As células dendríticas (DCs) são fundamentais no desencadeamento da resposta imune antitumoral. Mas, no microambiente tumoral há condições que impedem esta função imunoestimuladora das DCs. Este comprometimento funcional parece ser fruto da hiperativação de STAT-3. O presente estudo visou avaliar a capacidade de Ta1 de interferir na ativação de STAT-3. Então, monócitos e mo-DCs foram tratados ou não com Ta1 e comparados com o controle, o inibidor de STAT-3, JSI-124. Avaliou-se a expressão de moléculas de superfície e a capacidade de mo-DCs de estimular linfócitos T alogeneicos. Ta1 não interferiu na ativação de STAT-3. Além disso, Ta1 não reproduziu os efeitos encontrados em mo-DCs de pacientes com câncer. Já, o tratamento com JSI-124 levou a alterações nas mo-DCs fazendo com que exibissem perfil infamatório, com aumento de HLA-DR, CD86 e concomitante queda de PD-L1. Além disso, mostramos que STAT-3 está envolvido na expressão de leucointergrinas, uma vez que sua inibição proporcionou queda da expressão em nível proteico e gênico destas moléculas. / Dendritic cells ( DCs ) are critical in triggering antitumor immune response . But there are conditions in the tumor microenvironment that prevent this immunostimulatory function of DCs. This functional impairment appears to be the result of hyperactivation of STAT-3. The present study aimed to evaluate the ability of Ta1 to interfere in the activation of STAT-3. Then, monocytes and mo-DCs were treated or not with Ta1 and compared with the control, the STAT-3 inhibitor, JSI -124. We assessed the expression of surface molecules and the mo-DCs ability in stimulating allogeneic T lymphocytes. Ta1 did not affect the activation of STAT-3. In addition, Ta1 did not reproduce the effects found in mo-DCs from cancer patients. However, JSI -124 treatment led to changes in mo-DCs, that exhibited an inflammatory profile, with an increase of HLA-DR , CD86 and concomitant drop in PD- L1. Furthermore, we have shown that STAT-3 is involved in the expression of leukointergrins, since its inhibition resulted in down-regulation of expression level of this gene and protein molecules.

Jia, Lin. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 124-146). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

The mig-10 gene in C. elegans is required for proper axon guidance and/or cell migration of certain neurons during development. In mig-10 (ct41) mutant worms, there is incomplete migration of the anterior lateral microtubule cells (ALMs), hermaphrodite specific neuron (HSN), left coelomocyte cells (ccL), and canal associated neuron (CAN) (Manser and Wood, 1990). The mig-10 (ct41) mutation also causes axon guidance defects in the IL2 neurons, and it enhances unc-6 defects in the axon guidance of the anterior ventral microtubule cell (AVM) (Rusiecki, 1999; C. Quinn, personal communication). mig-10's function in axon guidance and neuronal migration is unknown, but is believed to be involved in a signal transduction pathway that uses a G-protein, such as ras. The two mig-10 transcripts discussed in this thesis, mig-10 A and mig-10 B, encode proteins that are similar to Grb-7 and Grb-10 proteins, which are also believed to function in a signal transduction pathway (Manser et al., 1997). One of these similarities is the presence of a proline-rich region, which may be used to bind another protein (Manser et al., 1997). The MIG-10 A protein has an additional proline region, compared to MIG-10 B, which may indicate that the MIG-10 A and B proteins are utilized in different cells, or at different developmental stages. As a first step in learning where MIG-10 is expressed, mig-10 (ct41) mutant worms containing a wild-type mig-10 B::GFP fusion were constructed. Rescue of the mutant phenotype would indicate that the expression pattern of the transgene was similar to that of the endogenous gene. As this experiment did not allow for rescue, even after integration of the construct, a strain of worms containing a mig-10 promoter::GFP transgene was used. Preliminary observations of this strain indicated that mig-10 is expressed in neuronal tissue. The AIY neurons were observed in wild-type and mig-10 (ct41) worms to determine if they are affected by the mig-10 mutation as previously reported (O. Hobert, personal communication). As no difference was detected, the AIYs were not used in any further experiments. In order to determine which cells require functional MIG-10 protein for the proper development/migration of neurons to occur, mig-10 (ct41) worms containing mec-3 promoter::mig-10 A or B cDNA transgenes were constructed. The mec-3 promoter drives expression of the mig-10 cDNA in the ALM neurons and other touch cells early in the development of the embryo. If these transgenes rescued the ALM migration defect, then mig-10 would be acting cell autonomously in ALM. Partial rescue was obtained, which may be due to the need for both of the mig-10 transcripts to be expressed in the same cell; alternatively, one or both transcripts may need to be expressed in a cell nonautonomous fashion in addition to being expressed cell autonomously. Low production of the rescuing protein, or expression of the protein at a later developmental stage than is needed for rescue to occur, may also have been the cause of the partial rescue. Future work in this area includes putting mig-10 promoter::mig-10 A or B cDNA in mig-10 (ct41) background to investigate if the different transcripts rescue different aspects of the mig-10 phenotype. The mig-10 A and mig-10 B cDNA constructs could also be expressed in the same worm in an attempt to correct for partial rescue that may be due to the lack of both MIG-10 proteins.

