Tiina J. Hilden

Specific integrin α and β chain phosphorylations regulate LFA-1 activation through affinity-dependent and -independent mechanisms

Integrins are adhesion receptors that are crucial to the functions of multicellular organisms. Integrin-mediated adhesion is a complex process that involves both affinity regulation and cytoskeletal coupling, but the molecular mechanisms behind this process have remained incompletely understood. In this study, we report that the phosphorylation of each cytoplasmic domain of the leukocyte function-associated antigen-1 integrin mediates different modes of integrin activation. α Chain phosphorylation on Ser1140 is needed for conformational changes in the integrin after chemokine- or integrin ligand–induced activation or after activation induced by active Rap1 (Rap1V12). In contrast, the β chain Thr758 phosphorylation mediates selective binding to 14-3-3 proteins in response to inside-out activation through the T cell receptor, resulting in cytoskeletal rearrangements. Thus, site-specific phosphorylation of the integrin cytoplasmic domains is important for the dynamic regulation of these complex receptors in cells.

Threonine Phosphorylation Sites in the β2 and β7 Leukocyte Integrin Polypeptides

The cytoplasmic domains of integrins play a key role in a variety of integrin-mediated events including adhesion, migration, and signaling. The molecular mechanisms that enhance integrin function are still incompletely understood. Because protein kinases are known to be involved in the signaling and the activation of integrins, the role of phosphorylation has been studied by several groups. The β2 leukocyte integrin subunit has previously been shown to become phosphorylated in leukocytes on cytoplasmic serine and functionally important threonine residues. We have now mapped the phosphorylated threonine residues in activated T cells. After phorbol ester stimulation, all three threonine residues (758–760) of the threonine triplet became phosphorylated but only two at a time. CD3 stimulation leads to a strong threonine phosphorylation of the β2 integrin, but differed from phorbol ester activation in that phosphorylation occurred only on threonine 758. The other leukocyte-specific integrin, β7, has also been shown to need the cytoplasmic domain and leukocyte-specific signal transduction elements for integrin activation. Cell activation with phorbol ester, and interestingly, through the TCR-CD3 complex, caused β7 integrin binding to VCAM-1. Additionally, cell activation led to increased phosphorylation of the β7 subunit, and phosphoamino acid analysis revealed that threonine residues became phosphorylated after cell activation. Sequence analysis by manual radiosequencing by Edman degradation established that threonine phosphorylation occurred in the same threonine triplet as in β2 phosphorylation.

Lck tyrosine kinase is important for activation of the CD11a/CD18‐integrins in human T lymphocytes

CD11a/CD18 (β2)‐integrins are expressed on leukocytes and are involved in cell adhesion and signaling. Despite extensive studies the signaling pathways and molecular mechanisms involved inintegrin regulation in T cells remain not completely understood. We have now studied the involvement of the tyrosine kinase Lck in the regulation of CD11a/CD18 function in Jurkat T cells. Using theSrc‐family kinase inhibitor PP2, we found that CD3 ligation‐induced adhesion to ICAM‐1 was inhibited by PP2 at the same concentration required for complete inhibition of the MAP kinase pathway, implicating a role for Lck in integrin activation. We therefore used the Lck‐deficient Jurkat cell line JCaM1.6 to further examine the involvement of Lck in integrin regulation. Interestingly, JCaM1.6 cells showed dramatically reduced levels of both CD3‐ and phorbol ester‐induced adhesion to coated ICAM‐1 as compared to normal Jurkat cells. By using flow cytometry and cell surface labeling, it was found that the surface expression of the CD11a/CD18‐integrins was significantly lower in Lck‐deficient T cells as compared to normal Jurkat cells. CD18 was expressed as a mature and an immaturely glycosylated form in Jurkat T cell lines, and predominantly the immature form, not associated with CD11a, was found in Lck‐deficient cells. Retransfection of human Lck in JCaM1.6 cells restored adhesion. Thus, Lck is involved in regulating CD11a/CD18‐integrins in T cells.

Interfering with leukocyte integrin activation—a novel concept in the development of anti-inflammatory drugs

Inflammation is a crucial response against invading pathogens, in which immune cells, including neutrophils and T cells, are recruited into tissue from the bloodstream to help clear infection. However, a prevailing inflammatory response where the immune cells attack healthy tissue is associated with many diseases, including asthma, rheumatoid arthritis, atherosclerosis and multiple sclerosis. Integrins are key players in the recruitment of immune cells from the bloodstream into tissues, and are thus therapeutic targets for intervention with inflammatory responses. Thus far, mainly extracellularly acting therapeutics (monoclonal antibodies) have been developed against integrins, targeting ligand binding sites in these heterodimeric adhesion receptors. However, since these therapeutics nonselectively block all integrin functions, some side effects are expected and have been observed. Therefore, novel concepts need to be developed in the therapeutic targeting of integrins. Recently, major advances have been made in the understanding of integrin biology. Integrin structures have been solved by X‐ray crystallography, revealing unexpected data about the activation mechanism of integrins in cells. Additionally, several intracellular factors in the integrin activation process have been identified, providing potential specific targets for therapeutic intervention. Here, we present key events and players in leukocyte integrin activation, and discuss potential new drug targets in the prevention of inflammatory disease.

Leukocyte Adhesion’an Integrated Molecular Process at the Leukocyte Plasma Membrane

Leukocyte adhesion is of pivotal functional importance, because most leukocyte functions depend on cell–cell contact. It must be strictly controlled, both at the level of specificity and strength of interaction, and therefore several molecular systems are involved. The most important leukocyte adhesion molecules are the selectins, the leukocyte-specific β2-integrins and the intercellular adhesion molecules. The selectins induce an initial weak contact between cells, whereas firm adhesion is achieved through integrin–intercellular adhesion molecular binding. Although studies during the past twenty years have revealed several important features of leukocyte adhesion much is still poorly understood, and further work dealing with several aspects of adhesion is urgently needed. In this short essay, we review some recent developments in the field.

Leukocyte adhesion - a fundamental process in leukocyte physiology

Leukocyte adhesion is of pivotal functional importance. The adhesion involves several different adhesion molecules, the most important of which are the leukocyte beta 2-integrins (CD11/CD18), the intercellular adhesion molecules, and the selectins. We and others have extensively studied the specificity and binding sites in the integrins and the intercellular adhesion molecules for their receptors and ligands. The integrins have to become activated to exert their functions but the possible mechanisms of activation remain poorly understood. Importantly, a few novel intercellular adhesion molecules have been recently described, which seem to function only in specific tissues. Furthermore, it is becoming increasingly apparent that changes in integrins and intercellular adhesion molecules are associated with a number of acute and chronic diseases.