Effect of posttranslational processing on the in vitro and in vivo activity of chemokines

The CXC and CC chemokine gene clusters provide an abundant number of chemotactic factors selectively binding to shared G protein-coupled receptors (GPCR). Hence, chemokines function in a complex network to mediate migration of the various leukocyte subsets, expressing specific GPCRs during the immune response. Further fine-tuning of the chemokine system is reached through specific posttranslational modifications of the mature proteins. Indeed, enzymatic processing of chemokines during an early phase of inflammation leads to activation of precursor molecules or cleavage into even more active or receptor specific chemokine isoforms. At a further stage, proteolytic processing leads to loss of GPCR signaling, thereby providing natural chemokine receptor antagonists. Finally, further NH₂-terminal cleavage results in complete inactivation to dampen the inflammatory response. During inflammatory responses, the two chemokines which exist in a membrane-bound form may be released by proteases from the cellular surface. In addition to proteolytic processing, citrullination and glycosylation of chemokines is also important for their biological activity. In particular, citrullination of arginine residues seems to reduce the inflammatory activity of chemokines in vivo. This goes along with other positive and negative regulatory mechanisms for leukocyte migration, such as chemokine synergy and scavenging by decoy receptors.     

Chemokine arrest signals to leukocyte integrins trigger bi-directional-occupancy of individual heterodimers by extracellular and cytoplasmic ligands

