Adaptor protein GULP is involved in stabilin-1-mediated phagocytosis

The clearance of apoptotic cells is critical during cellular homeostasis as well as inflammation resolution. Recently, we found that stabilin-1 is a phagocytic receptor that is involved in the clearance of apoptotic cells. However, the downstream signaling pathway of stabilin-1-mediated phagocytosis remains to be investigated. Here we identify that GULP is able to specifically interact with the NPxF/Y motif of stabilin-1 cytoplasmic region. The PTB domain of GULP is necessary for interaction with stabilin-1. GULP is enriched around PS-coated beads for phagocytosis and co-localized with stabilin-1. Downregulation of endogenous GULP expression decreased stabilin-1-mediated phagocytosis. Thus, these results indicate that GULP functions as an adaptor protein for stabilin-1-mediated phagocytosis.     

Cross talk between engulfment receptors stabilin-2 and integrin αvβ5 orchestrates engulfment of phosphatidylserine-exposed erythrocytes

Efficient cell corpse clearance is critical for health in organisms. Apoptotic cells displaying phosphatidylserine (PS) are recognized by engulfment receptors and ingested through two conserved pathways. In one pathway, engulfment receptor brain-specific angiogenesis inhibitor 1 (BAI-1) or integrin functions upstream of ELMO/DOCK180 and activate the small GTPase Rac1. In the other pathway, engulfment receptor CED-1 or stabilin-2 acts in concert with the adaptor protein GULP to activate Rac1. Stabilin-2, a PS receptor, facilitates phagocytosis of apoptotic cells and mediates the production of anti-inflammatory cytokines. Here, we propose that the stabilin-2 extracellular domain consisting of integrin-binding fasciclin 1 (FAS1) domains coordinates the activities of the two phagocytic pathways via direct interactions with integrin. Interactions between stabilin-2 and integrin were determined using biochemical assays, including coimmunoprecipitation and fluorescence resonance energy transfer (FRET). These interactions appear to have functional relevance, since knockdown of endogenous αvβ5 expression or treatment with a function-blocking αvβ5 antibody significantly decreased stabilin-2-mediated phagocytosis in the absence of soluble factors. Our data collectively suggest that the engulfment receptors of the two phagocytic pathways communicate with each other to orchestrate engulfment of damaged erythrocytes. Coordinated phagocytic signaling would be advantageous for physiological and pathological circumstances that require rapid clearance of abnormal (apoptotic or aged) cells.     

Interaction of CED-6/GULP, an adapter protein involved in engulfment of apoptotic cells with CED-1 and CD91/low density lipoprotein receptor-related protein (LRP).

The prompt clearance of cells undergoing apoptosis is critical during embryonic development, normal tissue turnover, as well as inflammation and autoimmunity. The molecular details of the engulfment of apoptotic cells are not fully understood. ced-6 and its human homologue gulp, encode an adapter protein, whose function in engulfment is highly evolutionarily conserved; however, the upstream and downstream components of CED-6 mediated signaling are not known. Recently, ced-1 has been shown to encode a transmembrane protein on phagocytic cells, with two functional sequence motifs in its cytoplasmic tail that are important for engulfment. In this study, using a combination of biochemical approaches and yeast two-hybrid analysis, we present evidence for a physical interaction between GULP/CED-6 and one of the two motifs (NPXY motif) in the cytoplasmic tail of CED-1. The phosphotyrosine binding domain of GULP was necessary and sufficient for this interaction. Since the precise mammalian homologue of CED-1 is not known, we undertook a database search for human proteins that contain the motifs shown to be important for CED-1 function and identified CD91/LRP (low density lipoprotein receptor-related protein) as one candidate. Interestingly, recent studies have also identified CD91/LRP as a receptor involved in the phagocytosis of apoptotic cells in mammals. The GULP phosphotyrosine binding domain was able to specifically interact with one specific NPXY motif in the CD91 cytoplasmic tail. During these studies we have also identified the mouse GULP sequence. These studies suggest a physical link between CED-1 or CD91/LRP and the adapter protein CED-6/GULP during engulfment of apoptotic cells and further elucidate the pathway suggested by the genetic studies.     

