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1.  A shortcut from GPCR signaling to Rac-mediated actin cytoskeleton through an ELMO/DOCK complex 
Small GTPases  2012;3(3):183-185.
Chemotaxis, chemoattractant-guided directional cell migration, plays major roles in human innate immunity and in development of a model organism Dictyostelium discoideum. Human leukocytes and D. disscoideum share remarkable similarities in the molecular mechanisms that control chemotaxis. These cells use G-Protein-Coupled Receptors (GPCRs), such as chemokine receptors, to control a signaling network that carries out chemotactic gradient sensing and directs cell migration. Diverse chemokines bind to their receptors to activate small G protein Rac through an evolutionarily conserved mechanism. Elmo and Dock180 proteins form ELMO/Dock180 complexes functioning as guanine nucleotide exchange factors (GEFs) for Rac activation. However, the linkage between GPCR to Elmo/Dock180 for Rac activation that controls F-actin dynamics remained unclear. Recently, we discovered a novel function of an ELMO protein in Dictyostelium discoideum linking GPCR signaling from Gβ to actin dynamics through regulating Rac activation during chemotaxis.
doi:10.4161/sgtp.20271
PMCID: PMC3442806  PMID: 22647486
ELMO protein; G-protein-coupled receptor; Gβ subunit; Rac; chemotaxis; guanine nucleotide exchange factor (GEF); heterotrimeric G protein; small G protein; the actin cytoskeleton
2.  Chemotaxis, chemokine receptors and human disease 
Cytokine  2008;44(1):1-8.
Cell migration is involved in diverse physiological processes including embryogenesis, immunity, and diseases such as cancer and chronic inflammatory disease. The movement of many cell types is directed by extracellular gradients of diffusible chemicals. This phenomenon, referred to as "chemotaxis", was first described in 1888 by Leber who observed the movement of leukocytes toward sites of inflammation. We now know that a large family of small proteins, chemokines, serves as the extracellular signals and a family of G-protein-coupled receptors (GPCRs), chemokine receptors, detects gradients of chemokines and guides cell movement in vivo. Currently, we still know little about the molecular machineries that control chemokine gradient sensing and migration of immune cells. Fortunately, the molecular mechanisms that control these fundamental aspects of chemotaxis appear to be evolutionarily conserved, and studies in lower eukaryotic model systems allowed us to form concepts, uncover molecular components, develop new techniques, and test models of chemotaxis. These studies have helped our current understanding of this complicated cell behavior. In this review, we wish to mention landmark discoveries in the chemotaxis research field that shaped our current understanding of this fundamental cell behavior and lay out key questions that remain to be addressed in the future.
doi:10.1016/j.cyto.2008.06.017
PMCID: PMC2613022  PMID: 18722135
chemotaxis; chemokine; GPCR; actin; inflammation
3.  Phosphorylation of chemoattractant receptors regulates chemotaxis, actin reorganization and signal relay 
Journal of Cell Science  2013;126(20):4614-4626.
Summary
Migratory cells, including mammalian leukocytes and Dictyostelium, use G-protein-coupled receptor (GPCR) signaling to regulate MAPK/ERK, PI3K, TORC2/AKT, adenylyl cyclase and actin polymerization, which collectively direct chemotaxis. Upon ligand binding, mammalian GPCRs are phosphorylated at cytoplasmic residues, uncoupling G-protein pathways, but activating other pathways. However, connections between GPCR phosphorylation and chemotaxis are unclear. In developing Dictyostelium, secreted cAMP serves as a chemoattractant, with extracellular cAMP propagated as oscillating waves to ensure directional migratory signals. cAMP oscillations derive from transient excitatory responses of adenylyl cyclase, which then rapidly adapts. We have studied chemotactic signaling in Dictyostelium that express non-phosphorylatable cAMP receptors and show through chemotaxis modeling, single-cell FRET imaging, pure and chimeric population wavelet quantification, biochemical analyses and TIRF microscopy, that receptor phosphorylation is required to regulate adenylyl cyclase adaptation, long-range oscillatory cAMP wave production and cytoskeletal actin response. Phosphorylation defects thus promote hyperactive actin polymerization at the cell periphery, misdirected pseudopodia and the loss of directional chemotaxis. Our data indicate that chemoattractant receptor phosphorylation is required to co-regulate essential pathways for migratory cell polarization and chemotaxis. Our results significantly extend the understanding of the function of GPCR phosphorylation, providing strong evidence that this evolutionarily conserved mechanism is required in a signal attenuation pathway that is necessary to maintain persistent directional movement of Dictyostelium, neutrophils and other migratory cells.
doi:10.1242/jcs.122952
PMCID: PMC3795335  PMID: 23902692
GPCR; cAMP; PI3K; TORC2; AKT; Oscillations; Heterotrimeric G proteins; ERK2
4.  The Downstream Regulation of Chemokine Receptor Signalling: Implications for Atherosclerosis 
Mediators of Inflammation  2013;2013:459520.
