Johannes A. Eble

The C-type lectin receptors CLEC-2 and Dectin-1, but not DC-SIGN, signal via a novel YXXL-dependent signaling cascade.

The two lectin receptors, CLEC-2 and Dectin-1, have been shown to signal through a Syk-dependent pathway, despite the presence of only a single YXXL in their cytosolic tails. In this study, we show that stimulation of CLEC-2 in platelets and in two mutant cell lines is dependent on the YXXL motif and on proteins that participate in signaling by immunoreceptor tyrosine-based activation motif receptors, including Src, Syk, and Tec family kinases, and on phospholipase Cγ. Strikingly, mutation of either Src homology (SH) 2 domain of Syk blocks signaling by CLEC-2 despite the fact that it has only a single YXXL motif. Furthermore, signaling by CLEC-2 is only partially dependent on the BLNK/SLP-76 family of adapter proteins in contrast to that of immunoreceptor tyrosine-based activation motif receptors. The C-type lectin receptor, Dectin-1, which contains a YXXL motif preceded by the same four amino acids as for CLEC-2 (DEDG), signals like CLEC-2 and also requires the two SH2 domains of Syk and is only partially dependent on the BLNK/SLP-76 family of adapters. In marked contrast, the C-type lectin receptor, DC-SIGN, which has a distinct series of amino acids preceding a single YXXL, signals independent of this motif. A mutational analysis of the DEDG sequence of CLEC-2 revealed that the glycine residue directly upstream of the YXXL tyrosine is important for CLEC-2 signaling. These results demonstrate that CLEC-2 and Dectin-1 signal through a single YXXL motif that requires the tandem SH2 domains of Syk but is only partially dependent on the SLP-76/BLNK family of adapters.

Migration of human melanoma cells depends on extracellular pH and Na+/H+ exchange.

Their glycolytic metabolism imposes an increased acid load upon tumour cells. The surplus protons are extruded by the Na+/H+ exchanger (NHE) which causes an extracellular acidification. It is not yet known by what mechanism extracellular pH (pHe) and NHE activity affect tumour cell migration and thus metastasis. We studied the impact of pHe and NHE activity on the motility of human melanoma (MV3) cells. Cells were seeded on/in collagen I matrices. Migration was monitored employing time lapse video microscopy and then quantified as the movement of the cell centre. Intracellular pH (pHi) was measured fluorometrically. Cell–matrix interactions were tested in cell adhesion assays and by the displacement of microbeads inside a collagen matrix. Migration depended on the integrin α2β1. Cells reached their maximum motility at pHe∼7.0. They hardly migrated at pHe 6.6 or 7.5, when NHE was inhibited, or when NHE activity was stimulated by loading cells with propionic acid. These procedures also caused characteristic changes in cell morphology and pHi. The changes in pHi, however, did not account for the changes in morphology and migratory behaviour. Migration and morphology more likely correlate with the strength of cell–matrix interactions. Adhesion was the strongest at pHe 6.6. It weakened at basic pHe, upon NHE inhibition, or upon blockage of the integrin α2β1. We propose that pHe and NHE activity affect migration of human melanoma cells by modulating cell–matrix interactions. Migration is hindered when the interaction is too strong (acidic pHe) or too weak (alkaline pHe or NHE inhibition).

The structural bases of integrin-ligand interactions

Many extracellular matrix (ECM) proteins, particularly those in the vascular system, use their classical integrin-recognition motif Arg-Gly-Asp (RGD) to interact with integrins. The RGD motif is generally located in flexible peptide loops whose variable conformation enables the relatively few integrins with broad specificity, such as alpha(v)beta(3) and alpha(IIb)beta(3), to bind to a large variety of different ECM proteins. However, certain ECM constituents, such as collagens and laminins, interact with integrins in a conformation-dependent manner, in which both the linear structure and spatial arrangement of the polypeptides are important for the formation of active binding sites. These interactions provide high specificity for the communication of cells with distinct members of the ECM.