Regulation of Arf6 and ACAP1 signaling by the PTB-domain-containing adaptor protein GULP

The GTPase Arf6 regulates multiple cellular processes, including endocytosis, secretion, phagocytosis, cell adhesion, and cell migration [1, 2]. The Arf6-specific GAP ACAP1 is a negative regulator of Arf6-mediated signaling [3-7]. However, regulation of ACAP1- and Arf6-mediated signaling by other cellular proteins is not well understood. GULP/CED-6 is a phosphotyrosine binding (PTB)-domain-containing adaptor protein linked to engulfment of apoptotic cells [8-13] and to cholesterol homeostasis [14]. Here, we identify a novel role for GULP as a positive regulator of Arf6. Knockdown of GULP decreased cellular Arf6-GTP, whereas GULP overexpression increased cellular Arf6-GTP. At the mechanistic level, GULP influenced Arf6 at four levels. First, GULP bound directly to GDP-bound Arf6 via its PTB domain. Second, GULP associated with the Arf6-GAP ACAP1 at endogenous levels. Third, GULP reversed the Arf6-GTP decrease induced by ACAP1, and countered the ACAP1-mediated inhibition of cell migration. Fourth, GULP, ACAP1, and GDP-bound Arf6 were part of a tripartite complex, suggesting sequestration of ACAP1 as one mechanism of GULP action. Taken together, these data identify GULP as a modifier of cellular Arf6-GTP through regulation of ACAP1. Because PTB-domain-containing adaptor proteins influence endocytosis and trafficking of membrane proteins and cell migration [15, 16], our data support a model wherein PTB-domain-containing adaptor proteins regulate Arf family proteins.     

Thymosin beta4 is involved in stabilin-2-mediated apoptotic cell engulfment

Stabilin-2 was recently identified as a novel receptor for membrane phosphatidylserine of apoptotic cells. To identify proteins that were candidates for stabilin-2 cytoplasmic domain binding, we screened a human spleen cDNA library using the yeast two-hybrid system. We found that thymosin beta4 interacts with the stabilin-2 cytoplasmic domain and is co-localized with stabilin-2 at the phagocytic cup. Knockdown of thymosin beta4 significantly decreased the phagocytic activity of stabilin-2, whereas overexpression of thymosin beta4 increased this activity. Additionally, amino acids 2504-2514 of stabilin-2 cytoplasmic domain were found to be responsible for the interaction with thymosin beta4. Taken together, these results suggest that thymosin beta4 is a downstream molecule of stabilin-2 that plays a role in stabilin-2-mediated cell corpse clearance.     

Identification and characterization of a dimerization domain in CED-6, an adapter protein involved in engulfment of apoptotic cells

Phagocytosis of apoptotic cells is a key step in the completion of programmed cell death that occurs throughout life in multicellular organisms. The molecular events involved in clearance of apoptotic cells are just beginning to be elucidated. Recently, CED-6, an adapter protein involved in engulfment has been cloned in Caenorhabditis elegans and in humans. CED-6 is composed of a phosphotyrosine-binding (PTB) domain and a proline-rich C-terminal domain with no apparent catalytic domain. Since PTB domains, originally identified in Shc, mediate intracellular signaling downstream of cell surface receptors, CED-6 has also been proposed to mediate intracellular signals leading to engulfment. In this report, we demonstrate that CED-6 dimerizes through a leucine zipper domain that is immediately adjacent to the PTB domain. Several lines of evidence based on co-immunoprecipitation studies, yeast two-hybrid assays, and gel filtration studies suggest that CED-6 exists as a dimer in vivo. Through mutational analyses, we show that the leucine zipper is necessary and sufficient for CED-6 dimerization and that this dimerization is conserved among C. elegans, rodent, and human CED-6 proteins. We propose that dimerization may have unique implications for ligand binding via CED-6 and its function during the phagocytosis of apoptotic cells.     