Heterotrimeric G-protein-coupled receptors (GPCRs) are key mediators of intracellular signalling, control numerous physiological processes, and are one of the largest class of proteins to be pharmacologically targeted. Chemokine-induced macrophage recruitment into the vascular wall is an early pathological event in the progression of atherosclerosis. Leukocyte activation and chemotaxis during cell recruitment are mediated by chemokine ligation of multiple GPCRs. Regulation of GPCR signalling is critical in limiting vascular inflammation and involves interaction with downstream proteins such as GPCR kinases (GRKs), arrestin proteins and regulator of G-protein signalling (RGS) proteins. These have emerged as new mediators of atherogenesis by functioning in internalisation, desensitisation, and signal termination of chemokine receptors. Targeting chemokine signalling through these proteins may provide new strategies to alter atherosclerotic plaque formation and plaque biology.
doi:10.1155/2013/459520
PMCID: PMC3649756  PMID: 23690662
5.  Shaping the gradient by nonchemotactic chemokine receptors 
Cell Adhesion & Migration  2009;3(2):146-147.
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.
PMCID: PMC2679872  PMID: 19279397
Chemokine; chemokine receptor; leukocyte recruitment; chemotaxis; transcytosis
6.  T cell homeostasis requires G protein-coupled receptor-mediated access to trophic signals that promote growth and inhibit chemotaxis 
European journal of immunology  2005;35(3):786-795.
Signals that regulate T cell homeostasis are not fully understood. G protein-coupled receptors (GPCR), such as the chemokine receptors, may affect homeostasis by direct signaling or by guiding T cell migration to distinct location-restricted signals. Here, we show that blockade of Gαi-associated GPCR signaling by treatment with pertussis toxin led to T cell atrophy and shortened life-span in T cell-replete hosts and prevented T cell homeostatic growth and proliferation in T cell-deficient hosts. In vitro, however, neither GPCR inhibition nor chemokine stimulation affected T cell atrophy, survival, or proliferation. These findings suggest that GPCR signals are not trophic stimuli, but instead may be required for migration to distinct trophic signals, such as IL-7 or self-peptide/MHC. Surprisingly, while chemokines did not affect atrophy, atrophic T cells displayed increased chemokine-induced chemotaxis that was prevented by IL-7 and submitogenic anti-CD3 antibody treatment. This increase in migration was associated with increased levels of GTP-bound Rac and the ability to remodel actin. These data suggest a novel mechanism of T cell homeostasis wherein GPCR may promote T cell migration to distinct location-restricted homeostatic trophic cues for T cell survival and growth. Homeostatic trophic signals, in turn, may suppress chemokine sensitivity and cytoskeletal remodeling, to inhibit further migration.
doi:10.1002/eji.200425729
PMCID: PMC2628485  PMID: 15719365
G protein-coupled receptor; CXCR4; IL-7; Homeostasis; Rac
7.  WIP Regulates Persistence of Cell Migration and Ruffle Formation in Both Mesenchymal and Amoeboid Modes of Motility 
PLoS ONE  2013;8(8):e70364.
The spatial distribution of signals downstream from receptor tyrosine kinases (RTKs) or G-protein coupled receptors (GPCR) regulates fundamental cellular processes that control cell migration and growth. Both pathways rely significantly on actin cytoskeleton reorganization mediated by nucleation-promoting factors such as the WASP-(Wiskott-Aldrich Syndrome Protein) family. WIP (WASP Interacting Protein) is essential for the formation of a class of polarised actin microdomain, namely dorsal ruffles, downstream of the RTK for PDGF (platelet-derived growth factor) but the underlying mechanism is poorly understood. Using lentivirally-reconstituted WIP-deficient murine fibroblasts we define the requirement for WIP interaction with N-WASP (neural WASP) and Nck for efficient dorsal ruffle formation and of WIP-Nck binding for fibroblast chemotaxis towards PDGF-AA. The formation of both circular dorsal ruffles in PDGF-AA-stimulated primary fibroblasts and lamellipodia in CXCL13-treated B lymphocytes are also compromised by WIP-deficiency. We provide data to show that a WIP-Nck signalling complex interacts with RTK to promote polarised actin remodelling in fibroblasts and provide the first evidence for WIP involvement in the control of migratory persistence in both mesenchymal (fibroblast) and amoeboid (B lymphocytes) motility.
