CHAPTER 30 Cellular Adhesion

All cells interact with molecules in their environment, in many cases relying on cell surface adhesion proteins to bind these molecules. Multicellular organisms are particularly dependent on adhesion of cells to each other and the extracellular matrix (ECM). During development, carefully regulated genetic programs specify cell-cell and cell-matrix interactions that determine the architecture of each tissue and organ. Some adhesive interactions are stable. Muscle cells must adhere firmly to each other and to the connective tissue of tendons to transmit force to the skeleton (see Chapter 39). Skin cells must also bind tightly to each other and the underlying connective tissue to resist abrasion (see Fig. 35-6). On the other hand, many cellular interactions are transient and delicate. At sites of inflammation, leukocytes bind transiently to endothelial cells lining small blood vessels and then use transient interactions with the ECM to migrate through connective tissue (look ahead to Figs. 30-13 and 30-14).

Figure 30-13 five steps in the migration of a neutrophil from the blood to the connective tissue. Endothelial cells exposed to inflammatory agents like histamine expose selectins on their surface and snare mucins on neutrophils flowing in the bloodstream (1). As a neutrophil rolls along the surface (2), chemotactic factors activate their integrins (3), causing the neutrophil to bind tightly to Ig-CAMs on the endothelium (4). The neutrophil then migrates between the endothelial cells into the connective tissue (5).

(Reference: Springer T: Traffic signals for lymphocyte and leukocyte emigration: The multi-step paradigm. Cell 76:301–314, 1994.)

Figure 30-14 platelet activation and aggregation at the site of a defect in the endothelium. A–D, Steps in platelet activation and aggregation. E, Electron micrograph of a thin section of a platelet adhering to the basal lamina through a tiny defect in the endothelium. F, Resting platelets circulate in the blood without interacting with the intact endothelium lining the vessel. G, Platelets are activated in three ways: Binding of a₂b₁ integrins to collagen results in firm adhesion (1). Where the basal lamina is exposed, von Willebrand factor (vWF) binds the collagen; platelet GP1B-IX binds weakly to von Willebrand factor, allowing platelets to adhere to the exposed matrix (2). Thrombin activates seven-helix receptors (3). These interactions stimulate secretion of ADP, which binds seven-helix receptors and activates the a_IIbb₃ integrins; then a_IIbb₃ integrins bind dimeric fibrinogen and aggregate platelets together. Platelet proteins are not to scale.

Cells use a relatively small repertoire of adhesion mechanisms to interact with matrix molecules and each other. This conceptual breakthrough came when comparisons of amino acid sequences showed that most adhesion proteins fall into five large families (Fig. 30-1). Within each of these distinctive families, ancestral genes duplicated and diverged during evolution, giving rise to adhesion proteins with the many different specificities that are required for embryonic development, maintenance of organ structure, and migrations of cells of our defense systems. Common properties within each family allowed the appreciation of general mechanisms to emerge from characterizing a few examples. Several important adhesion proteins fall outside the five major families, and additional families may emerge from continued research.

Figure 30-1 the major classes of adhesion proteins. Ig-CAMs have one or more extracellular domains folded like an immunoglobulin domain (orange). Cadherins have five or more extracellular CAD domains (maroon) that typically bind the same class of cadherin on a neighboring cell. Ca²⁺ stabilizes the interaction of neighboring CAD domains. Their cytoplasmic tails bind β-catenin (blue) and other adapter proteins. Integrins are heterodimers of a and b subunits that establish a wide range of specificities for matrix molecules by pairwise combinations of 1 of 16 α-chains and 1 of 8 β-chains. Selectins have a Ca²⁺-dependent lectin (carbohydrate-binding) domain (green), an EGF-like domain (blue), and a variable number of complement regulatory domains (red). Mucins display multiple carbohydrates for interactions with other cells. CD numbers refer to the names of representative adhesion molecules based on the “clusters of differentiation” nomenclature (see the text). Single transmembrane segments attach all of these receptors to the plasma membrane. Adapter proteins link the cytoplasmic tails of most adhesion proteins to the actin cytoskeleton or, in the case of specialized cadherins and integrins, to intermediate filaments.

(Redrawn from van der Merwe PA, Barclay AN: Transient intercellular adhesion: The importance of weak protein-protein interactions. Trends Cell Biol 19:354–358, 1994.)