A Highlights from MBoC Selection: The adaptor protein GULP promotes Jedi-1–mediated phagocytosis through a clathrin-dependent mechanism

During the development of the peripheral nervous system, the large number of apoptotic neurons generated are phagocytosed by glial precursor cells. This clearance is mediated, in part, through the mammalian engulfment receptor Jedi-1. However, the mechanisms by which Jedi-1 mediates phagocytosis are poorly understood. Here we demonstrate that Jedi-1 associates with GULP, the mammalian homologue of CED-6, an adaptor protein required for phagocytosis mediated by the nematode engulfment receptor CED-1. Silencing GULP or mutating the NPXY motif in Jedi-1, which is required for GULP binding, prevents Jedi-1–mediated phagocytosis. How GULP promotes engulfment is not known. Of interest, we find that Jedi-1–induced phagocytosis requires GULP binding to clathrin heavy chain (CHC). During engulfment, CHC is tyrosine phosphorylated, which is required for Jedi-mediated engulfment. Both phosphoclathrin and actin accumulate around engulfed microspheres. Furthermore, knockdown of CHC in HeLa cells prevents Jedi-1–mediated engulfment of microspheres, and knockdown in glial precursors prevents the engulfment of apoptotic neurons. Taken together, these results reveal that Jedi-1 signals through recruitment of GULP, which promotes phagocytosis through a noncanonical phosphoclathrin-dependent mechanism.     

Rapid cell corpse clearance by stabilin-2, a membrane phosphatidylserine receptor

Rapid phagocytic clearance of apoptotic cells is crucial for the prevention of both inflammation and autoimmune responses. Phosphatidylserine (PS) at the external surface of the plasma membrane has been proposed to function as a general 'eat me' signal for apoptotic cells. Although several soluble bridging molecules have been suggested for the recognition of PS, the PS-specific membrane receptor that binds directly to the exposed PS and provides a tickling signal has yet to be definitively identified. In this study, we provide evidence that stabilin-2 is a novel PS receptor, which performs a key function in the rapid clearance of cell corpses. It recognizes PS on aged red blood cells and apoptotic cells, and mediates their engulfment. The downregulation of stabilin-2 expression in macrophages significantly inhibits phagocytosis, and anti-stabilin-2 monoclonal antibody provokes the release of the anti-inflammatory cytokine, transforming growth factor-beta. Furthermore, the results of time-lapse video analyses indicate that stabilin-2 performs a crucial function in the rapid clearance of aged and apoptotic cells. These data indicate that stabilin-2 is the first of the membrane PS receptors to provide tethering and tickling signals, and may also be involved in the resolution of inflammation and the prevention of autoimmunity.     

The apoptotic engulfment protein Ced-6 participates in clathrin-mediated yolk uptake in Drosophila egg chambers

Clathrin-mediated endocytosis and phagocytosis are both selective surface internalization processes but have little known mechanistic similarity or interdependence. Here we show that the phosphotyrosine-binding (PTB) domain protein Ced-6, a well-established phagocytosis component that operates as a transducer of so-called "eat-me" signals during engulfment of apoptotic cells and microorganisms, is expressed in the female Drosophila germline and that Ced-6 expression correlates with ovarian follicle development. Ced-6 exhibits all the known biochemical properties of a clathrin-associated sorting protein, yet ced-6-null flies are semifertile despite massive accumulation of soluble yolk precursors in the hemolymph. This is because redundant sorting signals within the cytosolic domain of the Drosophila vitellogenin receptor Yolkless, a low density lipoprotein receptor superfamily member, occur; a functional atypical dileucine signal binds to the endocytic AP-2 clathrin adaptor directly. Nonetheless, the Ced-6 PTB domain specifically recognizes the noncanonical Yolkless FXNPXA sorting sequence and in HeLa cells promotes the rapid, clathrin-dependent uptake of a Yolkless chimera lacking the distal dileucine signal. Ced-6 thus operates in vivo as a clathrin adaptor. Because the human Ced-6 orthologue GULP similarly binds to clathrin machinery, localizes to cell surface clathrin-coated structures, and is enriched in placental clathrin-coated vesicles, new possibilities for Ced-6/Gulp operation during phagocytosis must be considered.     