doi:10.1371/journal.pone.0070364
PMCID: PMC3737202  PMID: 23950925
8.  The Signaling Mechanisms Underlying Cell Polarity and Chemotaxis 
Chemotaxis—the directed movement of cells in a gradient of chemoattractant—is essential for neutrophils to crawl to sites of inflammation and infection and for Dictyostelium discoideum (D. discoideum) to aggregate during morphogenesis. Chemoattractant-induced activation of spatially localized cellular signals causes cells to polarize and move toward the highest concentration of the chemoattractant. Extensive studies have been devoted to achieving a better understanding of the mechanism(s) used by a neutrophil to choose its direction of polarity and to crawl effectively in response to chemoattractant gradients. Recent technological advances are beginning to reveal many fascinating details of the intracellular signaling components that spatially direct the cytoskeleton of neutrophils and D. discoideum and the complementary mechanisms that make the cell's front distinct from its back.
Spatially localized signaling allows cells to polarize and move toward chemoattractants. G proteins and products of PI 3-kinase control the necessary actin rearrangements.
doi:10.1101/cshperspect.a002980
PMCID: PMC2773618  PMID: 20066099
9.  GrlJ, a Dictyostelium GABAB-like receptor with roles in post-aggregation development 
Background
The G-protein-coupled receptor (GPCR) family represents the largest and most important group of targets for chemotherapeutics. They are extremely versatile receptors that transduce signals as diverse as biogenic amines, purins, odorants, ions and pheromones from the extracellular compartment to the interior via biochemical processes involving GTP-binding proteins. Until recently, the cyclic AMP receptors (cARs) were the only known G protein coupled receptors in Dictyostelium discoideum. The completed genome sequence revealed the presence of several families of GPCRs in Dictyostelium, among them members of the family 3 of GPCRs, the GABAB/glutamate like receptor family, which in higher eukaryotes is involved in neuronal signaling.
Results
D. discoideum has seventeen Family 3 members of GPCRs, denoted GrlA through GrlR. Their transcripts are detected throughout development with increased levels during early and late development. We have examined here GrlJ. GFP-tagged GrlJ localises to the plasmamembrane and to internal membranes. Inactivation of the grlJ gene leads to precocious development, and the mutant completes development ~6 hours earlier. Alterations were also noted at the slug stage and in spore formation. grlJ- slugs were longer and broke apart several times on their way to culmination forming smaller but proportionate fruiting bodies. Spores from grlJ- fruiting bodies were malformed and less viable, although the spore differentiation factors were synthesized and sensed normally. Expression of a GFP-tagged full length GrlJ rescued the phenotype.
Conclusion
Our data suggest that GrlJ acts at several stages of Dictyostelium development and that it is a negative regulator in Dictyostelium development.
doi:10.1186/1471-213X-7-44
PMCID: PMC1885808  PMID: 17501984
10.  A Synthetic Biology Approach Reveals a CXCR4-G13-Rho Signaling Axis Driving Transendothelial Migration of Metastatic Breast Cancer Cells 
Science signaling  2011;4(191):ra60.
Tumor cells can co-opt the pro-migratory activity of chemokines and their cognate G protein-coupled receptors (GPCRs) to metastasize to regional lymph nodes or distant organs. Indeed, the migration toward SDF-1 (stromal cell-derived factor-1) of tumor cells bearing CXCR4 [chemokine (C-X-C motif) receptor 4] has been implicated in the lymphatic and organ-specific metastasis of various human malignancies. Here, we used chimeric G proteins and GPCRs activated solely by artificial ligands to selectively activate the signaling pathways downstream of specific G proteins, and showed that CXCR4-mediated chemotaxis and transendothelial migration of metastatic basal-like breast cancer cells required activation of members of the Gα12/13 G protein family and of the small guanosine trisphosphatase Rho. Multiple complementary experimental strategies, including synthetic biology approaches, indicated that signaling-selective inhibition of the CXCR4-Gα13-Rho axis prevents the metastatic spread of basal-like breast cancer cells.
doi:10.1126/scisignal.2002221
PMCID: PMC3429372  PMID: 21934106
11.  FRAP Analysis of Chemosensory Components of Dictyostelium 
Methods in molecular biology (Clifton, N.J.)  2009;571:10.1007/978-1-60761-198-1_24.