Integrin heterodimers acquire high affinity to endothelial ligands by extensive conformational changes in both their α and β subunits. These heterodimers are maintained in an inactive state by inter-subunit constraints. Changes in the cytoplasmic interface of the integrin heterodimer (referred to as inside-out integrin activation) can only partially remove these constraints. Full integrin activation is achieved when both inter-subunit constraints and proper rearrangements of the integrin headpiece by its extracellular ligand (outside-in activation) are temporally coupled. A universal regulator of these integrin rearrangements is talin1, a key integrin-actin adaptor that regulates integrin conformation and anchors ligand-occupied integrins to the cortical cytoskeleton. The arrest of rolling leukocytes at target vascular sites depends on rapid activation of their α4 and β2 integrins at endothelial contacts by chemokines displayed on the endothelial surface. These chemotactic cytokines can signal within milliseconds through specialized Gi-protein coupled receptors (GPCRs) and Gi-triggered GTPases on the responding leukocytes. Some chemokine signals can alter integrin conformation by releasing constraints on integrin extension, while other chemokines activate integrins to undergo conformational activation mainly after ligand binding. Both of these modalities involve talin1 activation. In this opinion article, I propose that distinct chemokine signals induce variable strengths of associations between talin1 and different target integrins. Weak interactions of the integrin cytoplasmic tail with talin1 (the cytoplasmic integrin ligand) dissociate unless the extracellular ligand can simultaneously occupy the integrin headpiece and transmit, within milliseconds, proper allosteric changes across the integrin heterodimer back to the tail-talin1 complex. The fate of this bi-directional occupancy of integrins by both their extracellular and intracellular ligands is likely to benefit from immobilization of both ligands to cortical cytoskeletal elements. To properly anchor talin1 onto the integrin tail, a second integrin partner, Kindlin-3 may be also required, although an evidence that both partners can simultaneously bind the same integrin heterodimer is still missing. Once linked to the cortical actin cytoskeleton, the multi-occupied integrin complex can load weak forces, which deliver additional allosteric changes to the integrin headpiece resulting in further bond strengthening. Surface immobilized chemokines are superior to their soluble counterparts in driving this bi-directional occupancy process, presumably due to their ability to facilitate local co-occupancy of individual integrin heterodimers with talin1, Kindlin-3 and surface-bound extracellular ligands.Key words: adhesion, migration, endothelium, cytoskeleton, shear stress, immunityFirm adhesion of leukocytes to blood vessels is tightly regulated by integrins and their cognate ligands.^1,2 These include the α₄ integrins, VLA-4 (α₄β₁) and α₄β₇, and the β₂ integrins, LFA-1 (α_Lβ₂) and Mac-1 (α_Mβ₂). Accumulated data suggest that these counter-receptors are structurally adapted to operate under disruptive blood-derived shear forces.³ A remarkable feature of leukocyte integrins is that their affinity state and microclustering can be regulated within fractions of seconds.^4,5 The most robust signals for leukocyte integrins are transduced by chemoattractants, mostly chemokines displayed on the vessel wall.⁶ A growing body of evidence suggests that the Gi protein coupled receptors of these endothelial chemokines elicit diverse signaling pathways in distinct leukocyte subtypes,^2,22 which use two common downstream elements to coactive all leukocyte integrins: talin1 and Kindlin-3.⁷ In this review, I will describe a model explaining how chemokine signals to these elements regulate the conformation of all leukocyte integrins by facilitating a coupled bi-directional occupancy and activation via both their cytoplasmic and headpiece domains.Recent structural and biophysical studies suggest that leukocyte integrins can alternate between inactive bent conformers, which are clasped heterodimers, and variable unclasped heterodimers with extended ectodomains exhibiting intermediate and high affinity to ligand.⁵ Most leukocyte integrins are maintained in an inactive resting state,² whereas in situ chemokine-stimulated integrins unfold and extend 10–25 nm above the cell surface, allowing their headpiece to readily recognize immobilized ligand on a counter surface.⁸ These extended integrins must undergo extensive rearrangements of their headpiece I-domains induced by external endothelial-displayed ligands in order to arrest rolling leukocytes on blood vessel walls. In leukocytes, these two canonical switches are very short-lived, implying the necessity for a stabilization. It is therefore likely that any type of robust integrin activation must involve bi-directional occupancy of the integrin by both its extracellular ligand and one or more cytoplasmic partners.⁹The main cytoplasmic integrin-activating adaptor in leukocytes and platelets is talin1.^10,11 Talin knock down in multiple cell types results in nearly total loss of integrin activation.^12,13 This actin binding adaptor binds different integrin β subunit tails with low affinity,¹⁴ which can be locally increased by in situ generated PI(4,5)P2 (PIP2). This phosphoinositide presumably binds to the FERM domain within the talin head and thereby enhances talin binding to a membrane proximal NPXY motif on the β integrin tail, a key event in integrin heterodimer unclasping.^15,16 Recent studies suggest, however, that mere talin association may be insufficient to unclasp and activate the integrin heterodimer. Thus, the beta subunit tail may need to get co-occupied by the integrin co-activator, Kindlin, in order to optimize talin association with this integrin subunit.^17,18 In leukocytes, Talin1 and the Kindlin family member, Kindlin 3 co-activate both VLA-4 and LFA-1 and this co-activation is dramatically enhanced by multiple chemokine triggered effectors, the nature of which has begun to unfold¹⁹ (Fig. 1). I would like to propose that talin1-Kindlin-3 co-binding to the β tails of these and other leukocyte integrins is insufficient to switch these integrins to a conformation able to bind their soluble extracellular ligands due to fast dissocia-tion of PIP2-activated talin1 from the integrin cytoplasmic tail complex. This short lived talin-integrin complex may, on the other hand, get stabilized, if the integrin headpiece can simultaneously bind an immobilized extracellular ligand and undergo immediate outside-in activation, before the talin1 has dissociated from the integrin beta tail (Fig. 1). Such full confor-mational switch can result in additional allosteric changes in the integrin-bound talin which may expose vinculin binding sites and further increase talin-actin associations that reinforce this bi-directional allosteric integrin activation.