Anna Aragay | CSIC (Consejo Superior de Investigaciones Científicas-Spanish National Research Council) (original) (raw)

Papers by Anna Aragay

Trabajo presentado en la Molecular Pharmacology. Gordon Conference, celebrada en Ventura, CA, US,... more Trabajo presentado en la Molecular Pharmacology. Gordon Conference, celebrada en Ventura, CA, US, del 1 al 6 de febrero de 2015

Journal of Cell Science, 2016

Proper endosomal trafficking of ligand-activated G-protein-coupled receptors (GPCRs) is essential... more Proper endosomal trafficking of ligand-activated G-protein-coupled receptors (GPCRs) is essential to spatiotemporally tune their physiological responses. For the monocyte chemoattractant receptor 2 (CCR2B; one of two isoforms encoded by CCR2), endocytic recycling is important to sustain monocyte migration, whereas filamin A (FLNa) is essential for CCL2-induced monocyte migration. Here, we analyze the role of FLNa in the trafficking of CCR2B along the endocytic pathway. In FLNa-knockdown cells, activated CCR2B accumulated in enlarged EEA-1-positive endosomes, which exhibited slow movement and fast fluorescence recovery, suggesting an imbalance between receptor entry and exit rates. Utilizing super-resolution microscopy, we observed that FLNa-GFP, CCR2B and β2-adrenergic receptor (β2AR) were present in actin-enriched endosomal microdomains. Depletion of FLNa decreased CCR2B association with these microdomains and concomitantly delayed CCR2B endosomal traffic, without apparently affecting the number of microdomains. Interestingly, CCR2B and β2AR signaling induced phosphorylation of FLNa at residue S2152, and this phosphorylation event was contributes to sustain receptor recycling. Thus, our data strongly suggest that CCR2B and β2AR signals to FLNa to stimulate its endocytosis and recycling to the plasma membrane.

Journal of Biological Chemistry, Dec 1, 1992

eLife, Apr 25, 2023

Kazrin is a protein widely expressed in vertebrates whose depletion causes a myriad of developmen... more Kazrin is a protein widely expressed in vertebrates whose depletion causes a myriad of developmental defects, in part derived from altered cell adhesion and migration, as well as failure to undergo epidermal to mesenchymal transition. However, the primary molecular role of kazrin, which might contribute to all these functions, has not been elucidated yet. We previously identified one of its isoforms, kazrin C, as a protein that potently inhibits clathrin-mediated endocytosis when overexpressed. We now generated kazrin knockout mouse embryonic fibroblasts to investigate its endocytic function. We found that kazrin depletion delays juxtanuclear enrichment of internalized material, indicating a role in endocytic traffic from early to recycling endosomes. Consistently, we found that the C-terminal domain of kazrin C, predicted to be an intrinsically disordered region, directly interacts with several early endosome (EE) components, and that kazrin depletion impairs retrograde motility of these organelles. Further, we noticed that the N-terminus of kazrin C shares homology with dynein/dynactin adaptors and that it directly interacts with the dynactin complex and the dynein light intermediate chain 1. Altogether, the data indicate that one of the primary kazrin functions is to facilitate endocytic recycling by promoting dynein/dynactin-dependent transport of EEs or EE-derived transport intermediates to the recycling endosomes. Editor's evaluation In their paper, Hernandez-Perez et al. perform a detailed and solid analysis of kazrin, a widely expressed protein that appears to be involved in many diverse cellular processes, but whose exact function is unknown. The authors employ mouse embryonic fibroblasts and biochemistry to investigate the function of Kazrin and determine that Kazrin promotes the dynein/dynactin-dependent transport of early endosomes. These valuable findings will be of interest to those in the field of intracellular transport.

Journal of Biological Chemistry, Jun 1, 2015

Background: Rgnef (ArhGEF28) is activated downstream of gastrin and the cholecystokinin receptor ... more Background: Rgnef (ArhGEF28) is activated downstream of gastrin and the cholecystokinin receptor to promote colon carcinoma tumor progression. Results: Rgnef activation by G␣ 13 triggers FAK and paxillin tyrosine phosphorylation in response to gastrin. A C-terminal Rgnef region is necessary for linkage to G␣ 13. Conclusion: Rgnef is an effector of G␣ 13 signaling. Significance: G␣ 13 and Rgnef are implicated in colon carcinoma. The guanine nucleotide exchange factor Rgnef (also known as ArhGEF28 or p190RhoGEF) promotes colon carcinoma cell motility and tumor progression via interaction with focal adhesion kinase (FAK). Mechanisms of Rgnef activation downstream of integrin or G protein-coupled receptors remain undefined. In the absence of a recognized G protein signaling homology domain in Rgnef, no proximal linkage to G proteins was known. Utilizing multiple methods, we have identified Rgnef as a new effector for G␣ 13 downstream of gastrin and the type 2 cholecystokinin receptor. In DLD-1 colon carcinoma cells depleted of G␣ 13 , gastrin-induced FAK Tyr(P)-397 and paxillin Tyr(P)-31 phosphorylation were reduced. RhoA GTP binding and promoter activity were increased by Rgnef in combination with active G␣ 13. Rgnef co-immunoprecipitated with activated G␣ 13 Q226L but not G␣ 12 Q229L. The Rgnef C-terminal (CT, 1279-1582) region was sufficient for co-immunoprecipitation, and Rgnef-CT exogenous expression prevented G␣ 13-stimulated SRE activity. A domain at the C terminus of the protein close to the FAK binding domain is necessary to bind to G␣ 13. Point mutations of Rgnef-CT residues disrupt association with active G␣ 13 but not G␣ q. These results show that Rgnef functions as an effector of G␣ 13 signaling and that this linkage may mediate FAK activation in DLD-1 colon carcinoma cells.