Summary
Dictyostelium discoideum is a useful cell model for studying protein–protein interactions and deciphering complex signaling pathways similar to those found in mammalian systems. Many of these interactions were analyzed using classical in vitro biochemical techniques. However, with the accessibility of fluorescently tagged proteins, extensive protein networks are now being mapped out in living cells using a variety of microscopic techniques. One such technique, fluorescent recovery after photobleaching (FRAP), has been used in Dictyostelium to investigate a number of cellular processes including actin and cytoskeleton dynamics during chemotaxis and cytokinesis (J. Muscle Res. Cell Motil. 23:639–649, 2002; Biophys. J. 81:2010–2019, 2001; Mol. Biol. Cell 16:4256–4266, 2005), to follow trafficking of proteins to organelles such as the membrane, nucleus, and endoplasmic reticulum (Development 130:797–804, 2003; J. Cell Biol. 154:137–146, 2001), and to understand the role of proteins in cell adhesion during motility and division (Mol. Biol. Cell 18:4074–4084, 2007; J. Cell Sci. 120:4302–4309, 2007). FRAP is a powerful tool that should provide a vast amount of information on the mobility of a number of proteins, not only in Dictyostelium, but in many organisms. This study will lay out the methods of conducting FRAP experiments in Dictyostelium and discuss the large amount of knowledge which can be gained by adopting this as a common technique.
doi:10.1007/978-1-60761-198-1_24
PMCID: PMC3839318  PMID: 19763979
Dictyostelium; Photobleaching; Fluorescent proteins; FRAP; PTEN; PLC
12.  Amoeboid Chemotaxis 
Cell Adhesion & Migration  2007;1(4):165-170.
Chemotaxis is the directed movement of a cell towards a gradient of chemicals such as chemokines or growth factors. This phenomenon can be studied in organisms ranging from bacteria to mammalian cells, and here we will focus on eukaryotic amoeboid chemotaxis. Chemotactic responses are mediated by two major classes of receptors: GPCR's and RTK's, with multiple pathways signaling downstream of them, certain ones functioning in parallel. In this review we address two important features of amoeboid chemotaxis that will be important for further advances in the field. First, the application of in vivo imaging will be critical for providing insight into the functional requirements for chemotactic responses. We will briefly cover a number of systems in which in vivo imaging is providing new insights. Second, due to the network-type design of signaling pathways of eukaryotic chemotaxis, more refined phenotypic analysis will be necessary, and we will discuss recent analyses of the role of the phosphoinositide 3-kinase pathway in this light. We will close with some speculations regarding future applications of more detailed in vivo analysis and mechanistic understanding of eukaryotic amoeboid chemotaxis.
PMCID: PMC2634101  PMID: 19262145
chemotaxis; signaling; in vivo models; development; phospholipase; phosphoinositide 3-kinase
13.  Tumor progression locus 2 regulates signal-induced calcium release from the endoplamic reticulum and cell migration 
Science Signaling  2011;4(187):ra55.
The mitogen-activated protein kinase kinase kinase (MAPKKK) tumor progression locus 2 (Tpl2) is required for the transduction of signals initiated by the thrombin-activated G protein-coupled receptor (GPCR) protease activated receptor-1 (PAR1), which promote reorganization of the actin cytoskeleton and cell migration. Here, we show that Tpl2 is activated through Gαi2-transduced GPCR signals. Activated Tpl2 promotes the phosphorylation and activation of phospholipase C beta 3 (PLCβ3) and, concsequently, Tpl2 is required for thrombin-dependent production of inositol 1,4,5-triphosphate (IP3), the upregulation of cytoplasmic Ca2+, and the activation of classical and novel members of the protein kinase C (PKC) family. A PKC feedback loop facilitates extracellular signal-regulated kinase (ERK) activation in response to Tpl2 and contributes to the coordinate regulation of the ERK and Ca2+ signaling pathways. Pharmacological and genetic studies revealed that stimulation of cell migration by Tpl2 depends on both of these pathways. Tpl2 also promoted Ca2+ signals and cell migration from Gαi-coupled GPCRs other than PAR1, and from the IL-1β receptor. Our data provide new insights into the role of Tpl2 in GPCR-mediated Ca2+ signaling and cell migration. In addition, they enhance our understanding of the fundamental role of Tpl2 in innate and adaptive immunity, cancer and inflammation.
