Integrins—mediators of cell-matrix and cell-cell adhesion

By around 1900, scientists had ascertained that animal tissues are not random clumps of cells, but rather, highly ordered assemblies. Without adhesion, our bodies would disintegrate, and we now know that cells exist in intimate association with the ECM. Mid-century experiments established the importance of adhesion in development, and the topic converged with cancer as well, yet its molecular basis remained elusive.

Surface explorations, a penetrating idea

By the early 1970s, tumor cells were known to go rogue in the lab as well as the body. Unlike normal cells, they can grow without clinging to culture dishes; furthermore, they ball up and detach instead of spreading out. These unusual properties pointed to the cell surface as a key to some of their aberrant behavior.

Hynes and Ruoslahti, with his collaborator Antti Vaheri (University of Helsinki), independently decided to identify proteins that differentially appear on the surface of normal and malignant cells. The researchers took different tacks but both groups obtained a single abundant protein that coats normal cells and is minimal or absent on cancer cells. Others showed that this protein, which was eventually named fibronectin, fosters the attachment of cells to components of the ECM, and biologists came to appreciate that fibronectin is itself a bona fide member of that material.

Fibronectin addition to cancer cells reversed several of their malignant features. The protein caused them to flatten onto culture dishes, and it restored their internal actin filaments, a major constituent of the molecular skeleton that maintains cell shape. Visualization of actin or fibronectin with fluorescent antibodies demonstrated that these two proteins produce superimposable patterns—with actin inside the cell and fibronectin outside. These observations, from Hynes and others, suggested that some membrane-spanning link connects the molecules. This conjecture raised provocative questions. What was this presumptive molecular hookup and how might it yoke events in the environment to those inside cells?

Dynamic duo

In the meantime, Springer was working in the distant field of immunology. Numerous investigators were racing to track down the T-cell molecule that recognizes infected or malignant cells, which announce themselves by displaying chunks of invaders or suspicious molecules. Certain perplexing observations led Springer to infer that a factor in addition to the canonical T cell receptor was required for this interaction, and he set out to find it. He raised antibodies to surface molecules on cytolytic T cells and tested whether they blocked these cells’ ability to kill their quarry. In 1981, he reported that one such antibody had exactly this effect and thus defined an entity, lymphocyte-function-associated antigen (LFA-1), that executes a vital T-cell task. Further results suggested that LFA-1 helps T cells embrace their targets.

LFA-1 is composed of two distinct proteins, called α and β, and Springer noticed that their sizes correspond to those of another protein, Mac-1, which he had previously identified on a different immune cell, the macrophage. These observations hinted that LFA-1 and Mac-1 might be related. In 1982, he found that each protein’s α and β chain physically associate to form heterodimers—“hetero” because the components differ from each other and “dimer” because there are two. Structural analysis established that the smaller, β subunits of Mac-1 and LFA-1 are close kin or the same, whereas the larger, α subunits differed. Springer soon added a third protein, p150,95, to this growing molecular family. In 1985, he sequenced the first 18 amino acids of the LFA-1 and Mac-1 αs; this fine-tuned examination revealed similarities that implied a relationship in which a single ancestral gene had given rise to multiple αs during evolution.

Springer also discovered that Mac-1 was a previously known receptor that allows macrophages to grasp and engulf microbes and damaged cells. The notion that LFA-1 and Mac-1 perform essential immune functions gained support from observations by Springer and others that a group of individuals with recurrent life-threatening bacterial infections have scant quantities of Mac-1, LFA-1, and p150,95. The underlying condition, Leukocyte Adhesion Deficiency, arises because the common β chain is flawed or underproduced. Consequently, macrophages and other white blood cells cannot appropriately lodge on vessel walls, so they fail to exit the bloodstream and reach infection sites.

Sticky questions, smooth progress

Ruoslahti had been homing in on the part of fibronectin that attaches to cells, a venture that would wind up enabling discovery of the element that ties fibronectin to the cell’s innards. In 1984, he narrowed down this site to an amino-acid stretch that contains an essential arginine-glycine-aspartic acid (RGD in single-letter nomenclature) core. Remarkably, only three of fibronectin’s 1008 amino acids are sufficient to mediate cell attachment.

The following year, he used the molecular tools he had created, including a small peptide that contains RGD, to isolate a protein that binds fibronectin and appeared to be embedded in the cell membrane. Soon afterward, he nabbed a second receptor that tethers a different RGD-containing protein, vitronectin.