Many adhesion proteins were named before they were classified into families. Tables 30-1 through 30-5 are designed to help the reader with the nomenclature. Many adhesion proteins are named “CD” followed by a number. This stands for “clusters of differentiation,” a term that is used to classify cell surface antigens recognized by monoclonal antibodies, independent of any knowledge about the structure or function of the antigen. Hence, members of the four major families of adhesion proteins have CD numbers.

Table 30-1 CELL ADHESION MOLECULES: IMMUNOGLOBULIN FAMILY^*

Table 30-2 EXAMPLES OF CELL ADHESION MOLECULES: CADHERIN FAMILY^*

Table 30-3 CELL ADHESION MOLECULES: INTEGRIN FAMILY^*

Table 30-4 CELL ADHESION MOLECULES: SELECTIN FAMILY (LEC-CAM)

Table 30-5 OTHER CELL ADHESION MOLECULES

This chapter first highlights some general features of adhesion proteins and then introduces four major families: immunoglobulin–cell adhesion molecules (Ig-CAMs), cadherins, integrins, and selectins. While learning about each family, the reader should not lose track of an important point: These receptors rarely act alone. Rather, they usually function as parts of multicomponent systems. Two examples at the end of the chapter illustrate the cooperation that is required for leukocytes to respond to inflammation and for platelets to repair damage to blood vessels. Chapter 31 on intercellular junctions, Chapter 32 on specialized connective tissues, and Chapter 38 on cellular motility provide more examples of cellular adhesion.

General Principles of Cellular Adhesion

First Principle of Adhesion

Cells define their capacity for adhesive interactions by selectively expressing plasma membrane receptors (cell adhesion molecules, or CAMs) with limited ligand-binding activity. Generally, expression of the proper mix of receptors is part of a genetic program for the differentiation of the cell. In some cases, extracellu-lar stimuli control expression of adhesion recep-tors. For example, endothelial cells produce E-selectin only when stimulated by inflammatory hormones or endotoxin.

Second Principle of Adhesion

Many adhesion proteins bind one main ligand, and many ligands bind a single type of receptor (refer to Tables 30-1 through 30-5). If this one-to-one pairing were the rule, adhesion would be simple indeed. However, many exceptions exist, particularly in the integrin family of receptors (Table 30-3). These receptors generally bind more than one ligand, and some ligands, such as fibronectin, bind more than one integrin. One can generalize about the ligands for the several families of cell adhesion molecules:

• Most cadherins prefer to bind themselves, so they promote the adhesion of like cells. These homophilic interactions (association of like receptors on two cells) require Ca²⁺.

• Selectins bind anionic polysaccharides like those on mucins. Generally, such interactions bind together two different types of cells.

• Most Ig-CAMs bind other cell surface adhesion proteins. These heterophilic interactions (association of unlike receptors on two cells) may occur between the same or different cell types.

• Integrins stand apart because they bind a variety of ligands: matrix macromolecules, such as fibronectin (see Fig. 29-15) and laminin (see Fig. 39-9); soluble proteins, such as fibrinogen in blood; and adhesion proteins on the surface of other cells, including Ig-CAMs and one cadherin.

Third Principle of Adhesion

Cells modulate adhesion by controlling the surface density, state of aggregation, and state of activation of their adhesion receptors. Surface density reflects not only the level of synthesis but also the partitioning of adhesion molecules between the plasma membrane and intracellular storage compartments. For example, endothelial cells express P-selectin constitutively but store it internally in membranes of cytoplasmic vesicles. When inflammatory cytokines activate endothelial cells, these vesicles fuse with the plasma membrane, exposing P-selectin on the cell surface, where it binds white blood cells (Fig. 30-13). The importance of surface density is illustrated by an experiment in which cells that express different levels of the same cadherin are mixed together. Over time, they sort out from each other, the more adherent cells forming a cluster surrounded by the less adherent cells (Fig. 30-2). Such differential expression of cadherin determines the position of the oocyte in Drosophila egg follicles. Intracellular signals control the extracellular binding activity of integrins and cadherins. A variety of extracellular stimuli activate intracellular signaling pathways in lymphocytes, platelets, and other cells, which enhance or inhibit the ligand-binding activity of integrins already located on the cell surface. Integrin activation also regulates cellular interactions during development.