doi:10.1126/scisignal.2002006
PMCID: PMC3340127  PMID: 21868363
14.  Myosin I 
Phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) is a key signaling molecule in chemotaxis, a directed cell migration toward chemoattractants. PtdIns(3,4,5)P3 is transiently generated by chemotactic stimulation and activates reorganization of the actin cytoskeleton at the leading edge of migrating cells. In a recent study, we demonstrated that PtdIns(3,4,5)P3 directly binds to three members of the actin-based motor protein myosin I (myosin ID, IE and IF) in Dictyostelium discoideum and recruits these proteins to the plasma membrane of the leading edge. The PtdIns(3,4,5)P3-regulated membrane recruitment of myosin I induced chemoattractant-stimulated actin polymerization and was therefore required for chemotaxis. Similarly, human myosin IF was translocated to the plasma membrane through interactions with PtdIns(3,4,5)P3 upon chemotactic stimulation in a neutrophil cell line. Interestingly, we also found that the three PtdIns(3,4,5)P3-binding myosin I proteins function in phagocytosis, which involves both PtdIns(3,4,5)P3 signaling and actin cytoskeleton remodeling. Our findings provide an evolutionarily conserved mechanism by which class I myosin transmits PtdIns(3,4,5)P3 signals to the actin cytoskeleton.
doi:10.4161/cib.19892
PMCID: PMC3419119  PMID: 22896797
chemotaxis; phagocytosis; PIP3; myosin I; actin; Dictyostelium
15.  Similarities and Differences in RANTES- and (AOP)-RANTES–triggered Signals: Implications for Chemotaxis  
The Journal of Cell Biology  1999;144(4):755-765.
Chemokines are a family of proinflammatory cytokines that attract and activate specific types of leukocytes. Chemokines mediate their effects via interaction with seven transmembrane G protein–coupled receptors (GPCR). Using CCR5-transfected HEK-293 cells, we show that both the CCR5 ligand, RANTES, as well as its derivative, aminooxypentane (AOP)- RANTES, trigger immediate responses such as Ca2+ influx, receptor dimerization, tyrosine phosphorylation, and Gαi as well as JAK/STAT association to the receptor. In contrast to RANTES, (AOP)-RANTES is unable to trigger late responses, as measured by the association of focal adhesion kinase (FAK) to the chemokine receptor complex, impaired cell polarization required for migration, or chemotaxis. The results are discussed in the context of the dissociation of the late signals, provoked by the chemokines required for cell migration, from early signals.
PMCID: PMC2132943  PMID: 10037796
chemokines; receptor dimerization; inflammation; HIV-1
16.  Expression of Actin-interacting Protein 1 Suppresses Impaired Chemotaxis of Dictyostelium Cells Lacking the Na+-H+ Exchanger NHE1 
Molecular Biology of the Cell  2010;21(18):3162-3170.
Dictyostelium cells lacking the intracellular pH regulator NHE1 have defective chemotaxis. A modifier screen and reconstitution studies show expression of recombinant actin interacting protein 1 (Aip1) suppresses the Ddnhe1-phenotype. Aip1 promotes cofilin-dependent actin remodeling, which is likely a major determinant in pH-dependent chemotaxis.
Increased intracellular pH is an evolutionarily conserved signal necessary for directed cell migration. We reported previously that in Dictyostelium cells lacking H+ efflux by a Na+-H+ exchanger (NHE; Ddnhe1−), chemotaxis is impaired and the assembly of filamentous actin (F-actin) is attenuated. We now describe a modifier screen that reveals the C-terminal fragment of actin-interacting protein 1 (Aip1) enhances the chemotaxis defect of Ddnhe1− cells but has no effect in wild-type Ax2 cells. However, expression of full-length Aip1 mostly suppresses chemotaxis defects of Ddnhe1− cells and restores F-actin assembly. Aip1 functions to promote cofilin-dependent actin remodeling, and we found that although full-length Aip1 binds cofilin and F-actin, the C-terminal fragment binds cofilin but not F-actin. Because pH-dependent cofilin activity is attenuated in mammalian cells lacking H+ efflux by NHE1, our current data suggest that full-length Aip1 facilitates F-actin assembly when cofilin activity is limited. We predict the C-terminus of Aip1 enhances defective chemotaxis of Ddnhe1− cells by sequestering the limited amount of active cofilin without promoting F-actin assembly. Our findings indicate a cooperative role of Aip1 and cofilin in pH-dependent cell migration, and they suggest defective chemotaxis in Ddnhe1− cells is determined primarily by loss of cofilin-dependent actin dynamics.
