Reciprocal signaling by integrin and nonintegrin receptors during collagen activation of platelets

Activation of platelets by exposed collagen after vessel wall injury is a primary event in the pathogenesis of stroke and myocardial infarction. Two collagen receptors, integrin alpha2beta1 and glycoprotein VI (GPVI), are expressed at similar levels on human and mouse platelets, but their individual roles during collagen activation remain poorly defined. Recent genetic and pharmacologic experiments have revealed an essential role for GPVI but have failed to define the role of alpha2beta1 or explain how two structurally distinct collagen receptors might function together to mediate platelet collagen responses. Discriminating the roles of these two collagen receptors is complicated by evidence suggesting that GPVI and platelet integrins may activate a common intracellular signaling pathway. To determine how alpha2beta1 and GPVI activate platelets in response to collagen, we have (i) examined collagen signaling conferred by expression of these receptors in hematopoietic cell lines; (ii) determined the effect of blocking each receptor on the activation of human platelets by collagen; (iii) generated low-GPVI mice in which the alpha2beta1/GPVI receptor ratio has been altered from 1:1 to 50:1 to expose alpha2beta1 function; (iv) studied the collagen responses of mouse platelets lacking LAT, an adaptor protein critical for GPVI but not integrin signaling; and (v) addressed the mechanism by which soluble collagens activate wild-type platelets. These studies demonstrate that alpha2beta1 requires inside-out signals to participate in collagen signaling and that alpha2beta1 is required for collagen activation of platelets when GPVI signals are reduced by blocking anti-GPVI antibody, low receptor number, specific disruption of the GPVI signaling pathway, or forms of collagen that bind weakly to GPVI relative to alpha2beta1. We propose a reciprocal two-receptor model of collagen signaling in platelets in which the nonintegrin receptor GPVI provides the primary collagen signal that activates and recruits the integrin receptor alpha2beta1 to further amplify collagen signals and fully activate platelets through a common intracellular signaling pathway. This model explains many of the genetic and pharmacologic observations regarding collagen signaling in platelets and demonstrates a novel mechanism by which hematopoietic cells integrate signaling by structurally distinct receptors that share a common ligand.     

Release of integrin macroaggregates as a mechanism of rear detachment during keratinocyte migration

Cell-substrate adhesion can be mediated by the relatively short-lived focal complexes and focal adhesions and by the more stable hemidesmosomes. During cell migration both types of cell-substrate adhesions must be disrupted allowing the cell rear to detach. Rear detachment has been described to be accompanied by membrane ripping and the loss of cellular material in a variety of cell types including fibroblasts and chondrocytes, but also in fast moving cells such as keratinocytes. Here we show that migrating keratinocytes leave behind "migration tracks" of cellular remnants that can be classified due to their size, distribution and molecular composition. Type I macroaggregates appeared as spherical and tubular structures with a diameter of about 50-100 nm that were arranged like "pearls on a string". These structures apparently derived from fragmentation of long tubular extensions, the retracting fibers, at the cell rear and contained high amounts of beta1 integrin and different alpha integrins that are components of fibronectin and laminin receptors in migrating keratinocytes usually found in focal adhesions. Type II macroaggregates were recognized as spherical structures with a diameter of about 30 - 50 nm that were arranged in clusters scattered over the gaps between type I, macroaggregates. In contrast to type I type II macroaggregates contained high amounts of beta4 integrin and seemed to derive from former hemidesmosomes. Both types of macroaggregates were completely membrane covered, impermeable compartments devoid of cytosolic proteins. Our observations strongly support the concept that the release of macroaggregates represents a distinct cellular mechanism of rear detachment based on the loss of adhesive receptors embedded in membrane-covered cellular remnants.     

Altered velocity processing in schizophrenia during pursuit eye tracking

Smooth pursuit eye movements (SPEM) are needed to keep the retinal image of slowly moving objects within the fovea. Depending on the task, about 50%-80% of patients with schizophrenia have difficulties in maintaining SPEM. We designed a study that comprised different target velocities as well as testing for internal (extraretinal) guidance of SPEM in the absence of a visual target. We applied event-related fMRI by presenting four velocities (5, 10, 15, 20°/s) both with and without intervals of target blanking. 17 patients and 16 healthy participants were included. Eye movements were registered during scanning sessions. Statistical analysis included mixed ANOVAs and regression analyses of the target velocity on the Blood Oxygen Level Dependency (BOLD) signal. The main effect group and the interaction of velocity×group revealed reduced activation in V5 and putamen but increased activation of cerebellar regions in patients. Regression analysis showed that activation in supplementary eye field, putamen, and cerebellum was not correlated to target velocity in patients in contrast to controls. Furthermore, activation in V5 and in intraparietal sulcus (putative LIP) bilaterally was less strongly correlated to target velocity in patients than controls. Altered correlation of target velocity and neural activation in the cortical network supporting SPEM (V5, SEF, LIP, putamen) implies impaired transformation of the visual motion signal into an adequate motor command in patients. Cerebellar regions seem to be involved in compensatory mechanisms although cerebellar activity in patients was not related to target velocity.     

