Regulation of G protein-coupled receptor kinase subtypes by calcium sensor proteins

AbstractG protein-coupled receptor homologous desensitization is intrinsically related to the function of a class of S/T kinases named G protein-coupled receptor kinases (GRK). The GRK family is composed of six cloned members, named GRK1 to 6. Studies from different laboratories have demonstrated that different calcium sensor proteins (CSP) can selectively regulate the activity of GRK subtypes. In the presence of calcium, rhodopsin kinase (GRK1) is inhibited by the photoreceptor-specific CSP recoverin through direct binding. Several other recoverin homologues (including NCS 1, VILIP 1 and hippocalcin) are also able to inhibit GRK1. The ubiquitous calcium-binding protein calmodulin (CaM) can inhibit GRK5 with a high affinity (IC50=40–50 nM). A direct interaction between GRK5 and Ca2+/CaM was documented and this binding does not influence the catalytic activity of the kinase, but rather reduced GRK5 binding to the membrane. These studies suggest that CSP act as functional analogues in mediating the regulation of different GRK subtypes by Ca2+. This mechanism is, however, highly selective with respect to the GRK subtypes: while GRK1, but not GRK2 and GRK5, is regulated by recoverin and other NCS, GRK4, 5 and 6, that belong to the GRK4 subfamily, are potently inhibited by CaM, which had little or no effect on members of other GRK subfamilies

Interactions of Mast Cell Tryptase with Thrombin Receptors and PAR-2

Tryptase is a serine protease secreted by mast cells that is able to activate other cells. In the present studies we have tested whether these responses could be mediated by thrombin receptors or PAR-2, two G-protein-coupled receptors that are activated by proteolysis. When added to a peptide corresponding to the N terminus of PAR-2, tryptase cleaved the peptide at the activating site, but at higher concentrations it also cleaved downstream, as did trypsin, a known activator of PAR-2. Thrombin, factor Xa, plasmin, urokinase, plasma kallikrein, and tissue kallikrein had no effect. Tryptase also cleaved the analogous thrombin receptor peptide at the activating site but less efficiently. When added to COS-1 cells expressing either receptor, tryptase stimulated phosphoinositide hydrolysis. With PAR-2, this response was half-maximal at 1 nM tryptase and could be inhibited by the tryptase inhibitor, APC366, or by antibodies to tryptase and PAR-2. When added to human endothelial cells, which normally express PAR-2 and thrombin receptors, or keratinocytes, which express only PAR-2, tryptase caused an increase in cytosolic Ca2+. However, when added to platelets or CHRF-288 cells, which express thrombin receptors but not PAR-2, tryptase caused neither aggregation nor increased Ca2+. These results show that 1) tryptase has the potential to activate both PAR-2 and thrombin receptors; 2) for PAR-2, this potential is realized, although cleavage at secondary sites may limit activation, particularly at higher tryptase concentrations; and 3) in contrast, although tryptase clearly activates thrombin receptors in COS-1 cells, it does not appear to cleave endogenous thrombin receptors in platelets or CHRF-288 cells. These distinctions correlate with the observed differences in the rate of cleavage of the PAR-2 and thrombin receptor peptides by tryptase. Tryptase is the first protease other than trypsin that has been shown to activate human PAR-2. Its presence within mast cell granules places it in tissues where PAR-2 is expressed but trypsin is unlikely to reach

Fucans, but Not Fucomannoglucuronans, Determine the Biological Activities of Sulfated Polysaccharides from Laminaria saccharina Brown Seaweed

Sulfated polysaccharides from Laminaria saccharina (new name: Saccharina latissima) brown seaweed show promising activity for the treatment of inflammation, thrombosis, and cancer; yet the molecular mechanisms underlying these properties remain poorly understood. The aim of this work was to characterize, using in vitro and in vivo strategies, the anti-inflammatory, anti-coagulant, anti-angiogenic, and anti-tumor activities of two main sulfated polysaccharide fractions obtained from L. saccharina: a) L.s.-1.0 fraction mainly consisting of O-sulfated mannoglucuronofucans and b) L.s.-1.25 fraction mainly composed of sulfated fucans. Both fractions inhibited leukocyte recruitment in a model of inflammation in rats, although L.s.-1.25 appeared to be more active than L.s.-1.0. Also, these fractions inhibited neutrophil adhesion to platelets under flow. Only fraction L.s.-1.25, but not L.s.-1.0, displayed anticoagulant activity as measured by the activated partial thromboplastin time. Investigation of these fractions in angiogenesis settings revealed that only L.s.-1.25 strongly inhibited fetal bovine serum (FBS) induced in vitro tubulogenesis. This effect correlated with a reduction in plasminogen activator inhibitor-1 (PAI-1) levels in L.s.-1.25-treated endothelial cells. Furthermore, only parent sulfated polysaccharides from L. saccharina (L.s.-P) and its fraction L.s.-1.25 were powerful inhibitors of basic fibroblast growth factor (bFGF) induced pathways. Consistently, the L.s.-1.25 fraction as well as L.s.-P successfully interfered with fibroblast binding to human bFGF. The incorporation of L.s.-P or L.s.-1.25, but not L.s.-1.0 into Matrigel plugs containing melanoma cells induced a significant reduction in hemoglobin content as well in the frequency of tumor-associated blood vessels. Moreover, i.p. administrations of L.s.-1.25, as well as L.s.-P, but not L.s.-1.0, resulted in a significant reduction of tumor growth when inoculated into syngeneic mice. Finally, L.s.-1.25 markedly inhibited breast cancer cell adhesion to human platelet-coated surfaces. Thus, sulfated fucans are mainly responsible for the anti-inflammatory, anticoagulant, antiangiogenic, and antitumor activities of sulfated polysaccharides from L. saccharina brown seaweed