doi:10.1091/mbc.E09-12-1058
PMCID: PMC2938382  PMID: 20668166
17.  A Continuum Model of Actin Waves in Dictyostelium discoideum 
PLoS ONE  2013;8(5):e64272.
Actin waves are complex dynamical patterns of the dendritic network of filamentous actin in eukaryotes. We developed a model of actin waves in PTEN-deficient Dictyostelium discoideum by deriving an approximation of the dynamics of discrete actin filaments and combining it with a signaling pathway that controls filament branching. This signaling pathway, together with the actin network, contains a positive feedback loop that drives the actin waves. Our model predicts the structure, composition, and dynamics of waves that are consistent with existing experimental evidence, as well as the biochemical dependence on various protein partners. Simulation suggests that actin waves are initiated when local actin network activity, caused by an independent process, exceeds a certain threshold. Moreover, diffusion of proteins that form a positive feedback loop with the actin network alone is sufficient for propagation of actin waves at the observed speed of . Decay of the wave back can be caused by scarcity of network components, and the shape of actin waves is highly dependent on the filament disassembly rate. The model allows retraction of actin waves and captures formation of new wave fronts in broken waves. Our results demonstrate that a delicate balance between a positive feedback, filament disassembly, and local availability of network components is essential for the complex dynamics of actin waves.
doi:10.1371/journal.pone.0064272
PMCID: PMC3669376  PMID: 23741312
18.  Chemokines, selectins and intracellular calcium flux: temporal and spatial cues for leukocyte arrest 
Leukocyte trafficking to acute sites of injury or infection requires spatial and temporal cues that fine tune precise sites of firm adhesion and guide migration to endothelial junctions where they undergo diapedesis to sites of insult. Many detailed studies on the location and gradient of chemokines such as IL-8 and other CXCR ligands reveal that their recognition shortly after selectin-mediated capture and rolling exerts acute effects on integrin activation and subsequent binding to their ligands on the endothelium, which directs firm adhesion, adhesion strengthening, and downstream migration. In this process, G-protein coupled receptor (GPCR) signaling has been found to play an integral role in activating and mobilizing intracellular stores of calcium, GTPases such as Rap-1 and Rho and cytokeletal proteins such as Talin and F-actin to facilitate cell polarity and directional pseudopod formation. A critical question remaining is how intracellular Ca2+ flux from CRAC channels such as Orai1 synergizes with cytosolic stores to mediate a rapid flux which is critical to the onset of PMN arrest and polarization. Our review will highlight a specific role for calcium as a signaling messenger in activating focal clusters of integrins bound to the cytoskeleton which allows the cell to attain a migratory phenotype. The precise interplay between chemokines, selectins, and integrins binding under the ubiquitous presence of shear stress from blood flow provides an essential cooperative signaling mechanism for effective leukocyte recruitment.
doi:10.3389/fimmu.2012.00188
PMCID: PMC3392659  PMID: 22787461
calcium; chemokine; cytoskeletal proteins; inflammation; integrin affinity; LFA-1; neutrophils; Orai1
19.  Regulation of the formation and trafficking of vesicles from Golgi by PCH Family Proteins During Chemotaxis 
Biochimica et biophysica acta  2009;1793(7):1199-1209.
Previous study demonstrated that WASP localizes on vesicles during Dictyostelium chemotaxis and these vesicles appear to be preferentially distributed at the leading and trailing edge of migrating cells. In this study, we have examined the role of PCH family proteins, Nwk/Bzz1p-like protein (NLP) and Syndapin-like protein (SLP), in the regulation of the formation and trafficking of WASP-vesicles during chemotaxis. NLP and SLP appear to be functionally redundant and deletion of both nlp and slp genes cause the loss of polararized F-actin organization and significant defects in chemotaxis. WASP and NLP are colocalized on vesicles and interactions between two molecules via the SH3 domain of NLP/SLP and the proline-rich repeats of WASP are required for vesicle formation from Golgi. Microtubules are required for polarized trafficking of these vesicles as vesicles showing high directed mobility are absent in cells treated with nocodazole. Our results suggest that interaction of WASP with NLP/SLP is required for the formation and trafficking of vesicles from Golgi to the membrane, which might play a central role in the establishment of cell polarity during chemotaxis.