Interaction of matrix with integrin receptors is required for optimal LPS-induced MAP kinase activation

Exposure of macrophages to endotoxin [lipopolysaccharide (LPS)] results in a cascade of events resulting in the release of multiple inflammatory and anti-inflammatory mediators. The Toll-like receptor (TLR) 4 complex is the major receptor that mediates LPS signaling. However, there is evidence that other surface molecules may play a complementary role in the TLR-induced events. Integrin receptors are one class of receptors that have been linked to LPS signaling. This study investigates the role of macrophage integrin receptors in the activation of mitogen-activated protein (MAP) kinases by LPS. In conditions where macrophages were not permitted to adhere to matrix or a tissue culture surface, we found a decrease in LPS signaling as documented by a marked reduction in tyrosine phosphorylation of whole cell proteins. This was accompanied by a significant decrease in extracellular signal-regulated kinase and c-Jun NH(2)-terminal kinase MAP kinase activation. Inhibition of integrin signaling, with EDTA or RGD peptides, decreased LPS-induced MAP kinase activity. The functional consequence of blocking integrin signaling was demonstrated by decreased LPS-induced tumor necrosis factor-alpha production. These observations demonstrate that, in addition to the TLR receptor complex, optimal LPS signaling requires complementary signals from integrin receptors.     

Talin与整合蛋白的激活

整合蛋白的激活是通过细胞内信号来调控的,通过对整合蛋白胞质结构域的作用,诱导整合蛋白胞外结构域构象产生变化,从而导致整合蛋白对配体亲和力的增加。近来的研究结果表明talin与整合蛋白β尾部的结合是整合蛋白激活通路中最重要的环节。     

整合素的活化调控

整合素家族是介导细胞与细胞外基质作用的最主要分子,不仅可以识别细胞外环境将信号传到细胞内,还可以通过来自细胞内的信号调节整合素和配体的亲和力,这个过程也就是整合素的活化。本文主要阐述了整合素的活化在生理过程中的重要作用、整合素活性调节的结构基础以及细胞内的信号通路和结合蛋白对整合素活性的影响。     

Signal transduction by integrin receptors for extracellular matrix: cooperative processing of extracellular information.

Adhesion receptors allow cells to interact with a dynamic and information-rich environment of extracellular matrix molecules. The integrin family of adhesion receptors transduces signals from the extracellular matrix that regulate growth, gene expression and differentiation, as well as cell shape, motility and cytoskeletal architecture. Recent data support the hypothesis that integrins transduce signals cooperatively with other classes of adhesion receptors or with growth factor receptors. Furthermore, the ability of integrins to interact with the cytoskeleton appears to be fundamental to their mechanism for signal transduction.     

The tail of integrin activation

Integrins are essential adhesion receptors found on the surfaces of all metazoan cells. As regulators of cell migration and extracellular matrix assembly, these membrane-spanning heterodimers are critical for embryonic development, tissue repair and immune responses. Signals transmitted by integrins from outside to inside the cell promote cell survival and proliferation, but integrin affinity for extracellular ligands can also be controlled by intracellular cues. This bidirectional signaling is mediated by the short cytoplasmic tails of the two integrin subunits. Recent structural and functional studies of various integrin fragments and complexes between the cytoplasmic tails and intracellular proteins, such as talin, have provided new insight into the signaling processes centered around the tails, particularly inside-out integrin activation.     