Journal of Biological Chemistry, Oct 1, 1993

Nucleotide binding properties of the G protein a subunit Goa were probed by mutational analysis i... more Nucleotide binding properties of the G protein a subunit Goa were probed by mutational analysis in recombinant Escherichia coli. Thousands of random mutations generated by polymerase chain reaction were screened by in situ [35S]GTPyS (guanosine 5'43-0-thio)triphosphate) binding on the colony lifts following transformation of bacteria with modified G,(Y cDNA. Clones that did not bind the nucleotide under these conditions were characterized by DNA sequence analysis, and the nucleotide binding properties were further studied in crude bacterial extracts. A number of novel mutations reducing the affinity of Goa for GTPyS or Mg2' were identified. Some of the mutations substitute amino acid residues homologous to those known to interact with guanine nucleotides in ~2 1 "~ proteins. Other mutations show that previously unstudied residues also participate in the nucleotide binding. Several mutants lost GTP@ binding but retained the capacity to interact with the Py subunit complex as determined by pertussis toxin-mediated ADP-ribosylation. One of these, mutant S47C, was functionally expressed in Xenopus laevis oocytes along with the G protein-coupled thyrotropin-releasing hormone (TRH) receptor. Whereas wild-type Goa increased TRH-promoted chloride currents, S47C significantly decreased the hormone-induced C1response, suggesting that this mutation resulted in a dominant negative phenotype. Heterotrimeric GTP-binding proteins (G proteins)* link the excitation of cell-surface receptors to the activity of intracellular effector enzymes or ion channels. G protein specificity is thought to be determined at least in part by the a subunits that are most divergent and contain the GTP binding site. The protein-protein interactions of the a subunit with the /3y subunit complex, receptor, and effector depend upon a cycle of binding and hydrolysis of the nucleotide. In the GDP-bound form, the protein is essentially in a resting state primed to receive the incoming signal. In the GTP-bound active form, the G protein can interact with its effector starting a chain of downstream intracellular events. Activation of G protein is a complicated process that includes receptor-catalyzed dissociation of the prebound GDP and binding of GTP and M 8 + , which triggers a strong change in a subunit conformation and dissociation of the aPy heterotrimer (for reviews, see Refs. 14). Much of the understanding of the mechanisms of G protein function has come * The costs of publication of this article were defrayed in part by the

Journal of Immunology, Jul 15, 1998

IL-2-regulated expression of the monocyte chemotactic protein-1 receptor (CCR2) in human NK cells... more IL-2-regulated expression of the monocyte chemotactic protein-1 receptor (CCR2) in human NK cells: characterization of a predominant 3.4-kilobase transcript containing CCR2B and CCR2A sequences.

Comunicación presentada en The 43 FEBS Congress, celebrado en Praga (República Checa) del 7 al 12... more Comunicación presentada en The 43 FEBS Congress, celebrado en Praga (República Checa) del 7 al 12 de julio de 2018

Poster presentado en el XXXVI Congreso de la Sociedad Espanola de Bioquimica y Biologia Molecular... more Poster presentado en el XXXVI Congreso de la Sociedad Espanola de Bioquimica y Biologia Molecular SEBBM, celebrada del 3 al 6 de septiembre de 2013 en Madrid (Espana)

Trabajo presentado en el XXXIX Congreso de la SEBBM, celebrado en Salamanca, Espana, del 5 al 8 d... more Trabajo presentado en el XXXIX Congreso de la SEBBM, celebrado en Salamanca, Espana, del 5 al 8 de septiembre de 2016

Antioxidants

All processes in human physiology relies on homeostatic mechanisms which require the activation o... more All processes in human physiology relies on homeostatic mechanisms which require the activation of specific control circuits to adapt the changes imposed by external stimuli. One of the critical modulators of homeostatic balance is autophagy, a catabolic process that is responsible of the destruction of long-lived proteins and organelles through a lysosome degradative pathway. Identification of the mechanism underlying autophagic flux is considered of great importance as both protective and detrimental functions are linked with deregulated autophagy. At the mechanistic and regulatory levels, autophagy is activated in response to diverse stress conditions (food deprivation, hyperthermia and hypoxia), even a novel perspective highlight the potential role of physical forces in autophagy modulation. To understand the crosstalk between all these controlling mechanisms could give us new clues about the specific contribution of autophagy in a wide range of diseases including vascular disor...