Around the same time and within months of each other, Hynes and Ruoslahti reported that they had uncovered yet another receptor that binds RGD-containing proteins, including fibronectin and others involved in clotting. This receptor resides on platelets, cell fragments that slow bleeding and help wounds heal, and it turned out to be a known entity called platelet membrane glycoprotein gpIIb/gpIIIa. David R. Phillips (Gladstone Institute, San Francisco) had established that gpIIb/gpIIIa was, like LFA-1, Mac-1, and p150,95, a heterodimer; he also had demonstrated that platelets from patients with a bleeding disorder called Glanzmann thrombasthenia carry unusually small quantities of gpIIb/gpIIIa. Hynes’s and Ruoslahti’s finding was therefore exciting because it attributed a crucial physiological function to the family of proteins that adhere to the ECM.

Collide and conquer

By then, scientists knew that two components unite to form the fibronectin and the vitronectin receptors. In 1986, Ruoslahti obtained partial sequence of the DNA that encodes these proteins’ α subunits and found that they resemble not only each other, but also the α subunits of LFA-1 and Mac-1.

The likeness did not end there. The scientists would soon find that the β subunits of these proteins were also related. That same year, Hynes isolated DNA that encodes the β subunit of the fibronectin receptor, and a satisfying feature jumped out: It contained a sequence that characterizes transmembrane anchors. These results suggested that the protein was the molecule he had imagined that bridges the cell’s exterior and the actin filaments inside. Hynes named it integrin because it is an integral membrane protein complex that couples the ECM to the cytoskeleton (Figure 1A).

Within months, Springer reported that a single gene encodes the β chains of LFA-1, Mac-1, and p150,95, and that the integrin β chain was a molecular sibling. He also identified the protein on cells that binds LFA-1 and showed that it defined a new type of partner, as it lacks RGD and belongs to a group of immunoglobulin-like proteins, well known for their roles at the cell surface. The protein that Springer unearthed, intercellular adhesion molecule 1 (ICAM-1), dwells on the endothelial cells that line blood vessels and its residence there explains how LFA-1 foments immune-cell escape from the circulation.

Subsequent work has revealed that humans carry 18 genes for α subunits and eight for β subunits. Various combinations generate 24 known integrins, a name that now applies to all members of the protein family (Figure 1B). Approximately one third of them recognize RGD. These adhesion proteins operate in a plethora of biological enterprises, including the most fundamental processes of cell growth, migration, survival, and proliferation; they likely enabled the evolution of multicellular animals from our unicellular forebears.

Cementing discoveries

Scientists have added many layers to our understanding of how integrin-based contact translates into behavioral changes. For example, integrin stimulation spurs a classic molecular event that turns activities on and off: addition of a phosphate group to a particular amino acid on an intracellular protein. This observation, simultaneously reported in 1991 by Hynes and R. L. Juliano (University of North Carolina, Chapel Hill), suggested a means by which integrins might govern physiological affairs and propelled a multitude of studies on their chemical signaling mechanism.

Furthermore, integrins themselves can be activated. Many of these receptors must seize their molecular partners only under particular circumstances. Otherwise, platelets would prompt clotting as they circulate in the bloodstream and T cells would permanently fasten onto cells rather than scan the body for trouble. In 1989, Springer showed that LFA-1 tightens its grip only when the T cell detects another cell that bears foreign proteins and, in response, triggers intracellular events that tune LFA-1’s binding properties. This system allows T cells to circulate until they encounter evidence of infection or other disturbances, at which point the T cell hugs its target.

Burgeoning insights into integrin contributions to health and disease have spawned novel therapeutic strategies for several illnesses. For instance, the eyedrop lifitegrast (Xiida ®) soothes dry eye disease by obstructing LFA-1, one of the integrins that Springer discovered; consequently, white blood cell activity falls, which reduces ocular inflammation. The blockbuster drug vedolizumab (Entyvio ®) also had its origins in Springer’s work, although less directly. This humanized monoclonal antibody selectively thwarts an integrin that promotes immune-cell migration into the gut, thereby quieting ulcerative colitis and Crohn’s disease, both of which result from inflammation of the digestive tract. The agent, which was approved by the FDA for both conditions in 2014, binds an integrin that belongs to the immunoglobulin-like subfamily that Springer first defined. Other compounds, such as tirofiban (Aggrastat ®), aim at cell-ECM rather than cell-cell interactions. This medication inhibits gpIIb/gpIIIa and is used to hamper clotting in certain cardiovascular conditions.