Nck-interacting Ste20 kinase couples Eph receptors to c-Jun N-terminal kinase and integrin activation

The mammalian Ste20 kinase Nck-interacting kinase (NIK) specifically activates the c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase module. NIK also binds the SH3 domains of the SH2/SH3 adapter protein Nck. To determine whether Nck functions as an adapter to couple NIK to a receptor tyrosine kinase signaling pathway, we determined whether NIK is activated by Eph receptors (EphR). EphRs constitute the largest family of receptor tyrosine kinases (RTK), and members of this family play important roles in patterning of the nervous and vascular systems. In this report, we show that NIK kinase activity is specifically increased in cells stimulated by two EphRs, EphB1 and EphB2. EphB1 kinase activity and phosphorylation of a juxtamembrane tyrosine (Y594), conserved in all Eph receptors, are both critical for NIK activation by EphB1. Although pY594 in the EphB1R has previously been shown to bind the SH2 domain of Nck, we found that stimulation of EphB1 and EphB2 led predominantly to a complex between NIK/Nck, p62(dok), RasGAP, and an unidentified 145-kDa tyrosine-phosphorylated protein. Tyrosine-phosphorylated p62(dok) most probably binds directly to the SH2 domain of Nck and RasGAP and indirectly to NIK bound to the SH3 domain of Nck. We found that NIK activation is also critical for coupling EphB1R to biological responses that include the activation of integrins and JNK by EphB1. Taken together, these findings support a model in which the recruitment of the Ste20 kinase NIK to phosphotyrosine-containing proteins by Nck is an important proximal step in the signaling cascade downstream of EphRs.     

Altered phosphorylation and activation of pp60c-src during fibroblast mitosis

At least half the pp60c-src in NIH 3T3-derived c-src overexpresser cells in modified by novel threonine and, possibly, serine phosphorylation within its amino 16 kd region during mitosis. At the same time, the tryptic phosphopeptide containing Ser 17, the site of cyclic AMP-dependent phosphorylation, is either modified or dephosphorylated. While the amount of pp60c-src is not significantly altered, the in vitro-specific kinase activity of modified pp60c-src is enhanced 4- to 7-fold. Modified pp60c-src has the same tyrosine-containing tryptic phosphopeptides as pp60c-src from unsynchronized cells, indicating that activation is independent of Tyr 416/Tyr 527 phosphorylation. Electrophoretic mobility retardations indicated that endogenous pp60c-src and pp60v-src are similarly modified during mitosis. The modifications and enhanced activity disappear near the time of cell division. These results suggest that pp60c-src is regulated by and, in turn, may regulate mitosis-specific events in fibroblasts.     

Evidence that cadherins play a role in the downregulation of integrin expression that occurs during keratinocyte terminal differentiation

In epidermis the onset of terminal differentiation normally coincides with inhibition of integrin function and expression, thereby ensuring that differentiating cells are selectively expelled from the basal layer. However, when stratification of cultured human epidermal keratinocytes is prevented by reducing the calcium concentration of the medium to 0.1 mM, keratinocytes initiate terminal differentiation while still attached to the culture substrate. We have examined the mechanism by which differentiating keratinocytes adhere to extracellular matrix proteins in low calcium medium and the consequences of inducing stratification by raising the calcium ion concentration to 1.8 mM (Standard Medium). In low calcium medium keratinocytes co-expressed integrins and terminal differentiation markers such as involucrin and peanut lectin-binding glycoproteins: differentiating cells contained integrin mRNA, synthesized integrin proteins de novo and expressed functional mature integrins. There were no differences in integrin synthesis, maturation or break down in low calcium or standard medium, although the level of beta 1 integrins on the surface of proliferating cells was higher in standard medium. Within 6 h of transfer from low calcium to standard medium integrin mRNA was no longer detectable in terminally differentiating cells, integrins were being lost from the cell surface, and selective migration out of the basal layer had begun. Antibodies to P- and E-cadherin, which block calcium-induced stratification, prevented the selective loss of integrin mRNA and protein from terminally differentiating cells. This suggests that cadherins may play a role in the down-regulation of integrin expression that is associated with terminal differentiation.     

Structural basis of integrin transmembrane activation

Integrins are cell adhesion receptors that transmit bidirectional signals across plasma membrane and are crucial for many biological functions. Recent structural studies of integrin transmembrane (TM) and cytoplasmic domains have shed light on their conformational changes during integrin activation. A structure of the resting state was solved based on Rosetta computational modeling and experimental data using intact integrins on mammalian cell surface. In this structure, the α_IIb GXXXG motif and their β₃ counterparts of the TM domains associate with ridge‐in‐groove packing, and the α_IIb GFFKR motif and the β₃ Lys‐716 in the cytoplasmic segments play a critical role in the α/β association. Comparing this structure with the NMR structures of the monomeric α_IIb and β₃ (represented as active conformations), the α subunit helix remains similar after dissociation whereas β subunit helix is tilted by embedding additional 5–6 residues into the lipid bilayer. These conformational changes are critical for integrin activation and signaling across the plasma membrane. We thus propose a new model of integrin TM activation in which the recent NMR structure of the α_IIbβ₃ TM/cytoplasmic complex represents an intermediate or transient state, and the electrostatic interaction in the cytoplasmic region is important for priming the initial α/β association, but not absolutely necessary for the resting state. J. Cell. Biochem. 109: 447–452, 2010. © 2009 Wiley‐Liss, Inc.     

