(a) Focal adhesion

Diversity of Ligand-Integrin Interactions Contributes to Numerous Biological Processes

Although most cells express several distinct integrins that bind the same ligand or different ligands, many integrins are expressed predominantly in certain types of cells. Table 6-2 lists a few of the numerous integrin-mediated interactions with ECM components or CAMs or both. Not only do many integrins bind more than one ligand, but several of their lig-ands bind to multiple integrins.

All integrins appear to have evolved from two ancient general subgroups: those that bind RGD-containing molecules (e.g., fibronectin) and those that bind laminin. For example, a5p1 integrin binds fibronectin, whereas the widely expressed a1p1 and a2p1 integrins, as well as the a6p4 in-tegrin expressed by epithelial cells, bind laminin. The a1, a2, and several other integrin a subunits contain a distinctive inserted domain, the I-domain. The I-domain in some integrins (e.g., a1p1 and a2p1) mediates binding to various collagens. Other integrins containing a subunits with I-domains are expressed exclusively on leukocytes and hematopoietic cells; these integrins recognize cell-adhesion molecules on other cells, including members of the Ig superfamily (e.g., ICAMs, VCAMs), and thus participate in cell-cell adhesion.

The diversity of integrins and their ECM ligands enables integrins to participate in a wide array of key biological processes, including the migration of cells to their correct locations in the formation the body plan of an embryo (morphogenesis) and in the inflammatory response. The importance of integrins in diverse processes is highlighted by the defects exhibited by knockout mice engineered to have mutations in each of almost all of the integrin subunit genes. These defects include major abnormalities in development, blood vessel formation, leukocyte function, the response to infection (inflammation), bone remodeling, and hemostasis.

Cell-Matrix Adhesion Is Modulated by Changes in the Binding Activity and Numbers of Integrins

Cells can exquisitely control the strength of integrin-mediated cell-matrix interactions by regulating the ligand-binding activity of integrins or their expression or both. Such regulation is critical to the role of these interactions in cell migration and other functions.

Many, if not all, integrins can exist in two conformations: a low-affinity (inactive) form and a high-affinity (active) form (Figure 6-28). The results of structural studies and experiments investigating the binding of ligands by integrins have provided a model of the changes that take place when integrins are activated. In the inactive state, the ap het-erodimer is bent, the conformation of the ligand-binding site at the tip of the molecule allows only low-affinity ligand binding, and the cytoplasmic C-terminal tails of the two sub-units are closely bound together. In the "straight," active state, alterations in the conformation of the domains that form the binding site permit tighter (high-affinity) ligand binding, and the cytoplasmic tails separate.

These structural models also provide an attractive explanation for the ability of integrins to mediate outside-in and inside-out signaling. The binding of certain ECM molecules or CAMs on other cells to the bent, low-affinity structure would force the molecule to straighten and consequently separate the cytoplasmic tails. Intracellular adapters could "sense" the separation of the tails and, as a result, either bind or dissociate from the tails. The changes in these adapters

M FIGURE 6-28 Model for integrin activation. (Left) The molecular model is based on the x-ray crystal structure of the extracellular region of avp3 integrin in its inactive, low-affinity ("bent") form, with the a subunit in shades of blue and the p subunit in shades of red. The major ligand-binding sites are at the tip of the molecule where the p propeller domain (dark blue) and pA domain (dark red) interact. An RGD peptide ligand is shown in yellow. (Right) Activation of integrins is thought to be due to conformational changes that include straightening of the molecule, key movements near the p propeller and pA domains, which increases the affinity for ligands, and separation of the cytoplasmic domains, resulting in altered interactions with adapter proteins. See text for further discussion. [Adapted from M. Arnaout et al., 2002, Curr. Opin. Cell Biol. 14:641, and R. O. Hynes, 2002, Cell 110:673.]

could then alter the cytoskeleton and activate or inhibit in-tracellular signaling pathways. Conversely, changes in the metabolic state of the cells (e.g., changes in the platelet cy-toskeleton that accompany platelet activation; see Figure 19-5) could cause intracellular adapters to bind to the tails or to dissociate from them and thus force the tails to either separate or associate. As a consequence, the integrin would either bend (inactivate) or straighten (activate), thereby altering its interaction with the ECM or other cells.

Platelet function provides a good example of how cell-matrix interactions are modulated by controlling inte-grin binding activity. In its basal state, the aIIbp3 integrin present on the plasma membranes of platelets normally cannot bind tightly to its protein ligands (e.g., fibrinogen, fibronectin), all of which participate in the formation of a blood clot, because it is in the inactive (bent) conformation. The binding of a platelet to collagen or thrombin in a forming clot induces from the cytoplasm an activating conforma-tional change in aIIbp3 integrin that permits it to tightly bind clotting proteins and participate in clot formation. Persons with genetic defects in the p3 integrin subunit are prone to excessive bleeding, attesting to the role of this integrin in the formation of blood clots.