Epidermal growth factor-like domain repeat of stabilin-2 recognizes phosphatidylserine during cell corpse clearance

Exposure of phosphatidylserine (PS) on the cell surface occurs early during apoptosis and serves as a recognition signal for phagocytes. Clearance of apoptotic cells by a membrane PS receptor is one of the critical anti-inflammatory functions of macrophages. However, the PS binding receptors and their recognition mechanisms have not been fully investigated. Recently, we reported that stabilin-2 is a PS receptor that mediates the clearance of apoptotic cells, thus releasing the anti-inflammatory cytokine, transforming growth factor β. In this study, we showed that epidermal growth factor (EGF)-like domain repeats (EGFrp) in stabilin-2 can directly and specifically recognize PS. The EGFrps also competitively impaired apoptotic cell uptake by macrophages in in vivo models. We also showed that calcium ions are required for stabilin-2 to mediate phagocytosis via EGFrp. Interestingly, at least four tandem repeats of EGF-like domains were required to recognize PS, and the second atypical EGF-like domain in EGFrp was critical for calcium-dependent PS recognition. Considering that PS itself is an important target molecule for both apoptotic cells and nonapoptotic cells during various cellular processes, our results should help elucidate the molecular mechanism by which apoptotic cell clearance in the human body occurs and also have implications for targeting PS externalization of nonapoptotic cells.     

Integrin α PAT-2/CDC-42 signaling is required for muscle-mediated clearance of apoptotic cells in Caenorhabditis elegans

Clearance of apoptotic cells by engulfment plays an important role in the homeostasis and development of multicellular organisms. Despite the fact that the recognition of apoptotic cells by engulfment receptors is critical in inducing the engulfment process, the molecular mechanisms are still poorly understood. Here, we characterize a novel cell corpse engulfment pathway mediated by the integrin α subunit PAT-2 in Caenorhabditis elegans and show that it specifically functions in muscle-mediated engulfment during embryogenesis. Inactivation of pat-2 results in a defect in apoptotic cell internalization. The PAT-2 extracellular region binds to the surface of apoptotic cells in vivo, and the intracellular region may mediate signaling for engulfment. We identify essential roles of small GTPase CDC-42 and its activator UIG-1, a guanine-nucleotide exchange factor, in PAT-2-mediated cell corpse removal. PAT-2 and CDC-42 both function in muscle cells for apoptotic cell removal and are co-localized in growing muscle pseudopods around apoptotic cells. Our data suggest that PAT-2 functions through UIG-1 for CDC-42 activation, which in turn leads to cytoskeletal rearrangement and apoptotic cell internalization by muscle cells. Moreover, in contrast to PAT-2, the other integrin α subunit INA-1 and the engulfment receptor CED-1, which signal through the conserved signaling molecules CED-5 (DOCK180)/CED-12 (ELMO) or CED-6 (GULP) respectively, preferentially act in epithelial cells to mediate cell corpse removal during mid-embryogenesis. Our results show that different engulfing cells utilize distinct repertoires of receptors for engulfment at the whole organism level.     

The conserved histidine in epidermal growth factor-like domains of stabilin-2 modulates pH-dependent recognition of phosphatidylserine in apoptotic cells