Trabajo presentado en la Gordon Conference on Molecular Pharmacology, celebrada en Lucca, Italia,... more Trabajo presentado en la Gordon Conference on Molecular Pharmacology, celebrada en Lucca, Italia, del 28 de abril al 3 de mayo de 2013

Endocrinology, 1996

PTH-related protein activates a G protein-coupled PTWPTHrP receptor in many cell types and produc... more PTH-related protein activates a G protein-coupled PTWPTHrP receptor in many cell types and produces diverse biological actions. To study the signal transduction events associated with biological activity of the PTH/F'THrP receptor in vascular smooth muscle, a principal PTHrP-responsive tissue, rat aortic smooth muscle cells (AlO) were stably transfected with a plasmid encoding a PTWPTHrP receptor and tested for ligand binding, PTHrP-(l-34)-induced CAMP levels, inositol phosphate production, and cytosolic calcium transients. Of nineteen G418-resistant lines recovered, all exhibited high affinity binding I-dissociation constant (K,) >lO I") of iodinated [Tyr""lhPTHrP(l-36)NH, and ligand-induced CAMP accumulation (2-to loo-fold), which was directly proportional to PTIWPTHrP reP

Trabajo presentado en la Gordon Research Conference (GRC) Molecular Pharmacology, celebrada en Ve... more Trabajo presentado en la Gordon Research Conference (GRC) Molecular Pharmacology, celebrada en Ventura, California (Estados Unidos) del 10 al 15 de febrero de 2019.

Biochemical and Biophysical Research Communications, 2014

Proceedings of the National Academy of Sciences, 1998

Monocyte chemoattractant protein 1 (MCP-1) is a member of the chemokine cytokine family, whose ph... more Monocyte chemoattractant protein 1 (MCP-1) is a member of the chemokine cytokine family, whose physiological function is mediated by binding to the CCR2 and CCR4 receptors, which are members of the G protein-coupled receptor family. MCP-1 plays a critical role in both activation and migration of leukocytes. Rapid chemokine receptor desensitization is very likely essential for accurate chemotaxis. In this report, we show that MCP-1 binding to the CCR2 receptor in Mono Mac 1 cells promotes the rapid desensitization of MCP-1-induced calcium flux responses. This desensitization correlates with the Ser/Thr phosphorylation of the receptor and with the transient translocation of the G protein-coupled receptor kinase 2 (GRK2, also called β-adrenergic kinase 1 or βARK1) to the membrane. We also demonstrate that GRK2 and the uncoupling protein β-arrestin associate with the receptor, forming a macromolecular complex shortly after MCP-1 binding. Calcium flux responses to MCP-1 in HEK293 cells e...

Molecular Pharmacology, 2003

G protein-coupled receptors (GPCRs) initiate diverse downstream signaling events in response to l... more G protein-coupled receptors (GPCRs) initiate diverse downstream signaling events in response to ligand stimulation, as rapid activation of the extracellular signal-regulated kinase ERK1 and ERK2. The chemokine monocyte chemoattractant protein-1 (MCP-1) is the agonist for several chemokine receptors that belong to the GPCR superfamily, CCR2 being the most important. Stimulation of mitogen-activated protein kinases (MAPKs) by MCP-1 has been implicated in integrin activation and chemotaxis, but the molecular pathways downstream of the receptors remain unclear. To dissect the cascade of events leading to MAPK activation upon CCR2 receptor stimulation, several specific inhibitors and mutants of signal transduction proteins were used in monocytic cells endogenously expressing CCR2 and/or in human embryonic kidney-293 cells transfected with CCR2B receptors and epitope-tagged ERK1. We show that ERK activation by MCP-1 involves heterotrimeric G i protein subunits, protein kinase C, phosphoinositide-3-kinase, and Ras. On the other hand, the activity of cytosolic tyrosine kinases, epidermal growth factor receptor transactivation, or variations in intracellular calcium levels are not required for the mitogenic activation elicited by MCP-1. In addition, we find that internalization of CCR2B itself is not necessary for efficient MCP-1-induced activation of ERK, although a dynamin mutant partially inhibits ERK stimulation. These results suggest that different parallel pathways are being activated that lead to the full activation of the mitogen-activated protein kinase cascade and that internalization of other signaling proteins but not of the receptor is required for complete ERK activation.