Phosphorylation of RIAM by src promotes integrin activation by unmasking the PH domain of RIAM

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Chemokine triggered integrin activation and actin remodeling events guiding lymphocyte migration across vascular barriers

Chemokine signals activate leukocyte integrins and actin remodeling machineries critical for leukocyte adhesion and motility across vascular barriers. The arrest of leukocytes at target blood vessel sites depends on rapid conformational activation of their α4 and β2 integrins by the binding of endothelial-displayed chemokines to leukocyte Gi-protein coupled receptors (GPCRs). A universal regulator of this event is the integrin-actin adaptor, talin1. Chemokine-stimulated GPCRs can transmit within fractions of seconds signals via multiple Rho GTPases, which locally raise plasma membrane levels of the talin activating phosphatidyl inositol, PtdIns(4,5)P2 (PIP2). Additional pools of GPCR stimulated Rac-1 and Rap-1 GTPases together with GPCR stimulated PLC and PI3K family members regulate the turnover of focal contacts of leukocyte integrins, induce the collapse of leukocyte microvilli, and promote polarized leukocyte crawling in search of exit cues. Concomitantly, other leukocyte GTPases trigger invasive protrusions into and between endothelial cells in search of basolateral chemokine exit cues. We will review here major findings and open questions related to these sequential guiding activities of endothelial presented chemokines, focusing mainly on lymphocyte-endothelial interactions as a paradigm for other leukocytes.     

Rap1 controls activation of the αMβ2 integrin in a talin‐dependent manner

The small GTPase Rap1 and the cytoskeletal protein talin regulate binding of C3bi‐opsonised red blood cells (RBC) to integrin α_Mβ₂ in phagocytic cells, although the mechanism has not been investigated. Using COS‐7 cells transfected with α_Mβ₂, we show that Rap1 acts on the β₂ and not the α_M chain, and that residues 732–761 of the β₂ subunit are essential for Rap1‐induced RBC binding. Activation of α_Mβ₂ by Rap1 was dependent on W747 and F754 in the β₂ tails, which are required for talin head binding, suggesting a link between Rap1 and talin in this process. Using talin1 knock‐out cells or siRNA‐mediated talin1 knockdown in the THP‐1 monocytic cell line, we show that Rap1 acts upstream of talin but surprisingly, RIAM knockdown had little effect on integrin‐mediated RBC binding or cell spreading. Interestingly, Rap1 and talin influence each other's localisation at phagocytic cups, and co‐immunoprecipitation experiments suggest that they interact together. These results show that Rap1‐mediated activation of α_Mβ₂ in macrophages shares both common and distinct features from Rap1 activation of α_IIbβ₃ expressed in CHO cells. J. Cell. Biochem. 111: 999–1009, 2010. © 2010 Wiley‐Liss, Inc.     

Crystal structure of Lamellipodin implicates diverse functions in actin polymerization and Ras signaling

The adapter protein Lamellipodin (Lpd) plays an important role in cell migration. In particular, Lpd mediates lamellipodia formation by regulating actin dynamics via interacting with Ena/VASP proteins. Its RA-PH tandem domain confi guration suggests that like its paralog RIAM, Lpd may also mediate particular Ras GTPase signaling. We determined the crystal structures of the Lpd RA-PH domains alone and with an N-terminal coiled-coil region (cc-RA-PH). These structures reveal that apart from the anticipated coiled-coil interaction, Lpd may also oligomerize through a second intermolecular contact site. We then validated both oligomerization interfaces in solution by mutagenesis. A fluorescence-polarization study demonstrated that Lpd binds phosphoinositol with low affinity. Based on our crystallographic and biochemical data, we propose that Lpd and RIAM serve diverse functions: Lpd plays a predominant role in regulating actin polymerization, and its function in mediating Ras GTPase signaling is largely suppressed compared to RIAM.     

RIAM and Vinculin Binding to Talin Are Mutually Exclusive and Regulate Adhesion Assembly and Turnover

Talin activates integrins, couples them to F-actin, and recruits vinculin to focal adhesions (FAs). Here, we report the structural characterization of the talin rod: 13 helical bundles (R1–R13) organized into a compact cluster of four-helix bundles (R2–R4) within a linear chain of five-helix bundles. Nine of the bundles contain vinculin-binding sites (VBS); R2R3 are atypical, with each containing two VBS. Talin R2R3 also binds synergistically to RIAM, a Rap1 effector involved in integrin activation. Biochemical and structural data show that vinculin and RIAM binding to R2R3 is mutually exclusive. Moreover, vinculin binding requires domain unfolding, whereas RIAM binds the folded R2R3 double domain. In cells, RIAM is enriched in nascent adhesions at the leading edge whereas vinculin is enriched in FAs. We propose a model in which RIAM binding to R2R3 initially recruits talin to membranes where it activates integrins. As talin engages F-actin, force exerted on R2R3 disrupts RIAM binding and exposes the VBS, which recruit vinculin to stabilize the complex.     