The attachment of cells to ECM components can also be modulated by altering the number of integrin molecules exposed on the cell surface. The a4p1 integrin, which is found on many hematopoietic cells (precursors of red and white blood cells), offers an example of this regulatory mechanism. For these hematopoietic cells to proliferate and differentiate, they must be attached to fibronectin synthesized by supportive ("stromal") cells in the bone marrow. The a4p1 integrin on hematopoietic cells binds to a Glu-Ile-Leu-Asp-Val (EILDV) sequence in fibronectin, thereby anchoring the cells to the matrix. This integrin also binds to a sequence in a CAM called vascular CAM-1 (VCAM-1), which is present on stromal cells of the bone marrow. Thus hematopoietic cells directly contact the stromal cells, as well as attach to the matrix. Late in their differentiation, hematopoietic cells decrease their expression of a4p1 integrin; the resulting reduction in the number of a4p1 integrin molecules on the cell surface is thought to allow mature blood cells to detach from the matrix and stromal cells in the bone marrow and subsequently enter the circulation.

Molecular Connections Between the ECM and Cytoskeleton Are Defective in Muscular Dystrophy

The importance of the adhesion receptor-mediated linkage between ECM components and the cyto-skeleton is highlighted by a set of hereditary muscle-wasting diseases, collectively called muscular dystrophies. Duchenne muscular dystrophy (DMD), the most common type, is a sex-linked disorder, affecting 1 in 3300 boys, that results in cardiac or respiratory failure in the late teens or early twenties. The first clue to understanding the molecular basis of this disease came from the discovery that persons with DMD carry mutations in the gene encoding a protein named dystrophin. This very large protein was found to be a cytosolic adapter protein, binding to actin filaments and to an adhesion receptor called dystroglycan. I

Dystroglycan is synthesized as a large glycoprotein precursor that is proteolytically cleaved into two subunits. The a subunit is a peripheral membrane protein, and the p subunit is a transmembrane protein whose extracellular domain associates with the a subunit (Figure 6-29). Multiple O-linked oligosaccharides are attached covalently to side-chain hydroxyl groups of serine and threonine residues in the a subunit. These O-linked oligosaccharides bind to various basal lamina components, including the multiadhesive matrix protein laminin and the proteoglycans perlecan and

▲ FIGURE 6-29 Schematic model of the dystrophin glycoprotein complex (DGC) in skeletal muscle cells. The

DGC comprises three subcomplexes: the a,p dystroglycan subcomplex; the sarcoglycan/sarcospan subcomplex of integral membrane proteins; and the cytosolic adapter subcomplex comprising dystrophin, other adapter proteins, and signaling molecules. Through its O-linked sugars, p-dystroglycan binds to components of the basal lamina, such as laminin. Dystrophin— the protein defective in Duchenne muscular dystrophy—links p-dystroglycan to the actin cytoskeleton, and a-dystrobrevin links dystrophin to the sarcoglycan/sarcospan subcomplex. Nitric oxide synthase (NOS) produces nitric oxide, a gaseous signaling molecule, and GRB2 is a component of signaling pathways activated by certain cell-surface receptors (Chapter 14). See text for further discussion. [Adapted from S. J. Winder, 2001, Trends Biochem. Sci. 26:118, and D. E. Michele and K. P Campbell, 2003, J. Biol. Chem.]

agrin. The neurexins, a family of adhesion molecules expressed by neurons, also are bound by the a subunit.

The transmembrane segment of the dystroglycan p subunit associates with a complex of integral membrane proteins; its cytosolic domain binds dystrophin and other adapter proteins, as well as various intracellular signaling proteins. The resulting large, heterogeneous assemblage, the dystrophin glycoprotein complex (DGC), links the extracellular matrix to the cytoskeleton and signaling pathways within muscle cells (see Figure 6-29). For instance, the signaling enzyme nitric oxide synthase (NOS) is associated through syntrophin with the cytosolic dystrophin subcom-plex in skeletal muscle. The rise in intracellular Ca2+ during muscle contraction activates NOS to produce nitric oxide (NO), which diffuses into smooth muscle cells surrounding nearby blood vessels. By a signaling pathway described in Chapter 13, NO promotes smooth muscle relaxation, leading to a local rise in the flow of blood supplying nutrients and oxygen to the skeletal muscle.

Mutations in dystrophin, other DGC components, laminin, or enzymes that add the O-linked sugars to dystro-glycan disrupt the DGC-mediated link between the exterior and the interior of muscle cells and cause muscular dystrophies. In addition, dystroglycan mutations have been shown to greatly reduce the clustering of acetylcholine receptors on muscle cells at the neuromuscular junctions (Chapter 7), which also is dependent on the basal lamina proteins laminin and agrin. These and possibly other effects of DGC defects apparently lead to a cumulative weakening of the mechanical stability of muscle cells as they undergo contraction and relaxation, resulting in deterioration of the cells and muscular dystrophy.