CLEC-2 is a phagocytic activation receptor expressed on murine peripheral blood neutrophils

CLEC-2 is a member of the “dectin-1 cluster” of C-type lectin-like receptors and was originally thought to be restricted to platelets. In this study, we demonstrate that murine CLEC-2 is also expressed by peripheral blood neutrophils, but only weakly by bone marrow or elicited inflammatory neutrophils. On circulating neutrophils, CLEC-2 can mediate phagocytosis of Ab-coated beads and the production of proinflammatory cytokines, including TNF-α, in response to the CLEC-2 ligand, rhodocytin. CLEC-2 possesses a tyrosine-based cytoplasmic motif similar to that of dectin-1, and we show using chimeric analyses that the activities of this receptor are dependent on this tyrosine. Like dectin-1, CLEC-2 can recruit the signaling kinase Syk in myeloid cells, however, stimulation of this pathway does not induce the respiratory burst. These data therefore demonstrate that CLEC-2 expression is not restricted to platelets and that it functions as an activation receptor on neutrophils.

α2β1 Integrin Is Not Recognized by Rhodocytin but Is the Specific, High Affinity Target of Rhodocetin, an RGD-independent Disintegrin and Potent Inhibitor of Cell Adhesion to Collagen

We have recombinantly expressed a soluble form of human α2β1 integrin that lacks the membrane-anchoring transmembrane domains as well as the cytoplasmic tails of both integrin subunits. This soluble α2β1 integrin binds to its collagen ligands the same way as the wild-type α2β1integrin. Furthermore, like the wild-type form, it can be activated by manganese ions and an integrin-activating antibody. However, it does not bind to rhodocytin, a postulated agonist of α2β1 integrin from the snake venom ofCalloselasma rhodostoma, which elicits platelet aggregation. Taking advantage of the recombinantly expressed, soluble α2β1 integrin, an inhibition assay was established in which samples can be tested for their capability to inhibit binding of soluble α2β1 integrin to immobilized collagen. Thus, by scrutinizing the C. rhodostoma snake venom in this protein-protein interaction assay, we found a component of the snake venom that inhibits the interaction of soluble α2β1 integrin to type I collagen efficiently. N-terminal sequences identified this inhibitor as rhodocetin, a recently published antagonist of collagen-induced platelet aggregation. We could demonstrate that its inhibitory effect bases on its strong and specific binding to α2β1 integrin, proving that rhodocetin is a disintegrin. Standing apart from the growing group of RGD-dependent snake venom disintegrins, rhodocetin interacts with α2β1 integrin in an RGD-independent manner. Furthermore, its native conformation, which is stabilized by disulfide bridges, is indispensibly required for its inhibitory activity. Rhodocetin does not contain any major collagenous structure despite its high affinity to α2β1integrin, which binds to collagenous molecules much more avidly than to noncollagenous ligands, such as laminin. Blocking α2β1 integrin as the major collagen receptor on platelets, rhodocetin is responsible for hampering collagen-induced, α2β1 integrin-mediated platelet activation, leading to hemorrhages and bleeding disorders of the snakebite victim. Moreover, having a widespread tissue distribution, α2β1 integrin also mediates cell adhesion, spreading, and migration. We showed that rhodocetin is able to inhibit α2β1 integrin-mediated adhesion of fibrosarcoma cells to type I collagen completely.

CLEC-2 activates Syk through dimerization.

The C-type lectin receptor CLEC-2 activates platelets through Src and Syk tyrosine kinases, leading to tyrosine phosphorylation of downstream adapter proteins and effector enzymes, including phospholipase-C gamma2. Signaling is initiated through phosphorylation of a single conserved tyrosine located in a YxxL sequence in the CLEC-2 cytosolic tail. The signaling pathway used by CLEC-2 shares many similarities with that used by receptors that have 1 or more copies of an immunoreceptor tyrosine-based activation motif, defined by the sequence Yxx(L/I)x(6-12)Yxx(L/I), in their cytosolic tails or associated receptor chains. Phosphorylation of the conserved immunoreceptor tyrosine-based activation motif tyrosines promotes Syk binding and activation through binding of the Syk tandem SH2 domains. In this report, we present evidence using peptide pull-down studies, surface plasmon resonance, quantitative Western blotting, tryptophan fluorescence measurements, and competition experiments that Syk activation by CLEC-2 is mediated by the cross-linking through the tandem SH2 domains with a stoichiometry of 2:1. In support of this model, cross-linking and electron microscopy demonstrate that CLEC-2 is present as a dimer in resting platelets and converted to larger complexes on activation. This is a unique mode of activation of Syk by a single YxxL-containing receptor.