Synthetic lactulose amines: novel class of anticancer agents that induce tumor-cell apoptosis and inhibit galectin-mediated homotypic cell aggregation and endothelial cell morphogenesis

Galectins, a family of structurally related carbohydrate-binding proteins, contribute to different events associated with cancer biology, including apoptosis, homotypic cell aggregation, angiogenesis and tumor-immune escape. To interfere with galectin-carbohydrate interactions during tumor progression, a current challenge is the design of specific galectin inhibitors for therapeutic purposes. Here, we report the synthesis of three novel low molecular weight synthetic lactulose amines (SLA): (1) N-lactulose-octamethylenediamine (LDO), (2) N,N´-dilactulose-octamethylenediamine (D-LDO), and (3) N,N´-dilactulose-dodecamethylenediamine (D-LDD). These compounds showed a differential ability to inhibit binding of galectin-1 and/or galectin-3 to the highly glycosylated protein 90K in solid-phase assays. In addition, each compound demonstrated selective regulatory effects in different events linked to tumor progression including tumor-cell apoptosis, homotypic cell aggregation, and endothelial cell morphogenesis. Our results suggest that galectin inhibitors with subtle differences in their carbohydrate structures may be potentially used to specifically block different steps of tumor growth and metastasisFil: Rabinovich, Gabriel A.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Cumashi, Albana. Università degli Studi G. D´Annunzio; ItaliaFil: Bianco, German Ariel. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Ciavardelli, Domenico. Università degli Studi G. D´Annunzio; ItaliaFil: Iurisci, Ida. Università degli Studi G. D´Annunzio; ItaliaFil: D’Egidio, Maurizia. Università degli Studi G. D´Annunzio; ItaliaFil: Piccolo, Enza. Università degli Studi G. D´Annunzio; ItaliaFil: Tinari, Nicola. Università degli Studi G. D´Annunzio; ItaliaFil: Nifantiev, Nikolay. Academia de Ciencias Rusa; RusiaFil: Iacobelli, Stefano. Università degli Studi G. D´Annunzio; Itali

Synthetic inhibitors of galectin-1 and -3 selectively modulate homotypic cell aggregation and tumor cell apoptosis

Galectins have emerged as critical regulators of tumor progression and metastasis, by modulating different biological events including homotypic cell aggregation, apoptosis, migration, angiogenesis and immune escape. Therefore, galectin inhibitors might represent novel therapeutic agents for cancer. A series of structural analogs of the disaccharide methyl beta-lactosaminide were screened as potential galectin inhibitors by examining their capability to block binding of galectin-1 and/or galectin-3 to LGalS3BP in solid-phase assays. To demonstrate any functional role in vitro, oligosaccharides were characterized by their ability to regulate tumor cell apoptosis and LGalS3BP-induced homotypic cell aggregation.Fil: Iurisci, Ida. Fondazione Università G. d’Annunzio; Italia. Consorzio Interuniversitario Nazionale Per la Bio-Oncologia; ItaliaFil: Cumashi, Albana. Fondazione Università G. d’Annunzio; Italia. Consorzio Interuniversitario Nazionale Per la Bio-Oncologia; ItaliaFil: Sherman, Andrei. Russian Academy of Sciences; Rusia. Consorzio Interuniversitario Nazionale Per la Bio-Oncologia; ItaliaFil: Tsvetkov, Yuri E.. Russian Academy of Sciences; Rusia. Consorzio Interuniversitario Nazionale Per la Bio-Oncologia; ItaliaFil: Tinari, Nicola. Fondazione Università G. d’Annunzio; Italia. Consorzio Interuniversitario Nazionale Per la Bio-Oncologia; ItaliaFil: Piccolo, Enza. Fondazione Università G. d’Annunzio; Italia. Consorzio Interuniversitario Nazionale Per la Bio-Oncologia; ItaliaFil: D'Egidio, Maurizia. Fondazione Università G. d’Annunzio; Italia. Consorzio Interuniversitario Nazionale Per la Bio-Oncologia; ItaliaFil: Adamo, Vincenzo. Università degli Studi di Messina; Italia. Consorzio Interuniversitario Nazionale Per la Bio-Oncologia; ItaliaFil: Natoli, Clara. Fondazione Università G. d’Annunzio; Italia. Consorzio Interuniversitario Nazionale Per la Bio-Oncologia; ItaliaFil: Rabinovich, Gabriel Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Consorzio Interuniversitario Nazionale Per la Bio-Oncologia; ItaliaFil: Iacobelli, Stefano. Fondazione Università G. d’Annunzio; Italia. Consorzio Interuniversitario Nazionale Per la Bio-Oncologia; ItaliaFil: Nifantiev, Nikolay E.. Russian Academy of Sciences; Italia. Consorzio Interuniversitario Nazionale Per la Bio-Oncologia; Itali