Figure 30-2 two experiments on sorting of embryonic cells. A, When cells from different tissues are dissociated and mixed together, they spontaneously sort themselves into two layers, with the more adherent cells inside the less adherent cells. B, Cells with high concentrations of cadherin sort inside less adherent cells.

(Based on the work of M. Steinberg, Princeton University, Princeton, New Jersey.)

Fourth Principle of Adhesion

The rates of ligand binding and dissociation are important determinants of cellular adhesion. Many cell surface adhesion proteins (including members of the Ig-CAM, cadherin, integrin, and selectin families) bind their ligands weakly in comparison with other specific macromolecular interactions, such as the interaction of antigens and antibodies, hormones and receptors, or transcription factors and DNA. The measured dissociation equilibrium constants for these adhesion receptors are in the range of 1 to 100 mM, reflecting high rate constants (>1 s⁻¹) for dissociation of ligand. In some cases, this makes good biological sense. Rapidly reversible interactions allow white blood cells to roll along the endothelium of blood vessels (Fig. 30-13). Transient adhesion also allows fibroblasts to migrate through connective tissue. On the other hand, the interactions of cells in epithelia and muscle appear to be more stable, perhaps owing to multiple weak interactions between clustered adhesion proteins cooperating to stabilize adherens junctions and desmosomes (Fig. 31-7). The combined strength of these bonds is said to increase the “avidity” of the interaction.

Fifth Principle of Adhesion

Many adhesion receptors interact with the cytoskeleton inside the cell. Adapter proteins link cadherins and integrins to actin filaments or intermediate filaments. These interactions provide mechanical continuity from cell to cell in muscles and epithelia, allowing them to transmit forces and resist mechanical disruption.

Sixth Principle of Adhesion

Association of ligands with adhesion receptors can activate intracellular signal transduction pathways, leading to changes in gene expression, cellular differentiation, secretion, motility, receptor activation, and cell division. Signaling through adhesion receptors allows cells to respond appropriately to physical interactions with the surrounding matrix or cells.

Identification and Characterization of Adhesion Receptors

The ability of mixed populations of cells to sort into homogeneous aggregates revealed that cells have mechanisms that are designed to bind like cells together. Similar assays showed that cells also bind matrix macromolecules, such as fibronectin, laminin, collagen, and proteoglycans. Biochemical isolation of the responsible adhesion proteins was challenging, but it progressed rapidly once it was possible to produce monoclonal antibodies that inhibit adhesion. These antibodies provided assays for purification of adhesion proteins and cloning of their cDNAs. With representatives from each family in hand, the cloning of cDNAs for related proteins was straightforward.

The modular construction of adhesion receptors makes it possible to isolate proteolytic fragments or to express one or more domains of recombinant protein suitable for structural analysis. Given sequence homologies within each family, the structures of many extracellular domains can be approximated from crystal structures of other family members.

Insights about the functions of adhesion receptors have usually come in several steps. Localization of a protein on specific cells frequently provides the first clues. Typically, the expression of each protein is restricted to a subset of cells or to a specific time during embryonic development or both. Next, investigators use specific antibodies to test for the participation of the adhesion protein in cellular interactions in vitro or in tissues. Blistering skin diseases called pemphigus illustrate the serious consequences when pathological autoantibodies disrupt adhesion between skin cells expressing the antigen (see the sections “Desmosomes” and “Adhesion to the Extracellular Matrix” in Chapter 31). Both human genetic diseases and experimental genetic knockouts in mice and other organisms produce defects caused by the absence of adhesion proteins. In leukocyte adhesion deficiency, white blood cells lack the b₂ integrin that is required to bind the endothelial cells that line blood vessels. These defective white blood cells fail to bind to blood vessel walls or to migrate into connective tissue at sites of infection. Similarly, patients with a bleeding disorder called Bernard-Soulier syndrome lack one of the adhesion receptors for von Willebrand factor, a protein that promotes platelet aggregation. Loss of cadherins contributes to the spread of some cancer cells.

Immunoglobulin Family of Cell Adhesion Molecules

The Ig-CAM family contains hundreds of adhesion proteins, each with one to seven extracellular domains, similar to immunoglobulin domains, anchored to the plasma membrane by a single transmembrane helix (Fig. 30-3 and Table 30-1). Crystal structures established the antibody-like fold of the extracellular domains of several Ig-CAMS. These compact Ig domains consist of 90 to 115 residues folded into seven to nine β-strands in two sheets, usually stabilized by an intramolecular disulfide bond. The N- and C-termini are at opposite ends of these domains, allowing the formation of linear arrays of immunoglobulin domains.