By cracking open the study of integrin-based cell adhesion, Hynes, Ruoslahti, and Springer launched a field that touches countless processes whose mechanisms depend on interchanges among cells and the ECM (Figure 2). Their breakthroughs have illuminated the influence of integrins, which extends throughout the body and the animal world.

by Evelyn Strauss

Selected Publications – Discovery of Integrins

Cell-Matrix Interactions

Ruoslahti, E., Vaheri. A., Kuusela, P., and Linder, E. (1973). Fibroblast surface antigen: a new serum protein. Biochim. Biophys. Acta. 322, 352-358.

Hynes, R.O. (1973). Alteration of cell-surface proteins by viral transformation and by proteolysis. Proc. Natl. Acad. Sci. USA. 70, 3170-3174.

Pierschbacher, M.D., and Ruoslahti, E. (1984). Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule. Nature. 309, 30-33.

Pytela, R., Pierschbacher, M.D., and Ruoslahti, E. (1985). A 125/115-kDa cell surface receptor specific for vitronectin interacts with the arginine-glycine-aspartic acid adhesion sequence derived from fibronectin. Proc. Natl. Acad. Sci. USA. 82, 5766-5770.

Pytela, R., Pierschbacher, M.D., Ginsberg, M.H., Plow, E.F., and Ruoslahti, E. (1986). Platelet membrane glycoprotein IIb/IIIa: member of a family of Arg-Gly-Asp-specific adhesion receptors. Science. 231, 1559-1562.

Tamkun, J.W., DeSimone, D.W., Fonda, D., Patel, R.S., Buck, C., Horwitz, A.F., and Hynes, R.O. (1986). Structure of integrin, a glycoprotein involved in the transmembrane linkage between fibronectin and actin. Cell. 46, 271-282.

Hynes, R.O. (1987). Integrins: a family of cell surface receptors. Cell. 48, 549-554.

Ruoslahti, E. (1996). RGD and other recognition sequences for integrins. Annu. Rev. Cell Dev. Biol. 12, 697-715.

Hynes, R.O. (2002). Integrins: bidirectional allosteric signaling machines. Cell. 110, 673-687.

Cell-Cell Interactions

Kurzinger, K., Reynolds, T., Germain, R.N., Davignon, D., Martz, E., and Springer, T.A. (1981). A novel lymphocyte function-associated antigen (LFA-1): cellular distribution, quantitative expression, and structure. J. Immunol. 127, 596-600.

Sanchez-Madrid, F., Nagy, J., Robbins, E., Simon, P., and Springer, T.A. (1983). A human leukocyte differentiation antigen family with distinct alpha subunits and a common beta subunit: the lymphocyte function-associated antigen (LFA-1), the C3bi complement receptor (OKM1/Mac-1), and the p150,95 molecule. J. Exp. Med. 158, 1785-1789.

Thompson, W.S., Miller, L.J., Schmalstieg, F.C., Anderson, D.C., and Springer, T.A. (1984). Inherited deficiency of the Mac-1, LFA-1, p150,95 glycoprotein family and its molecular basis. J. Exp. Med. 160, 1901-1905.

Kishimoto, T.K., Lee, A., Roberts, T.M., and Springer, T.A. (1987). Cloning of the beta subunit of the leukocyte adhesion proteins: homology to an extra-cellular matrix receptor defines a novel supergene family. Cell. 48, 681-690.

Makgoba. M.W., Sanders, M.E., Luce, G.E.G., Dustin, M.L., Springer, T.A., Clark, E.A., Mannoni, P., and Shaw, S. (1988). ICAM-1 a ligand for LFA-1-dependent adhesion of B, T and myeloid cells. Nature. 331, 86-88.

Staunton, D.E., Dustin, M.L., and Springer, T.A. (1989). Functional cloning of ICAM-2, a cell adhesion ligand for LFA-1 homologous to ICAM-1. Nature. 339, 61-65.

Springer, T.A. (1990). Adhesion receptors of the immune system. Nature. 346, 425-434.

Lawrence, M.B., and Springer, T.A. (1991). Leukocytes roll on a selectin at physiologic flow rates: distinction from and prerequisite for adhesion through integrins. Cell. 65, 859-873.

Luo, B.-H., Carman, C.V., and Springer, T.A. (2007) Structural basis of integrin regulation and signaling. Annu. Rev. Immunol. 25, 619-647.

Li, J., Yan, J., and Springer, T.A. (2021). Low-affinity integrin states have faster ligand-binding kinetics than the high-affinity state. eLife. 10, 1-22. e73359. doi: 10.7554/eLife.73359.