Threonine 788 in integrin subunit beta1 regulates integrin activation

In the present study, the functional role of suggested phosphorylation of the conserved threonines in the cytoplasmic domain of integrin subunit beta1 was investigated. Mutants mimicking phosphorylated and unphosphorylated forms of beta1 were expressed in beta1 deficient GD25 cells. T788 in beta1 was identified as a site with major influence on integrin function. The mutation to A788 strongly reduced beta1-dependent cell attachment and exposure of the extracellular 9EG7 epitope, whereas replacement of T789 with alanine did not interfere with the ligand-binding ability. Talin has been shown to mediate integrin activation, but the talin head domain bound equally well to the wild-type beta1 and the mutants indicating that the T788A mutation caused defect integrin activation by another mechanism. The phosphorylation-mimicking mutation T788D was fully active in promoting cell adhesion. GD25 cells expressing beta1T788D accumulated increased number of focal contacts and migrated slowly compared to GD25 beta1 wild-type. An analogous phenotype is seen when focal adhesion kinase activation is abrogated. However, neither the beta1T788D nor the beta1T788A mutation failed to induce tyrosine phosphorylation of focal adhesion kinase. The results suggest that phosphorylation of T788 in integrin beta1 promotes inside-out receptor activation, as well as focal contact accumulation.     

Mechanisms of integrin activation and trafficking

Integrin adhesion receptors are essential for the normal function of most multicellular organisms, and defective integrin activation or integrin signaling is associated with an array of pathological conditions. Integrins are regulated by conformational changes, clustering, and trafficking, and regulatory mechanisms differ strongly between individual integrins and between cell types. Whereas integrins in circulating blood cells are activated by an inside-out-induced conformational change that favors high-affinity ligand binding, β1-integrins in adherent cells can be activated by force or clustering. In addition, endocytosis and recycling play an important role in the regulation of integrin turnover and integrin redistribution in adherent cells, especially during dynamic processes such as cell migration and invasion. Integrin trafficking is strongly regulated by their cytoplasmic tails, and the mechanisms are now being identified.     

Bi-directional signal transduction by integrin receptors

The integrin family of cell surface glycoproteins functions primarily as receptors for extracellular matrix ligands. There are now many well characterized integrin-ligand interactions which are known to influence many aspects of cell behaviour including cell morphology, cell adhesion, cell migration as well as cellular proliferation and differentiation. However, in fulfilling these functions, integrins are not simple adhesion receptors that physically mediate connections across the plasma membrane. Rather, integrin function itself is highly regulated, largely through the formation of specific associations with both structural and regulatory components within cells. It is these intracellular interactions which allow integrin function to effect many biochemical signalling pathways and therefore to impinge upon complex cellular activities. Recently, much research has focused on elucidating the molecular mechanisms which control integrin function and the molecular processes which transduce integrin-mediated signalling events. In this review, we discuss progress in the field of integrin signal transduction including, where applicable, potential therapeutic applications arising from the research.     

Sequential activation of Notch family receptors during mouse spermatogenesis

The expression pattern of Notch family receptors during mouse spermatogenesis was examined by immunohistochemistry. The entire cytoplasm of spermatogonia, spermatocytes and spermatids showed staining with antibodies against extracellular domains of Notch1, 2 and 4. In contrast, the nuclei of spermatogonia showed staining with an antibody against the intracellular domain of Notch3, and the nuclei of spermatocytes and spermatids showed staining with antibodies against the intracellular domains of Notch1 and 4. During regeneration of spermatogonia in busulfan-treated mice, the nuclei of all proliferating cells showed staining for the intracellular domain of Notch3. Western blot analysis showed that the molecular weights of the intracellular domains of Notch1 and 3 localizing in the nuclear fraction were smaller than those in the cytoplasmic fraction. This was consistent with the theory that the intracellular domain of Notch was cleaved in the cytoplasm and translocated to the nucleus. These results suggest that different Notch signals are sequentially activated during mouse spermatogenesis and control the proliferation and differentiation of spermatogenic stem cells.