²⁰Open in a separate windowFigure 1Bi-directional integrin activation requires simultaneous co-occupancy of the integrin heterodimer by extracellular and cytoplasmic ligands. A proposed scheme for chemokine-triggered integrin activation on leukocytes. Integrin conformation is allosterically modulated in a bidirectional manner by at least two sets of ligands, extracellular and cytoplasmic. The degree of activation is dictated primarily by unclasping of the integrin heterodimer, a process dependent on the binding of the activated talin FERM domain to a specific site on the integrin β tail. (1) Inactive integrin. (2–5) Four postulated integrin conformations triggered by distinct chemokine signals. (2) Talin FERM domain activation close to the target integrin is a rate limiting step in integrin activation. This activation is triggered by PIP2 locally generated by talin-associated PIP5Kγ (purple rectangle) stimulated by a nearby Gi-coupled chemokine receptor. (3) Kindlin-3 binding to the integrin β tail stabilizes the otherwise weak talin1-integrin tail complex. The activated integrin can bind a soluble extracellular ligand with a low affinity due to a high k_off of the soluble ligand from the integrin headpiece. (4) In the absence of Kindlin-3, chemokine triggered, PIP2-activated talin1 binds only transiently the integrin tail (High k_off). The semiactivated integrin, even if occupied by an immobilized extracellular ligand, cannot undergo full bi-directional activation. (5) When both the extracellular ligand and talin are properly anchored, their escape from the integrin is dramatically reduced, lowering the k_off. Low k_off increases the probability of simultaneous bi-directional occupancy of both the integrin headpiece by the extracellular ligand and of the integrin tail by talin1 and Kindlin-3. This results in optimal bi-directional integrin activation and unclasping of the heterodimer. Stable linkages also allow this bi-directionally occupied integrin to undergo extensive mechanical strengthening by low forces applied on the headpiece; this further activates the headpiece I domains, further separates the β and α subunits from each other, and maximally stabilizes the unclasped integrin. Force application through the high affinity-talin complex can stretch the talin rod domain and expose vinculin binding sites (VBS). Since integrin ligands are generally multivalent, rapid integrin dimerization can take place to further stabilize the focal adhesion (not shown). Additional cytoplasmic partners of specific leukocyte integrins like a-actinin, L-plastin and RAPL may further strengthen subsets of focal adhesions. These and other cytosolic partners bind different integrin targets with different affinities. Therefore the effect of each of these partners on both the kinetics and stability of the talin1-integrin tail complex may vary with the cell type, the integrin type, the strength of the chemokine signal and the proximity between the integrin and its stimulatory GPCR.How can such postulated simultaneous bi-directional occupancy of a leukocyte integrin can be so rapidly triggered by a chemokine signal encountered during leukocyte rolling on blood vessels? An attractive mechanism for in situ facilitation of talin1 binding to the integrin β tail by chemokine signals involves chemokine triggered Gi stimulated RhoA and Rac1 GTPases and their downstream target, the PIP2 generating enzyme PIP5Kγ in the vicinity of the in situ activated integrin¹⁹ (Fig. 1). Additional talin1 molecules may also be recruited to the vicinity of this initially stimulated integrin by RIAM,²¹ an effector that associates with activated Rap-1, one of the key chemokine stimulated GTPases involved in rapid integrin mediated activation in both leukocytes and platelets.^22,23 To bidirectionally bind and activate their integrin targets, both the cytoplasmic integrin ligands, Talin1 and Kindlin-3 and the extracellular integrin ligand may need to achieve low dissociation rates from the integrin tail and headpiece, respectively. Why would an immobilized extracellular ligand be superior to soluble extracellular ligand in capacity to bi-directionally bind and activate a leukocyte integrin? The probability that a given surface-bound ligand, rather than a soluble integrin ligand would escape from its cognate integrin receptor following its dissociation is very small, since reactants in viscous medium are more likely to recombine than to diffuse apart.²⁴ Thus, surface-immobilized single integrin ligands may rebind the integrins they recenty dissociated from much more frequently than their soluble counterparts. Similarly, the cytoplasmic ligands talin1 and Kindlin may need to remain immobile once occupying their target integrin tail. Such immobilization of talin1 can be optimized by talin anchoring to the cortical cytoskeleton.²⁵ Talin may be also restricted from immediate dissociation from the integrin tail by Kindlin-3. An optimal integrin activating chemokine signal would therefore not only need to recruit and induce talin1 association with the β subunit of the target integrin and opening of the integrin clasp, but also need to keep the talin in an immobile state, and thereby maintain its low dissociation rate from its integrin tail sites.As both the integrin headpiece and the integrin subunits are predicted to undergo faster opening and separation in the presence of applied forces,^26,27 another tradeoff of this postulated immobilization of both the intracellular and extracellular integrin ligands is optimal force sensing of the integrin heterodimer. Application of force on the bidirectionally occupied integrin and its cognate ligands would be possible only if the integrin, its extracellular ligand, and talin1 are all properly anchored.^3,28,29 Force transduction through the integrin-talin1 complex can transmit additional conformational changes on the integrin-occupied talin by exposing vinculin binding sites on the talin rod.³⁰ Additional chemokine signals may induce talin rod phosphorylation and other changes in actin-talin associations (Fig. 1) that may further facilitate talin anchorage to the cortical cytoskeleton and subsequent microclustering of adjacent ligand-occupied integrins. It is well recognized that ligand occupancy anchors integrins to the cortical cytoskeleton.³¹ Thus, the anchorage states of both the extracellular and the cytoplasmic ligands of a given integrin may facilitate bidirectional integrin occupancy and optimize force driven bi-directional activation of the integrin-ligand complex and subsequent dimerization of ligand-occupied integrins. The ability of different integrin-ligand complexes to undergo diverse mechanochemical rearrangements provides a broad spectrum of integrin-ligand bond strengths, accounting for the unique capacity of chemokine stimulated leukocyte integrins to support both firm and reversible adhesions of leukocytes to their endothelial ligands.     