doi:10.1016/j.bbamcr.2009.04.012
PMCID: PMC2703453  PMID: 19409937
WASP; PCH family protein; Vesicle trafficking; Actin; Cytoskeleton; Cell Polarity
20.  Genetic analysis of the role of G protein–coupled receptor signaling in electrotaxis 
The Journal of Cell Biology  2002;157(6):921-928.
Cells display chemotaxis and electrotaxis by migrating directionally in gradients of specific chemicals or electrical potential. Chemotaxis in Dictyostelium discoideum is mediated by G protein–coupled receptors. The unique Gβ is essential for all chemotactic responses, although different chemoattractants use different receptors and Gα subunits. Dictyostelium amoebae show striking electrotaxis in an applied direct current electric field. Perhaps electrotaxis and chemotaxis share similar signaling mechanisms? Null mutation of Gβ and cAMP receptor 1 and Gα2 did not abolish electrotaxis, although Gβ-null mutations showed suppressed electrotaxis. By contrast, G protein signaling plays an essential role in chemotaxis. G protein–coupled receptor signaling was monitored with PHcrac–green fluorescent protein, which translocates to inositol phospholipids at the leading edge of cells during chemotaxis. There was no intracellular gradient of this protein during electrotaxis. However, F-actin was polymerized at the leading edge of cells during electrotaxis. We conclude that reception and transduction of the electrotaxis signal are largely independent of G protein–coupled receptor signaling and that the pathways driving chemotaxis and electrotaxis intersect downstream of heterotrimeric G proteins to invoke cytoskeletal elements.
doi:10.1083/jcb.200112070
PMCID: PMC2174050  PMID: 12045182
Dictyostelium; cell migration; electrotaxis; electric fields; G protein–coupled receptor signaling
21.  The Evolution of the GPCR Signaling System in Eukaryotes: Modularity, Conservation, and the Transition to Metazoan Multicellularity 
Genome Biology and Evolution  2014;6(3):606-619.
The G-protein-coupled receptor (GPCR) signaling system is one of the main signaling pathways in eukaryotes. Here, we analyze the evolutionary history of all its components, from receptors to regulators, to gain a broad picture of its system-level evolution. Using eukaryotic genomes covering most lineages sampled to date, we find that the various components of the GPCR signaling pathway evolved independently, highlighting the modular nature of this system. Our data show that some GPCR families, G proteins, and regulators of G proteins diversified through lineage-specific diversifications and recurrent domain shuffling. Moreover, most of the gene families involved in the GPCR signaling system were already present in the last common ancestor of eukaryotes. Furthermore, we show that the unicellular ancestor of Metazoa already had most of the cytoplasmic components of the GPCR signaling system, including, remarkably, all the G protein alpha subunits, which are typical of metazoans. Thus, we show how the transition to multicellularity involved conservation of the signaling transduction machinery, as well as a burst of receptor diversification to cope with the new multicellular necessities.
doi:10.1093/gbe/evu038
PMCID: PMC3971589  PMID: 24567306
arrestin; phosducin; Ric8; GRK; heterotrimeric G protein complex
22.  G Protein–Coupled Receptor Sorting to Endosomes and Lysosomes 
The heptahelical G protein–coupled receptors (GPCRs) belong to the largest family of cell surface signaling receptors encoded in the human genome. GPCRs signal to diverse extracellular stimuli and control a vast number of physiological responses, making this receptor class the target of nearly half the drugs currently in use. In addition to rapid desensitization, receptor trafficking is crucial for the temporal and spatial control of GPCR signaling. Sorting signals present in the intracytosolic domains of GPCRs regulate trafficking through the endosomal-lysosomal system. GPCR internalization is mediated by serine and threonine phosphorylation and arrestin binding. Short, linear peptide sequences including tyrosine- and dileucine-based motifs, and PDZ ligands that are recognized by distinct endocytic adaptor proteins also mediate internalization and endosomal sorting of GPCRs. We present new data from bioinformatic searches that reveal the presence of these types of sorting signals in the cytoplasmic tails of many known GPCRs. Several recent studies also indicate that the covalent modification of GPCRs with ubiquitin serves as a signal for internalization and lysosomal sorting, expanding the diversity of mechanisms that control trafficking of mammalian GPCRs.
doi:10.1146/annurev.pharmtox.48.113006.094646
PMCID: PMC2869288  PMID: 17995450
GPCR; arrestin; ubiquitin; trafficking; clathrin; PDZ; bioinformatic
23.  Computational methods in drug design: Modeling G protein-coupled receptor monomers, dimers, and oligomers 
The AAPS Journal  2006;8(2):E322-E336.
