatory domains with a single transmembrane domain and a short cytoplasmic tail. The selectins are important in mediating heterotypic cell cell interactions such as between immune cells and platelets with endothelial cells during inflammatory events and wound healing. Much less is known about the signaling pathways that are activated by these interactions, but it is likely that the short cytoplasmic tail also serves to recruit signaling molecules and to interact with the actin cytoskeleton so that binding can initiate complex cellular behavior Conclusions Cell surface receptors play an essential and pivotal role in mediating the response of cells to external stimuli. The varied receptors allow cells to respond to a wide range of stimuli ranging from ions to large ECM proteins that lead to activation of specific signaling processes and changes in cellular behavior. Any mutation that leads to unregulated or inactive signaling can lead to pathologies. Thus, cell surface receptors are key to the mechanism of many chemical toxicants and serve as targets for the development of drugs. References Alberts, B.; Johnston, A.; Lewis, J.; Raff, M.; Roberts, K.; Walter,. Molecular Biology of the Cell; Garland Science: NewYork, Bache, K. G.; Slagsvold, T.; Stenmark, H. EMBO J. 2004, 23,

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