Clearance of apoptotic cells is involved in the resolution of inflammation, and this mechanism is controlled by the regulation of pro- and anti-inflammatory cytokine production during the ingestion of apoptotic cells. Inflamed areas show extracellular acidity, and low pH stimulates cellular functions of immune cells. However, little is known about the influence of extracellular acidic pH on the function of phagocytic cells. In this study, we showed that stabilin-2-mediated phagocytosis is activated in low pH media (pH 6.8) and examined the molecular mechanisms underlying this pH-dependent enhancement of phagocytic activity. Stabilin-2, which is expressed in human monocyte derived macrophages (HMDM), is a phosphatidylserine (PS) receptor that mediates phagocytosis of apoptotic cells, and releases the anti-inflammatory cytokine, TGF-β. The PS binding activity of stabilin-2 is enhanced in low pH, and a conserved histidine¹⁴⁰³ in close proximity to the PS binding loop is critical for pH-dependent activity. We propose that protonation of His¹⁴⁰³ may rearrange the PS binding loop to enhance binding affinity in low pH, indicating that acidic pH might act as a danger signal to stimulate stabilin-2-mediated phagocytosis to resolve inflammation. Considering that phosphatidylserine is an important target molecule for apoptotic cells in the acidic microenvironment of inflammation and tumors, our results also have implications for pH sensitive targeting of apoptotic cells.     

Essential diurnal Rac1 activation during retinal phagocytosis requires αvβ5 integrin but not tyrosine kinases focal adhesion kinase or Mer tyrosine kinase

Diurnal phagocytosis of shed photoreceptor outer-segment particles by retinal pigment epithelial (RPE) cells belongs to a group of conserved clearance mechanisms employing αv integrins upstream of tyrosine kinases and Rho GTPases. In this study, we tested the interdependence of the tyrosine kinases focal adhesion kinase (FAK) and Mer tyrosine kinase (MerTK) and Rho GTPases during engulfment. RPE cells activated and redistributed Rac1, but not RhoA or Cdc42, during phagocytosis. Toxin B, overexpression of dominant-negative Rac1, or decreasing Rac1 expression prevented particle engulfment. Fluorescence microscopy showed that Rac1 inhibition had no obvious effect on F-actin arrangement in resting RPE but prevented recruitment of F-actin to surface-bound phagocytic particles. Quantification of active GTP-Rac1 in wild-type and mutant RPE in culture and in vivo revealed that Rac1 activation during phagocytosis requires αvβ5 integrin and its ligand milk fat globule EGF factor-8 (MFG-E8) but not the receptor tyrosine kinase MerTK. Abolishing tyrosine kinase signaling downstream of αvβ5 toward MerTK by inhibiting FAK specifically or tyrosine kinases generally neither prevented Rac1 activation nor F-actin recruitment during phagocytosis. Likewise, inhibiting Rac1 had no effect on FAK or MerTK activation. We conclude that MerTK activation via FAK and F-actin recruitment via Rac1 both require MFG-E8-ligated αvβ5 integrin. Both pathways are independently activated and required for clearance phagocytosis.     

GULP1 is a novel APP-interacting protein that alters APP processing

Altered production of Aβ (amyloid-β peptide), derived from the proteolytic cleavage of APP (amyloid precursor protein), is believed to be central to the pathogenesis of AD (Alzheimer's disease). Accumulating evidence reveals that APPc (APP C-terminal domain)-interacting proteins can influence APP processing. There is also evidence to suggest that APPc-interacting proteins work co-operatively and competitively to maintain normal APP functions and processing. Hence, identification of the full complement of APPc-interacting proteins is an important step for improving our understanding of APP processing. Using the yeast two-hybrid system, in the present study we identified GULP1 (engulfment adaptor protein 1) as a novel APPc-interacting protein. We found that the GULP1-APP interaction is mediated by the NPTY motif of APP and the GULP1 PTB (phosphotyrosine-binding) domain. Confocal microscopy revealed that a proportion of APP and GULP1 co-localized in neurons. In an APP-GAL4 reporter assay, we demonstrated that GULP1 altered the processing of APP. Moreover, overexpression of GULP1 enhanced the generation of APP CTFs (C-terminal fragments) and Aβ, whereas knockdown of GULP1 suppressed APP CTFs and Aβ production. The results of the present study reveal that GULP1 is a novel APP/APPc-interacting protein that influences APP processing and Aβ production.     