Rac signaling in breast cancer: a tale of GEFs and GAPs

Rac GTPases, small G-proteins widely implicated in tumorigenesis and metastasis, transduce signals from tyrosine-kinase, G-protein-coupled receptors (GPCRs), and integrins, and control a number of essential cellular functions including motility, adhesion, and proliferation. Deregulation of Rac signaling in cancer is generally a consequence of enhanced upstream inputs from tyrosine-kinase receptors, PI3K or Guanine nucleotide Exchange Factors (GEFs), or reduced Rac inactivation by GTPase Activating Proteins (GAPs). In breast cancer cells Rac1 is a downstream effector of ErbB receptors and mediates migratory responses by ErbB1/EGFR ligands such as EGF or TGFα and ErbB3 ligands such as heregulins. Recent advances in the field led to the identification of the Rac-GEF P-Rex1 as an essential mediator of Rac1 responses in breast cancer cells. P-Rex1 is activated by the PI3K product PIP3 and Gβγ subunits, and integrates signals from ErbB receptors and GPCRs. Most notably, P-Rex1 is highly overexpressed in human luminal breast tumors, particularly those expressing ErbB2 and estrogen receptor (ER). The P-Rex1/Rac signaling pathway may represent an attractive target for breast cancer therapy.     

Leukocyte polarization in cell migration and immune interactions.

Cell migration plays a key role in a wide variety of biological phenomena. This process is particularly important for leukocyte function and the inflammatory response. Prior to migration leukocytes undergo polarization, with the formation of a lamellipodium at the leading edge and a uropod at the trailing edge. This cell shape allows them to convert cytoskeletal forces into net cell-body displacement. Leukocyte chemoattractants, including chemokines, provide directional cues for leukocyte motility, and concomitantly induce polarization. Chemoattractant receptors, integrins and other adhesion molecules, cytoskeletal proteins and intracellular regulatory molecules change their cellular localization during cell polarization. A complex system of signal transduction molecules, including tyrosine kinases, lipid kinases, second messengers and members of the Rho family of small GTPases is thought to regulate the cytoskeletal rearrangements underlying leukocyte polarization and migration. The elucidation of the mechanisms and signals that control this complex reorganization will lead to a better understanding of critical questions in cell biology of leukocyte migration and polarity.     

Cells on the run: shear-regulated integrin activation in leukocyte rolling and arrest on endothelial cells

The arrest of rolling leukocytes on various target vascular beds is mediated by specialized leukocyte integrins and their endothelial immunoglobulin superfamily (IgSF) ligands. These integrins are kept in largely inactive states and undergo in situ activation upon leukocyte-endothelial contact by both biochemical and mechanical signals from flow-derived shear forces. In vivo and in vitro studies suggest that leukocyte integrin activation involves conformational alterations through inside-out signaling followed by ligand-induced rearrangements accelerated by external forces. This activation process takes place within fractions of seconds by in situ signals transduced to the rolling leukocyte as it encounters specialized endothelial-displayed chemoattractants, collectively termed arrest chemokines. In neutrophils, selectin rolling engagements trigger intermediate affinity integrins to support reversible adhesions before chemokine-triggered arrest. Different leukocyte subsets appear to use different modalities of integrin activation during rolling and arrest at distinct endothelial sites.     

Chemokines act as phosphatidylserine-bound “find-me” signals in apoptotic cell clearance

Removal of apoptotic cells is essential for maintenance of tissue homeostasis. Chemotactic cues termed “find-me” signals attract phagocytes toward apoptotic cells, which selectively expose the anionic phospholipid phosphatidylserine (PS) and other “eat-me” signals to distinguish healthy from apoptotic cells for phagocytosis. Blebs released by apoptotic cells can deliver find-me signals; however, the mechanism is poorly understood. Here, we demonstrate that apoptotic blebs generated in vivo from mouse thymus attract phagocytes using endogenous chemokines bound to the bleb surface. We show that chemokine binding to apoptotic cells is mediated by PS and that high affinity binding of PS and other anionic phospholipids is a general property of many but not all chemokines. Chemokines are positively charged proteins that also bind to anionic glycosaminoglycans (GAGs) on cell surfaces for presentation to leukocyte G protein–coupled receptors (GPCRs). We found that apoptotic cells down-regulate GAGs as they up-regulate PS on the cell surface and that PS-bound chemokines, unlike GAG-bound chemokines, are able to directly activate chemokine receptors. Thus, we conclude that PS-bound chemokines may serve as find-me signals on apoptotic vesicles acting at cognate chemokine receptors on leukocytes.