Integrin-ligand interaction

The fate of cells is largely controled by the extracellular matrix. Cells recognize matrix constituents via integrins and they and their respective matrix ligands, as well as the subsequent signal transduction pathways and their influence on cell adhesion, migration and morphology, are the main subjects of this book. It emphasizes the importance of the three-dimensional structure and the conformation of the intrgrin recognition sites within the ligands.

Decorin Regulates Endothelial Cell Motility on Collagen I through Activation of Insulin-like Growth Factor I Receptor and Modulation of α2β1 Integrin Activity

The proteoglycan decorin is expressed by sprouting but not quiescent endothelial cells, and angiogenesis is dysregulated in its absence. Previously, we have shown that decorin core protein can bind to and activate insulin-like growth factor-I receptor (IGF-IR) in endothelial cells. In this study, we show that decorin promotes α2β1 integrin-dependent endothelial cell adhesion and migration on fibrillar collagen type I. We provide evidence that decorin modulates cell-matrix interaction in this context by stimulating cytoskeletal and focal adhesion reorganization through activation of the IGF-IR and the small GTPase Rac. Further, the glycosaminoglycan moiety of decorin interacts with α2β1, but not α1β1 integrin, at a site distinct from the collagen I-binding A-domain, to allosterically modulate collagen I-binding activity of the integrin. We propose that induction of decorin expression in angiogenic, as opposed to quiescent, endothelial cells promotes a motile phenotype in an interstitial collagen I-rich environment by both signaling through IGF-IR and influencing α2β1 integrin activity.

Integrin α2β1 is the required receptor for endorepellin angiostatic activity

Endorepellin, the C-terminal module of perlecan, has angiostatic activity. Here we provide definitive genetic and biochemical evidence that the functional endorepellin receptor is the α2β1 integrin. Notably, the specific endorepellin binding to the receptor was cation-independent and was mediated by the α2I domain. We show that the anti-angiogenic effects of endorepellin cannot occur in the absence of α2β1. Microvascular endothelial cells from α2β1-/- mice, but not those isolated from either wild-type or α1β1-/- mice, did not respond to endorepellin. Moreover, syngeneic Lewis lung carcinoma xenografts in α2β1-/- mice failed to respond to systemic delivery of endorepellin. In contrast, endorepellin inhibited tumor growth and angiogenesis in the wild-type mice expressing integrin α2β1. We conclude that the angiostatic effects of endorepellin in vivo are mediated by a specific interaction of endorepellin with the α2β1 integrin receptor.

Integrins in Cancer Treatment

Anchorage-independent growth, anoikis resistance, and most steps of metastasis formation are integrin-mediated or -dependent processes, which are characteristics of malignant tumor cells. Acting as oncogenes or tumor suppressor genes, integrins may be involved in the oncogenic transformation of normal cells and their growth into a primary tumor node. During tumorigenesis, a switch of integrin expression can be observed, inasmuch as growth-promoting and growth-attenuating integrins are up- and down-regulated, respectively. ECM-ligand binding to an integrin initiates signals, which eradiating from the integrins are transmitted via different yet interconnecting pathways and elicit various cell functions, such as morphological changes, adhesion, migration and gene activation. Any of these functions takes part in the metastatic cascade of tumor progression, such as epithelial-to-mesenchymal transition of carcinoma cells, tumor cell contact with the basement membrane, invasion into neighboring tissues as well as production and activation of ECM-degrading MMPs. Besides their direct involvement in tumor progression as cell surface molecules on tumor cells, integrins in normal cells surrounding a tumor, e.g. endothelial cells, can also determine various cancer characteristics, such as tumor-induced neoangiogenesis and immune resistance. Hence, integrins are relevant pharmacological targets in tumor biology. Spurred by the recent success to generate pharmaceutical mimetics of RGD-dependent integrins and by the integrins easy accessibility on the cell surface, the hope is rising that also RGD-independent integrins, such as the collagen- and laminin-binding integrins, can be pharmacologically manipulated to fight integrin-dependent functions of cancer cells, which are necessary and at least partially specific for their proliferation and progression.