A comparative study of the anti-inflammatory, anticoagulant, antiangiogenic, and antiadhesive activities of nine different fucoidans from brown seaweeds

The anti-inflammatory, antiangiogenic, anticoagulant, and antiadhesive properties of fucoidans obtained from nine species of brown algae were studied in order to examine the influence of fucoidan origin and composition on their biological activities. All fucoidans inhibited leucocyte recruitment in an inflammation model in rats, and neither the content of fucose and sulfate nor other structural features of their polysaccharide backbones significantly affected the efficacy of fucoidans in this model. In vitro evaluation of P-selectin-mediated neutrophil adhesion to platelets under flow conditions revealed that only polysaccharides from Laminaria saccharina, L. digitata, Fucus evanescens, F. serratus, F. distichus, F. spiralis, and Ascophyllum nodosum could serve as P-selectin inhibitors. All fucoidans, except that from Cladosiphon okamuranus carrying substantial levels of 2-O-alpha-D-glucuronopyranosyl branches in the linear (1-->3)-linked poly-alpha-fucopyranoside chain, exhibited anticoagulant activity as measured by activated partial thromboplastin time whereas only fucoidans from L. saccharina, L. digitata, F. serratus, F. distichus, and F. evanescens displayed strong antithrombin activity in a platelet aggregation test. The last fucoidans potently inhibited human umbilical vein endothelial cell (HUVEC) tubulogenesis in vitro and this property correlated with decreased levels of plasminogen-activator inhibitor-1 in HUVEC supernatants, suggesting a possible mechanism of fucoidan-induced inhibition of tubulogenesis. Finally, fucoidans from L. saccharina, L. digitata, F. serratus, F. distichus, and F. vesiculosus strongly blocked MDA-MB-231 breast carcinoma cell adhesion to platelets, an effect which might have critical implications in tumor metastasis. The data presented herein provide a new rationale for the development of potential drugs for thrombosis, inflammation, and tumor progression.Fil: Cumashi, Albana. Universita degli Studi G. D Annunzio; ItaliaFil: Ushakova, Natalia A.. Academia Rusa de Ciencias Médicas; RusiaFil: Preobrazhenskaya, Marina E.. Academia Rusa de Ciencias Médicas; RusiaFil: D'Incecco, Armida. Universita degli Studi G. D Annunzio; ItaliaFil: Piccoli, Antonio. Consorzio Mario Negri Sud; ItaliaFil: Totani, Licia. Consorzio Mario Negri Sud; ItaliaFil: Tinari, Nicola. Universita degli Studi G. D Annunzio; ItaliaFil: Morozevich, Galina E.. Academia Rusa de Ciencias Médicas; RusiaFil: Berman, Albert E.. Academia Rusa de Ciencias Médicas; RusiaFil: Bilan, María. Academia Rusa de Ciencias; RusiaFil: Usov, Anatolii I.. Academia Rusa de Ciencias; RusiaFil: Ustyuzhanina, Nadezhda E.. Academia Rusa de Ciencias; RusiaFil: Grachev, Alexey A.. Academia Rusa de Ciencias; RusiaFil: Sanderson, Craig J.. Scottish Association for Marine Sciences; Reino UnidoFil: Kelly, Maeve. Scottish Association for Marine Sciences; Reino UnidoFil: Rabinovich, Gabriel Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; ArgentinaFil: Iacobelli, Stefano. Universita degli Studi G. D Annunzio; ItaliaFil: Nifantiev, Nikolay E.. Academia Rusa de Ciencias; Rusi