Chemokines attract leukocytes by activating chemokine receptors, but many also bind anionic phospholipids. This study shows that phosphatidylserine-binding chemokines endow extracellular apoptotic bodies with “find-me” signals that trigger phagocyte migration for potential apoptotic cell clearance.     

Shaping the gradient by nonchemotactic chemokine receptors

Chemokines are a class of inflammatory mediators which main function is to direct leukocyte migration through the binding to G protein-coupled receptors (GPCRs). In addition to these functional, signal-transducing chemokine receptors other types of receptors belonging to the chemokine GPCR family were identified. They are called atypical or decoy chemokine receptors because they bind and degrade chemokines but do not transduce signals or activate cell migration. Here there is the summary of two recent papers that identified other nonchemotactic chemokine receptors: the Duffy antigen receptor for chemokines (DARC) that mediates trancytosis of chemokines from tissue to vascular lumen promoting chemokine-mediated leukocyte transmigration and chemokine (CC motif) receptor-like 2 (CCRL2) that neither internalizes its ligands nor transduces signals but presents bound ligands to functional signaling receptors improving their activity. Collectively these nonchemotactic chemokine receptors do not directly induce cell migration, but appear nonetheless to play a nonredundant role in leukocyte recruitment by shaping the chemoattractant gradient, either by removing, transporting or concentrating their cognate ligands.Key words: Chemokine, chemokine receptor, leukocyte recruitment, chemotaxis, transcytosis     

Chemokine stimulation of lymphocyte alpha 4 integrin avidity but not of leukocyte function-associated antigen-1 avidity to endothelial ligands under shear flow requires cholesterol membrane rafts

VLA-4 and LFA-1 are the major vascular integrins expressed on circulating lymphocytes. Previous studies suggested that intact cholesterol rafts are required for integrin adhesiveness in different leukocytes. We found the alpha(4) integrins VLA-4 and alpha(4)beta(7) as well as the LFA-1 integrin to be excluded from rafts of human peripheral blood lymphocytes. Disruption of cholesterol rafts with the chelator methyl-beta-cyclodextrin did not affect the ability of these lymphocyte integrins to generate high avidity to their respective endothelial ligands and to promote lymphocyte rolling and arrest on inflamed endothelium under shear flow. In contrast, cholesterol extraction abrogated rapid chemokine triggering of alpha(4)-integrin-dependent peripheral blood lymphocytes adhesion, a process tightly regulated by G(i)-protein activation of G protein-coupled chemokine receptors (GPCR). Strikingly, stimulation of LFA-1 avidity to intercellular adhesion molecule 1 (ICAM-1) by the same chemokines, although G(i)-dependent, was insensitive to raft disruption. Our results suggest that alpha(4) but not LFA-1 integrin avidity stimulation by chemokines involves rapid chemokine-induced GPCR rearrangement that takes place at cholesterol raft platforms upstream to G(i) signaling. Our results provide the first evidence that a particular chemokine/GPCR pair can activate different integrins on the same cell using distinct G(i) protein-associated machineries segregated within defined membrane compartments.     

Cell-surface enzymes in control of leukocyte trafficking

Leukocyte trafficking between the blood and the tissues is pivotal for normal immune responses. Cell-adhesion molecules (such as selectins and leukocyte integrins) and chemoattractants (such as chemokines) have well-established roles in supporting leukocyte exit from the blood. Emerging data now show that, for both leukocytes and endothelial cells, enzymatic reactions that are catalysed by cell-surface-expressed enzymes with catalytic domains outside the plasma membrane (known as ectoenzymes) also make crucial contributions to this process. Ectoenzymes can function physically as adhesion receptors and can regulate the recruitment of cells through their catalytic activities. Here, we provide new insights into how ectoenzymes--including nucleotidases, cyclases, ADP-ribosyltransferases, peptidases, proteases and oxidases--guide leukocyte traffic.     