The structure of Rap1 in complex with RIAM reveals specificity determinants and recruitment mechanism

The small GTPase Rap1 induces integrin activation via an inside-out signaling pathway mediated by the Rapl-interacting adaptor mol- ecule （RIAM）. Blocking this pathway may suppress tumor metastasis and other diseases that are related to hyperactive integrins. However, the molecular basis for the specific recognition of RIAM by Rap1 remains largely unknown. Herein we present the crystal structure of an active, GTP-bound GTPase domain of Rap1 in complex with the Ras association （RA）-pleckstrin homology （PH） structural module of RIAM at 1.65 A. The structure reveals that the recognition of RIAM by Rap1 is governed by side-chain interactions. Several side chains are critical in determining specificity of this recognition, particularly the Lys31 residue in Rap1 that is oppositely charged compared with the Glu31/Asp31 residue in other Ras GTPases. Lys31 forms a salt bridge with RIAM residue Glu212, making it the key specificity determinant of the interaction. We also show that disruption of these interactions results in reduction of Rapl：RIAM association, leadingto a loss of co-clustering and cell adhesion. Our findings elucidate the molecular mechanism by which RIAM med- iates Rapl-induced integrin activation. The crystal structure also offers new insight into the structural basis for the specific recruitment of RA-PH module-containing effector proteins by their smaU GTPase partners.     

Netrin, Slit and Wnt receptors allow axons to choose the axis of migration

One of the challenges to understanding nervous system development has been to establish how a fairly limited number of axon guidance cues can set up the patterning of very complex nervous systems. Studies on organisms with relatively simple nervous systems such as Drosophila melanogaster and C. elegans have provided many insights into axon guidance mechanisms. The axons of many neurons migrate along both the dorsal-ventral (DV) and the anterior-posterior (AP) axes at different phases of development, and in addition they may also cross the midline. Axon migration in the dorsal-ventral (DV) direction is mainly controlled by Netrins with their receptors; UNC-40/DCC and UNC-5, and the Slits with their receptors; Robo/SAX-3. Axon guidance in the anterior-posterior (AP) axis is mainly controlled by Wnts with their receptors; the Frizzleds/Fz. An individual axon may be subjected to opposing attractive and repulsive forces coming from opposite sides in the same axis but there may also be opposing cues in the other axis of migration. All the information from the cues has to be integrated within the growth cone at the leading edge of the migrating axon to elicit a response. Recent studies have provided insight into how this is achieved.Evidence suggests that the axis of axon migration is determined by the manner in which Netrin, Slit and Wnt receptors are polarized (localized) within the neuron prior to axon outgrowth. The same molecules are involved in both axon outgrowth and axon guidance, for at least some neurons in C. elegans, whether the cue is the attractive cue UNC-6/Netrin working though UNC-40/DCC or the repulsive cue SLT-1/Slit working though the receptor SAX-3/Robo (Adler et al., 2006, Chang et al., 2006, Quinn et al., 2006, 2008). The molecules involved in cell signaling in this case are polarized within the cell body of the neuron before process outgrowth and direct the axon outgrowth. Expression of the Netrin receptor UNC-40/DCC or the Slit receptor SAX-3/Robo in axons that normally migrate in the AP direction causes neuronal polarity reversal in a Netrin and Slit independent manner (Levy-Strumpf and Culotti 2007, Watari-Goshima et al., 2007). Localization of the receptors in this case is caused by the kinesin-related VAB-8L which appears to govern the site of axon outgrowth in these neurons by causing receptor localization. Therefore, asymmetric localization of axon guidance receptors is followed by axon outgrowth in vivo using the receptor's normal cue, either attractive, repulsive or unknown cues.     

黄河流域生态水文分区及优先保护级别

在分析相关分区研究的基础上,探讨流域生态水文分区的内涵;并结合黄河流域的实际情况,从生态重要性、水文特征、生态背景和人类活动影响等方面建立分区的指标体系;为便于管理,分区以县为基本单位,由指标层定量计算县的准则层指标值,根据准则层对应的级别确定各县的分区类型,将黄河划分成20个一级分区和51个二级分区;最后采用RIAM模型选取生态重要性指标,确定二级分区的优先保护级别,其中2个重点优先保护区、11个中等优先保护区需要重点保护。优先保护区集中分布在以下四个地区,分别是流域的源区、贺兰山以东和鄂尔多斯以西、三省(山西、陕西和河南)交界山区、河口三角洲。     

Inhibition of talin-induced integrin activation by a double-hit stapled peptide

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