The lipoprotein receptor-related protein-1 (LRP) adapter protein GULP mediates trafficking of the LRP ligand prosaposin, leading to sphingolipid and free cholesterol accumulation in late endosomes and impaired efflux

One of the conserved functional pathways linked to engulfment of apoptotic corpses involves two membrane proteins low density lipoprotein receptor-related protein-1 (LRP) and ABCA1 and the LRP adapter protein GULP. Because LRP and ABCA1 play roles in cellular lipid trafficking and efflux, here we addressed whether the third member, the LRP adapter protein GULP, also affects cellular lipid transport. Several lines of evidence show that overexpression of GULP causes glycosphingolipid and free cholesterol accumulation in the late endosome/lysosome compartment that is accompanied by down-regulation of ABCA1 and decreased efflux. Conversely, knockdown of endogenous GULP expression promoted cholesterol flux through the late endosomes and up-regulation of ABCA1, even in the context of a disease state such as Niemann-Pick Type C disease. Mechanistically, we were able to show that trafficking of the LRP ligands alpha2-macroglobulin and prosaposin, a protein cofactor necessary for glycosphingolipid degradation, are impaired in cells expressing full-length GULP protein, resulting in glycosphingolipid and free cholesterol accumulation in the late endosome/lysosome compartment. On the other hand, knockdown of endogenous GULP results in enhanced targeting of prosaposin and enhanced clearance of glycosphingolipids and cholesterol from the late endosomes. Taken together, these data reveal that GULP/LRP/ABCA1 represents a triad of molecules involved in engulfment and cellular lipid homeostasis.     

The Disabled 1 Phosphotyrosine-Binding Domain Binds to the Internalization Signals of Transmembrane Glycoproteins and to Phospholipids

Disabled gene products are important for nervous system development in drosophila and mammals. In mice, the Dab1 protein is thought to function downstream of the extracellular protein Reln during neuronal positioning. The structures of Dab proteins suggest that they mediate protein-protein or protein-membrane docking functions. Here we show that the amino-terminal phosphotyrosine-binding (PTB) domain of Dab1 binds to the transmembrane glycoproteins of the amyloid precursor protein (APP) and low-density lipoprotein receptor families and the cytoplasmic signaling protein Ship. Dab1 associates with the APP cytoplasmic domain in transfected cells and is coexpressed with APP in hippocampal neurons. Screening of a set of altered peptide sequences showed that the sequence GYXNPXY present in APP family members is an optimal binding sequence, with approximately 0.5 microM affinity. Unlike other PTB domains, the Dab1 PTB does not bind to tyrosine-phosphorylated peptide ligands. The PTB domain also binds specifically to phospholipid bilayers containing phosphatidylinositol 4P (PtdIns4P) or PtdIns4,5P2 in a manner that does not interfere with protein binding. We propose that the PTB domain permits Dab1 to bind specifically to transmembrane proteins containing an NPXY internalization signal.     

A role for mammalian diaphanous-related formins in complement receptor (CR3)-mediated phagocytosis in macrophages

Macrophages, dendritic cells, and neutrophils use phagocytosis to capture and clear off invading pathogens. The process is triggered by the interaction of ligands on the pathogens' surface with specific phagocytic receptors, including immunoglobulin (FcR) and complement C3bi (CR3) receptors (integrin alpha(M)beta2, Mac1) . Localized actin-filament assembly that acts as the driving force for particle engulfment is controlled by Rho-family small GTPases . RhoA regulates CR3-mediated phagocytosis through a mechanism that is still unclear . Mammalian Diaphanous-related (mDia) formins participate in the generation of a diverse set of actin-remodeling events downstream of RhoA , and mDia1 is recruited around fibronectin-coated beads in a RhoA-dependent manner in fibroblasts . Here, we set out to examine whether mDia proteins are involved in CR3-mediated phagocytosis in macrophages. We show that the RhoA effector mDia1 is recruited early during CR3-mediated phagocytosis and colocalizes with polymerized actin in the phagocytic cup. Interfering with mDia activity inhibits CR3-mediated phagocytosis while having no effect on FcR-mediated phagocytosis. These results indicate a new function for mDia proteins in the regulation of actin polymerization during CR3-mediated phagocytosis.     