The blood-brain barrier, chemokines and multiple sclerosis

The infiltration of leukocytes into the central nervous system (CNS) is an essential step in the neuropathogenesis of multiple sclerosis (MS). Leukocyte extravasation from the bloodstream is a multistep process that depends on several factors including fluid dynamics within the vasculature and molecular interactions between circulating leukocytes and the vascular endothelium. An important step in this cascade is the presence of chemokines on the vascular endothelial cell surface. Chemokines displayed along the endothelial lumen bind chemokine receptors on circulating leukocytes, initiating intracellular signaling that culminates in integrin activation, leukocyte arrest, and extravasation. The presence of chemokines at the endothelial lumen can help guide the movement of leukocytes through peripheral tissues during normal immune surveillance, host defense or inflammation. The expression and display of homeostatic or inflammatory chemokines therefore critically determine which leukocyte subsets extravasate and enter the peripheral tissues. Within the CNS, however, infiltrating leukocytes that cross the endothelium face additional boundaries to parenchymal entry, including the abluminal presence of localizing cues that prevent egress from perivascular spaces. This review focuses on the differential display of chemokines along endothelial surfaces and how they impact leukocyte extravasation into parenchymal tissues, especially within the CNS. In particular, the display of chemokines by endothelial cells of the blood brain barrier may be altered during CNS autoimmune disease, promoting leukocyte entry into this immunologically distinct site. Recent advances in microscopic techniques, including two-photon and intravital imaging have provided new insights into the mechanisms of chemokine-mediated capture of leukocytes within the CNS.     

The small GTPase, Rap1, mediates CD31-induced integrin adhesion

Integrin-mediated leukocyte adhesion is a critical aspect of leukocyte function that is tightly regulated by diverse stimuli, including chemokines, antigen receptors, and adhesion receptors. How cellular signals from CD31 and other adhesion amplifiers are integrated with those from classical mitogenic stimuli to regulate leukocyte function remains poorly understood. Here, we show that the cytoplasmic tail of CD31, an important integrin adhesion amplifier, propagates signals that induce T cell adhesion via beta1 (VLA-4) and beta2 (LFA-1) integrins. We identify the small GTPase, Rap1, as a critical mediator of this effect. Importantly, CD31 selectively activated the small Ras-related GTPase, Rap1, but not Ras, R-Ras, or Rap2. An activated Rap1 mutant stimulated T lymphocyte adhesion to intercellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM), as did the Rap1 guanine nucleotide exchange factor C3G and a catalytically inactive mutant of RapGAP. Conversely, negative regulators of Rap1 signaling blocked CD31-dependent adhesion. These findings identify a novel important role for Rap1 in regulating ligand-induced cell adhesion and suggest that Rap1 may play a more general role in coordinating adhesion-dependent signals during leukocyte migration and extravasation. Our findings also suggest an alternative mechanism, distinct from interference with Ras-proximal signaling, by which Rap1 might mediate transformation reversion.     

Endothelin suppresses cell migration via the JNK signaling pathway in a manner dependent upon Src kinase,Rac1, and Cdc42

Cell migration is a complex phenomenon that is stimulated by chemoattractive factors such as chemokines, a family of ligands for G protein-coupled receptors (GPCRs). In contrast, factors that suppress cell migration, and the mechanism of their action, remain largely unknown. In this study, we show that endothelin, a GPCR ligand, inhibits cell motility in a manner dependent upon signaling through the c-Jun N-terminal kinase (JNK) pathway. We further demonstrate that this effect is dependent upon Src kinase and small GTPases Rac1 and Cdc42. These findings provide new insight into GPCR-mediated regulation of cell migration.     

Structural basis for the interaction between the cytoplasmic domain of the hyaluronate receptor layilin and the talin F3 subdomain

Talin is a large cytoskeletal protein that is involved in coupling the integrin family of cell adhesion molecules to the actin cytoskeleton, colocalising with the integrins in focal adhesions (FAs). However, at the leading edge of motile cells, talin colocalises with the hyaluronan receptor layilin in what are thought to be transient adhesions, some of which subsequently mature into more stable FAs. During this maturation process, layilin is replaced with integrins, which are highly clustered in FAs, where localised production of PI(4,5)P₂ by type 1 phosphatidyl inositol phosphate kinase type 1γ (PIPK1γ) is thought to play a role in FA assembly. The talin FERM F3 subdomain binds both the integrin β-subunit cytoplasmic domain and PIPK1γ, and these interactions are understood in detail at the atomic level. The talin F3 domain also binds to short sequences in the layilin cytoplasmic domain, and here we report the structure of the talin/layilin complex, which shows that talin binds integrins, PIPK1γ and layilin in similar although subtly different ways. Based on structure comparisons, we designed a set of talin F3 mutations that selectively affected the affinity of talin for its targets, as determined by stopped-flow fluorescence measurements. Such mutations will help to assess the importance of the interactions between talin and its various ligands in cell adhesion and migration.     