Ca2+-Independent Cell-Cell Adhesion in Neuronal and Other Tissues Is Mediated by CAMs in the Immunoglobulin Superfamily

Numerous transmembrane proteins characterized by the presence of multiple immunoglobulin domains (repeats) in their extracellular regions constitute the Ig superfamily of CAMs, or IgCAMs. The Ig domain is a common protein motif, containing 70-110 residues, that was first identified in antibodies, the antigen-binding immunoglobulins. The human, D. melanogaster, and C. elegans genomes include about 765, 150, and 64 genes, respectively, that encode proteins containing Ig domains. Immunoglobin domains are found in a wide variety of cell-surface proteins including T-cell receptors produced by lymphocytes and many proteins that take part in adhesive interactions. Among the IgCAMs are neural CAMs; intercellular CAMs (ICAMs), which function in the movement of leukocytes into tissues; and junction adhesion molecules (JAMs), which are present in tight junctions.

As their name implies, neural CAMs are of particular importance in neural tissues. One type, the NCAMs, primarily mediate homophilic interactions. First expressed during morphogenesis, NCAMs play an important role in the differentiation of muscle, glial, and nerve cells. Their role in cell adhesion has been directly demonstrated by the inhibition of adhesion with anti-NCAM antibodies. Numerous NCAM isoforms, encoded by a single gene, are generated by alternative mRNA splicing and by differences in glycosylation. Other neural CAMs (e.g., L1-CAM) are encoded by different genes. In humans, mutations in different parts of the L1-CAM gene cause various neuropathologies (e.g., mental retardation, congenital hydrocephalus, and spasticity).

An NCAM comprises an extracellular region with five Ig repeats and two fibronectin type III repeats, a single membrane-spanning segment, and a cytosolic segment that interacts with the cytoskeleton (see Figure 6-2). In contrast, the extracellular region of L1-CAM has six Ig repeats and four fibronectin type III repeats. As with cadherins, cis (intracellular) interactions and trans (intercellular) interactions probably play key roles in IgCAM-mediated adhesion (see Figure 6-3).

The covalent attachment of multiple chains of sialic acid, a negatively charged sugar derivative, to NCAMs alters their adhesive properties. In embryonic tissues such as brain, poly-sialic acid constitutes as much as 25 percent of the mass of NCAMs. Possibly because of repulsion between the many negatively charged sugars in these NCAMs, cell-cell contacts are fairly transient, being made and then broken, a property necessary for the development of the nervous system. In contrast, NCAMs from adult tissues contain only one-third as much sialic acid, permitting more stable adhesions.

Movement of Leukocytes into Tissues Depends on a Precise Sequence of Combinatorially Diverse Sets of Adhesive Interactions

In adult organisms, several types of white blood cells (leukocytes) participate in the defense against infection caused by foreign invaders (e.g., bacteria and viruses) and tissue damage due to trauma or inflammation. To fight infection and clear away damaged tissue, these cells must move rapidly from the blood, where they circulate as unattached, relatively quiescent cells, into the underlying tissue at sites of infection, inflammation, or damage. We know a great deal about the movement into tissue, termed extravasation, of four types of leukocytes: neutrophils, which release several antibacterial proteins; monocytes, the precursors of macrophages, which can engulf and destroy foreign particles; and T and B lymphocytes, the antigen-recognizing cells of the immune system.

Extravasation requires the successive formation and breakage of cell-cell contacts between leukocytes in the blood and endothelial cells lining the vessels. Some of these contacts are mediated by selectins, a family of CAMs that mediate leukocyte-vascular cell interactions. A key player in these interactions is P-selectin, which is localized to the blood-facing surface of endothelial cells. All selectins contain a Ca2 +-dependent lectin domain, which is located at the distal end of the extracellular region of the molecule and recognizes oligosaccharides in glycoproteins or glyco-lipids (see Figure 6-2). For example, the primary ligand for P- and E-selectins is an oligosaccharide called the sialyl Lewis-x antigen, a part of longer oligosaccharides present in abundance on leukocyte glycoproteins and glycolipids.

Figure 6-30 illustrates the basic sequence of cell-cell interactions leading to the extravasation of leukocytes. Various inflammatory signals released in areas of infection or inflammation first cause activation of the endothelium. P-selectin exposed on the surface of activated endothelial cells mediates the weak adhesion of passing leukocytes. Because of the force of the blood flow and the rapid "on" and "off" rates of P-selectin binding to its ligands, these "trapped" leukocytes are slowed but not stopped and literally roll along the surface of the endothelium. Among the signals that promote activation of the endothelium are chemokines, a group of small secreted proteins (8-12 kDa) produced by a wide variety of cells, including endothelial cells and leukocytes.

For tight adhesion to occur between activated endothelial cells and leukocytes, p2-containing integrins on the surfaces of leukocytes also must be activated by chemokines or other local activation signals such as platelet-activating factor (PAF). Platelet-activating factor is unusual in that it is a phospholipid, rather than a protein; it is exposed on the surface of activated endothelial cells at the same time that P-selectin is exposed. The binding of PAF or other activators to their receptors on leukocytes leads to activation of the leukocyte integrins to their high-affinity form (see Figure 6-28). (Most of the receptors for chemokines and PAF are members of the G protein-coupled receptor superfamily discussed in Chapter 13.) Activated integrins on leukocytes then bind to each of two distinct IgCAMs on the surface of en-

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