Engulfment of apoptotic cells is negatively regulated by Rho-mediated signaling

The rapid and efficient phagocytosis of apoptotic cells plays a critical role in preventing secondary necrosis, inflammation as well as in tissue remodeling and regulating immune responses. However, the molecular details of engulfment are just beginning to be elucidated. Among the Rho family GTPases, previous studies have implicated a role for Rac and Cdc42 in the uptake of apoptotic cells by phagocytes, yet the role of Rho has remained unclear. Here, we present evidence that Rho-GTP levels decrease during engulfment. RhoA seems to negatively affect basal engulfment, such that inhibition of Rho-mediated signaling in phagocytes enhanced the uptake of apoptotic targets. Activation of endogenous Rho or overexpression of constitutively active forms of Rho also inhibited engulfment. By testing mutants of RhoA that selectively activate downstream effectors, the Rho-kinase seemed to be primarily responsible for this inhibitory effect. Taken together, these data suggest that inhibition of Rho- and Rho-kinase-mediated signaling might be important during engulfment, which could have important implications for several clinical trials involving inhibition of the Rho kinase.     

Phagocytosis of apoptotic cells is regulated by a UNC-73/TRIO-MIG-2/RhoG signaling module and armadillo repeats of CED-12/ELMO

BACKGROUND: Phagocytosis of cells undergoing apoptosis is essential during development, cellular turnover, and wound healing. Failure to promptly clear apoptotic cells has been linked to autoimmune disorders. C. elegans CED-12 and mammalian ELMO are evolutionarily conserved scaffolding proteins that play a critical role in engulfment from worm to human. ELMO functions together with Dock180 (a guanine nucleotide exchange factor for Rac) to mediate Rac-dependent cytoskeletal reorganization during engulfment and cell migration. However, the components upstream of ELMO and Dock180 during engulfment remain elusive. RESULTS: Here, we define a conserved signaling module involving the small GTPase RhoG and its exchange factor TRIO, which functions upstream of ELMO/Dock180/Rac during engulfment. Complementary studies in C. elegans show that MIG-2 (which we identify as the homolog of mammalian RhoG) and UNC-73 (the TRIO homolog) also regulate corpse clearance in vivo, upstream of CED-12. At the molecular level, we identify a novel set of evolutionarily conserved Armadillo (ARM) repeats within CED-12/ELMO that mediate an interaction with activated MIG-2/RhoG; this, in turn, promotes Dock180-mediated Rac activation and cytoskeletal reorganization. CONCLUSIONS: The combination of in vitro and in vivo studies presented here identify two evolutionarily conserved players in engulfment, TRIO/UNC73 and RhoG/MIG-2, and the TRIO --> RhoG signaling module is linked by ELMO/CED-12 to Dock180-dependent Rac activation during engulfment. This work also identifies ARM repeats within CED-12/ELMO and their role in linking RhoG and Rac, two GTPases that function in tandem during engulfment.     

Integrin β3 Phosphorylation Dictates Its Complex with the Shc Phosphotyrosine-binding (PTB) Domain

Adaptor protein Shc plays a key role in mitogen-activated protein kinase (MAPK) signaling pathway, which can be mediated through a number of different receptors including integrins. By specifically recognizing the tyrosine-phosphorylated integrin β₃, Shc has been shown to trigger integrin outside-in signaling, although the structural basis of this interaction remains nebulous. Here we present the detailed structural analysis of Shc phosphotyrosine-binding (PTB) domain in complex with the bi-phosphorylated β₃integrin cytoplasmic tail (CT). We show that this complex is primarily defined by the phosphorylation state of the integrin C-terminal Tyr⁷⁵⁹, which fits neatly into the classical PTB pocket of Shc. In addition, we have identified a novel binding interface which concurrently accommodates phosphorylated Tyr⁷⁴⁷ of the highly conserved NPXY motif of β₃. The structure represents the first snapshot of an integrin cytoplasmic tail bound to a target for mediating the outside-in signaling. Detailed comparison with the known Shc PTB structure bound to a target TrkA peptide revealed some significant differences, which shed new light upon the PTB domain specificity.