A ménage à trois made in heaven: G-protein-coupled receptors, lipids and TRP channels

Recent technical and conceptual advances in lipid analysis have given us a glimpse into the true versatility of the lipidome and the complexity of lipid signaling species. Progress alike in protein chemistry and genetics has presented us with new signal pathways and molecular mechanisms for the lipid actions. G-protein-coupled receptors (GPCR) appear to play a central role in the regulation of many lipid signals and are also themselves targets for some of these. TRP channels have recently been acknowledged as one of the most important GPCR effectors; in many cases the signals from GPCRs to TRPs are mediated via lipid signals. This review aims at presenting a view into the complex lipid signaling networks, their possible regulation by GPCRs and the signals transmitted to the TRP channels. Critical views and possible shortcomings in the composition of the studies are also presented.     

PECAM-1 isoform-specific activation of MAPK/ERKs and small GTPases: implications in inflammation and angiogenesis

Platelet-endothelial cell adhesion molecule-1 (PECAM-1/CD31) is expressed on the surface of endothelial cells (EC) and leukocytes. PECAM-1 plays an important role in endothelial-leukocyte and endothelial-endothelial cell-cell interactions. The anti-PECAM-1 antibody-mediated blockade of these interactions inhibits transendothelial migration (TEM) of leukocytes and angiogenesis. PECAM-1 may accommodate these processes through the regulation of cell adhesive and migratory mechanisms. How PECAM-1 regulates these dynamic processes remain unknown. Here we show that PECAM-1 transduces outside-in signals, which activate MAPK/ERKs and small GTPases. This occurs through PECAM-1-mediated formation of intracellular-signaling complexes, Shc/Grb2/SOS1 and/or Crkl/C3G, which is initiated by PECAM-1 engagement on the surface of leukocytes and/or EC. Src, SHP2, and alternative PECAM-1 pre-mRNA splicing play a regulatory role in these signaling events. Our findings reveal that PECAM-1 engagement on the cell surface can transduce "outside-in" signals and activate MAPK/ERKs and small GTPases, impacting both cadherin-mediated cell-cell and integrin-mediated cell-matrix interactions. Thus, we propose PECAM-1 is an important mediator of vascular barrier and regulator of leukocyte and EC adhesion and migration.     

Specific Phosphorylations Transmit Signals from Leukocyte β2 to β1 Integrins and Regulate Adhesion

The regulation of integrins expressed on leukocytes must be controlled precisely, and members of different integrin subfamilies have to act in concert to ensure the proper traffic of immune cells to sites of inflammation. The activation of β₂ family integrins through the T cell receptor or by chemokines leads to the inactivation of very late antigen 4. The mechanism(s) of this cross-talk has not been known. We have now elucidated in detail how the signals are transmitted from leukocyte function-associated antigen 1 and show that, after its activation, the signaling involves specific phosphorylations of β₂ integrin followed by interactions with cytoplasmic signaling proteins. This results in loss of β₁ phosphorylation and a decrease in very late antigen 4 binding to its ligand vascular cell adhesion molecule 1. Our results show how a member of one integrin family regulates the activity of another integrin. This is important for the understanding of integrin-mediated processes.     

GPR55 regulates cannabinoid 2 receptor-mediated responses in human neutrophils

The directional migration of neutrophils towards inflammatory mediators, such as chemokines and cannabinoids, occurs via the activation of seven transmembrane G protein coupled receptors (7TM/GPCRs) and is a highly organized process. A crucial role for controlling neutrophil migration has been ascribed to the cannabinoid CB(2) receptor (CB(2)R), but additional modulatory sites distinct from CB(2)R have recently been suggested to impact CB(2)R-mediated effector functions in neutrophils. Here, we provide evidence that the recently de-orphanized 7TM/GPCR GPR55 potently modulates CB(2)R-mediated responses. We show that GPR55 is expressed in human blood neutrophils and its activation augments the migratory response towards the CB(2)R agonist 2-arachidonoylglycerol (2-AG), while inhibiting neutrophil degranulation and reactive oxygen species (ROS) production. Using HEK293 and HL60 cell lines, along with primary neutrophils, we show that GPR55 and CB(2)R interfere with each other's signaling pathways at the level of small GTPases, such as Rac2 and Cdc42. This ultimately leads to cellular polarization and efficient migration as well as abrogation of degranulation and ROS formation in neutrophils. Therefore, GPR55 limits the tissue-injuring inflammatory responses mediated by CB(2)R, while it synergizes with CB(2)R in recruiting neutrophils to sites of inflammation.     