Figure 30-3 Molecular structure of representative Ig-CAMS. A, Domain maps of examples with their common names and CD numbers. B, Ribbon diagrams of the lymphocyte coreceptors CD4 (domains 1 and 2 on the left and domains 3 and 4 on the right) and CD8.

(A, Reference: Springer T: Traffic signals for lymphocyte and leukocyte emigration: The multi-step paradigm. Cell 76:301–314, 1994. PDB files: 3CD4, 1CID, and 1CD8.)

Some Ig-CAMs consist of a single polypeptide, but others are multimeric, with two (CD8) or four (see Fig. 27-8 for the T-cell receptor) subunits. Some nervous system Ig-CAMs have three or four fibronectin III (FN-III) domains between the immunoglobulin domains and the membrane anchor. The C-terminal cytoplasmic tails of these receptors vary in sequence and binding sites. The cytoplasmic domains of the lymphocyte accessory receptors CD4 and CD8 bind protein tyrosine kinases required for cellular activation (see Fig. 27-8). The cytoplasmic domains of neuronal Ig-CAMs bind PDZ domain proteins or membrane skeleton (see Fig. 7-10).

Differentiated metazoan cells express Ig-CAMs selectively, especially during embryonic development, when they may contribute to the specificity of cellular interactions required to form the organs. Neurons and glial cells express specific Ig-CAMs that guide the growth of neurites, mediate synapse formation and promote the formation of myelin sheaths. In adults, interaction of endothelial cell ICAM-1 with a white blood cell integrin is essential for adhesion and movement of the leukocytes into the connective tissue at sites of inflammation (Fig. 30-13).

Like other cell adhesion proteins, Ig-CAMs participate in signaling processes. Best understood are interactions of lymphocytes with antigen-presenting cells during immune responses. Ig-CAMs reinforce the interaction of antigen-specific T-cell receptors with major histocompatibility complex molecules carrying appropriate antigens on other cells (see Fig. 27-8). Although individual interactions are weak, the combination of specific (T-cell receptor) and nonspecific (CD2 and CD4) interactions with the target cell is sufficient to initiate signaling.

Cadherin Family of Adhesion Receptors

The complex architecture of organs in vertebrates depends on Ca²⁺-dependent associations between the cells mediated by more than 80 cadherins (Table 30-2). Their name derives from “calcium-dependent adhesion” protein. Genes for cadherin domains appeared in unicellular precursors of sponges, an early step toward the evolution of metazoan organisms.

Cadherins generally interact with like cadherins on the surfaces of other cells in a calcium-dependent fashion, but research is uncovering a growing list of examples of heterophilic interactions. Homophilic interactions of cadherins link epithelial and muscle cells to their neighbors, especially at specialized adhesive junc tions called adherens junctions and desmosomes (Fig. 30-4; also see Fig. 31-7). The cytoplasmic domains of cadherins interact with actin filaments or intermediate filaments to reinforce these junctions and maintain the physical integrity of tissues. Contacts mediated by cadherins also influence cellular growth and migration, including suppression of growth and invasion of tu-mors, as well as formation of synapses in the nervous system.

Figure 30-4 electron micrographs of rod-like cadherins connecting the plasma membranes of adjacent cells. Intestinal epithelial cells were prepared by rapid freezing, freeze-fracture, deep etching, and rotary shadowing. A, Desmosome with associated intermediate filaments in the cytoplasm. B, Adherens junction with associated actin filaments.

(Courtesy of N. Hirokawa, University of Tokyo, Japan. Reproduced from Hirokawa N, Heuser J: Quick-freeze, deep-etch visualization of the cytoskeleton beneath surface differentiations of intestinal epithelial cells. J Cell Biol 91:399–409, 1981, by copyright permission of The Rockefeller University Press.)

The structural hallmark of the cadherin family is the CAD domain (Figs. 30-5 and 30-6). CAD domains consist of about 110 residues folded into a sandwich of seven β-strands. This fold is similar to immunoglobulin and FN-III domains, but the limited sequence homology suggests independent origins and convergent evolution. N- and C-termini are on opposite ends of CAD domains. Ca²⁺ bound to three sites between adjacent CAD domains links them together into rigid rods. Without Ca²⁺, the domains rotate freely around their linker peptides.