G protein-coupled receptors (GPCRs) are membrane proteins that serve as very important links through which cellular signal transduction mechanisms are activated. Many vital physiological events such as sensory perception, immune defense, cell communication, chemotaxis, and neuro-transmission are mediated by GPCRs. Not surprisingly, GPCRs are major targets for drug development today. Most modeling studies in the GPCR field have focused upon the creation of a model of a single GPCR (ie, a GPCR monomer) based upon the crystal structure of the Class A GPCR, rhodopsin. However, the emerging concept of GPCR dimerization has challenged our notions of the monomeric GPCR as functional unit. Recent work has shown not only that many GPCRs exist as homo- and heterodimers but also that GPCR oligomeric assembly may have important functional roles. This review focuses first on methodology for the creation of monomeric GPCR models. Special emphasis is given to the identification of localized regions where the structure of a GPCR may diverge from that of bovine rhodopsin. The review then focuses on GPCR dimers and oligomers and the bioinformatics methods available for identifying homo- and heterodimer interfaces.
doi:10.1007/BF02854903
PMCID: PMC3231557  PMID: 16796383
GPCR modeling; GPCR dimer; GPCR oligomer
24.  Four key signaling pathways mediating chemotaxis in Dictyostelium discoideum 
The Journal of Cell Biology  2008;180(4):747-753.
Chemotaxis is the ability of cells to move in the direction of an external gradient of signaling molecules. Cells are guided by actin-filled protrusions in the front, whereas myosin filaments retract the rear of the cell. Previous work demonstrated that chemotaxis of unpolarized amoeboid Dictyostelium discoideum cells is mediated by two parallel pathways, phosphoinositide-3-kinase (PI3K) and phospholipase A2 (PLA2). Here, we show that polarized cells exhibit very good chemotaxis with inhibited PI3K and PLA2 activity. Using genetic screens, we demonstrate that this activity is mediated by a soluble guanylyl cyclase, providing two signals. The protein localizes to the leading edge where it interacts with actin filaments, whereas the cyclic guanosine monophosphate product induces myosin filaments in the rear of the cell. We conclude that chemotaxis is mediated by multiple signaling pathways regulating protrusions at the front and rear of the cell. Cells that express only rear activity are polarized but do not exhibit chemotaxis, whereas cells with only front signaling are unpolarized but undergo chemotaxis.
doi:10.1083/jcb.200709180
PMCID: PMC2265585  PMID: 18299345
25.  Select Neuropeptides and their G-Protein Coupled Receptors in Caenorhabditis Elegans and Drosophila Melanogaster 
The G-protein coupled receptor (GPCR) family is comprised of seven transmembrane domain proteins and play important roles in nerve transmission, locomotion, proliferation and development, sensory perception, metabolism, and neuromodulation. GPCR research has been targeted by drug developers as a consequence of the wide variety of critical physiological functions regulated by this protein family. Neuropeptide GPCRs are the least characterized of the GPCR family as genetic systems to characterize their functions have lagged behind GPCR gene discovery. Drosophila melanogaster and Caenorhabditis elegans are genetic model organisms that have proved useful in characterizing neuropeptide GPCRs. The strength of a genetic approach leads to an appreciation of the behavioral plasticity that can result from subtle alterations in GPCRs or regulatory proteins in the pathways that GPCRs control. Many of these invertebrate neuropeptides, GPCRs, and signaling pathway components serve as models for mammalian counterparts as they have conserved sequences and function. This review provides an overview of the methods to match neuropeptides to their cognate receptor and a state of the art account of neuropeptide GPCRs that have been characterized in D. melanogaster and C. elegans and the behaviors that have been uncovered through genetic manipulation.
doi:10.3389/fendo.2012.00093
PMCID: PMC3414713  PMID: 22908006
invertebrate neuropeptides; G-protein coupled receptor; insects; nematodes; Caenorhabditis elegans; Drosophila melanogaster

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