Delay of migrating leukocytes by the basement membrane deposited by endothelial cells in long-term culture

We investigated the migration of human leukocytes through endothelial cells (EC), and particularly their underlying basement membrane (BM). EC were cultured for 20 days on 3 μm-pore filters or collagen gels to form a distinct BM, and then treated with tumour necrosis factor-α, interleukin-1β or interferon-γ. Neutrophil migration through the cytokine-treated EC and BM was delayed for 20-day compared to 4-day cultures. The BM alone obstructed chemotaxis of neutrophils, and if fresh EC were briefly cultured on stripped BM, there was again a hold-up in migration. In studies with lymphocytes and monocytes, we could detect little hold-up of migration for 20-day versus 4-day cultures, in either the filter- or gel-based models. Direct microscopic observations showed that BM also held-up neutrophil migration under conditions of flow. Treatment of upper and/or lower compartments of filters with antibodies against integrins, showed that neutrophil migration through the endothelial monolayer was dependent on β₂-integrins, but not β1- or β₃-integrins. Migration from the subendothelial compartment was supported by β1- and β₂-integrins for all cultures, but blockade of β₃-integrin only inhibited migration effectively for 20-day cultures. Flow cytometry indicated that there was no net increase in expression of β1- or β3-integrins during neutrophil migration, and that their specific subendothelial function was likely dependent on turnover of integrins during migration. These studies show that BM is a distinct barrier to migration of human neutrophils, and that β₃-integrins are particularly important in crossing this barrier. The lesser effect of BM on lymphocytes and monocytes supports the concept that crossing the BM is a separate, leukocyte-specific, regulated step in migration.     

The inhibition of MAPK potentiates the anti-angiogenic efficacy of mTOR inhibitors

The mammalian target of rapamycin (mTOR) which is part of two functionally distinct complexes, mTORC1 and mTORC2, plays an important role in vascular endothelial cells. Indeed, the inhibition of mTOR with an allosteric inhibitor such as rapamycin reduces the growth of endothelial cell in vitro and inhibits angiogenesis in vivo. Recent studies have shown that blocking mTOR results in the activation of other prosurvival signals such as Akt or MAPK which counteract the growth inhibitory properties of mTOR inhibitors. However, little is known about the interactions between mTOR and MAPK in endothelial cells and their relevance to angiogenesis. Here we found that blocking mTOR with ATP-competitive inhibitors of mTOR or with rapamycin induced the activation of the mitogen-activated protein kinase (MAPK) in endothelial cells. Downregulation of mTORC1 but not mTORC2 had similar effects showing that the inhibition of mTORC1 is responsible for the activation of MAPK. Treatment of endothelial cells with mTOR inhibitors in combination with MAPK inhibitors reduced endothelial cell survival, proliferation, migration and tube formation more significantly than either inhibition alone. Similarly, in a tumor xenograft model, the anti-angiogenic efficacy of mTOR inhibitors was enhanced by the pharmacological blockade of MAPK. Taken together these results show that blocking mTORC1 in endothelial cells activates MAPK and that a combined inhibition of MAPK and mTOR has additive anti-angiogenic effects. They also provide a rationale to target both mTOR and MAPK simultaneously in anti-angiogenic treatment.     

Monitoring ligand-mediated internalization of G protein-coupled receptor as a novel pharmacological approach

Agonist activation of a G protein-coupled receptor (GPCR) results in the redistribution of the receptor protein away from the cell surface into internal cellular compartments through a process of endocytosis known as internalization. Visualization of receptor internalization has become experimentally practicable by using fluorescent reagents such as green fluorescent protein (GFP). In this study, we examined whether the ligand-mediated internalization of a GPCR can be exploited for pharmacological evaluations. We acquired fluorescent images of cells expressing GFP-labeled GPCRs and evaluated the ligand-mediated internalization quantitatively by image processing. Using beta2-adrenoceptor and vasopressin V1a receptor as model GPCRs that couple to Gs and Gq, respectively, we first examined whether these GFP-tagged GPCRs exhibited appropriate pharmacology. The rank order of receptor internalization potency for a variety of agonists and antagonists specific to each receptor corresponded well with that previously observed in ligand binding studies. In addition to chemical ligand-induced internalization, this cell-based fluorescence imaging system successfully monitored the internalization of the proton-sensing GPCR TDAG8, and that of the free fatty acid-sensitive GPCR GPR120. The results show that monitoring receptor internalization can be a useful approach for pharmacological characterization of GPCRs and in fishing for ligands of orphan GPCRs.