Une réponse immunitaire adéquate nécessite la participation coordonnée de plusieurs populations de cellules immunitaires, comme les lymphocytes T et B, les macrophages, les cellules dendritiques ou les cellules NK. L’activation de ces types cellulaires est modulée par différents récepteurs membranaires dont la fonction est de déclencher une cascade de signalisation.L’activation des lymphocytes T, acteurs cruciaux de la mise en place de la réponse immunitaire adaptative, s’initie par l’engagement du récepteur T (TCR). Plusieurs autres types de récepteurs participent à la modulation des réponses cellulaires. Ainsi, les récepteurs aux facteurs de croissance, aux cytokines et aux chimiokines ainsi que les molécules d’adhésion et les récepteurs de la famille SLAM (pour Signaling Lymphocyte Activation Molecule) influencent l’activation cellulaire. Des travaux récents ont montré que l’activation des récepteurs SLAM induit leur association avec les membres de la famille SAP et est nécessaire à l’induction d’une réponse humorale, au développement des cellules NKT ainsi qu’à la cytotoxicité médiée par les lymphocytes T CD8 et les cellules NK. L’altération du gène sh2d1a codant pour SAP conduit à l’apparition du syndrome lymphoprolifératif lié à l’X-1 (XLP-1). Les patients atteints de ce syndrome développent trois principaux phénotypes cliniques qui sont une mononucléose infectieuse fulminante, une dysgammaglobulinémie, et des désordres lymphoprolifératifs.L’objectif de mon travail de thèse a été d’étudier les étapes précoces d’activation des lymphocytes T et de comprendre comment la protéine SAP, associée à d’autres protéines ou domaines protéiques intracellulaires, est impliquée dans la régulation de ces mécanismes d’activation. Mon travail s’est donc orienté vers l’identification de nouveaux partenaires de SAP, autres que les récepteurs SLAM, et qui nous permettraient de mieux définir la fonction de SAP dans la signalisation T. Par une approche de biochimie, mon travail a permis de démontrer que SAP s’associe directement à la chaîne CD3 du complexe TCR-CD3, régule la signalisation induite par l’activation du récepteur T et permet la sécrétion de cytokines. Enfin, par une approche de double hybride, nous avons identifié Pecam-1 comme nouveau partenaire de SAP. Nous avons par la suite observé que l’association de SAP avec Pecam-1 régule l’adhérence des lymphocytes T. Par ces deux études, mon travail de thèse a permis de démontrer l’implication de SAP dans de nouvelles voies de signalisation et permet de mieux comprendre les mécanismes dérégulés lors de l’absence de SAP. / Immune responses need a coordinate involvement between different immune cell populations, as T and B cells, macrophages, dendritic cells or NK cells. Activation of these different cell populations is mediated by different receptors whose function is to initiate a signal transduction cascade. T cell activation, a crucial event in adaptive immune response, begins with T cell receptor (TCR) triggering. A large number of receptors can modulate T cell responses. Thus, cytokines, chimiokines and growth factors receptors, adhesion molecules and SLAM (for Signaling Lymphocyte Activation Molecule) family receptors regulate cell activation. Recent works have shown that SLAM receptors triggering induce their association with SAP (for SLAM-Associated Protein) family members and is vital for humoral immunity, NKT cell development and T CD8+ and NK cells cytotoxicity. Mutations in sh2d1a gene, which code for SAP, are responsible of X-linked Lymphoproliferative-1 (XLP-1) syndrome. Patients, who suffer from this syndrome, develop three main clinical manifestations: a fulminant infectious mononucleosis, dysgammaglobulinemia and lymphoproliferative syndromes. My thesis work was to study early steps of T cell activation and to understand how the SAP protein, associated with its partners, regulates these cellular mechanisms. Thus, my work was to identify new SAP partners, others than SLAM receptors, in order to better understand SAP function in T cell signaling. With a biochemical approach, my work has demonstrated that SAP directly associates with CD3 chain of TCR-CD3 complex, regulates cell signaling and cytokines secretion following TCR triggering. Finally, with a two-hybrid assay, we have identified the adhesion molecule Pecam-1 as a new SAP partner. Then, we have observed that SAP directly interacts with Pecam-1 to regulate T cell adhesion. During my thesis work, we have identified new cellular signaling pathways that are regulated by SAP and permit to better understand the cellular mechanisms that are affected when SAP is absent.

Os mastócitos participam do sistema imunológico liberando mediadores farmacologicamente ativos. A principal via de ativação dos mastócitos é através do receptor de alta afinidade para a imunoglobulina E (FcRI). A ativação dos mastócitos via FcRI culmina com a liberação de mediadores. A enzima PLD atua sobre fosfolipídios hidrolisando a fosfatidilcolina em ácido fosfatídico e colina. A PLD é ativada após o estímulo via FcRI e possui um papel importante na transdução do sinal em mastócitos. Existem duas isoformas da enzima PLD, a PLD1 e a PLD2 que são expressas, diferentemente, de acordo com o tipo celular. Ambas as isoformas podem estar expressas numa mesma célula, apenas uma ou nenhuma. Neste estudo foram utilizadas células RBL-2H3 transfectadas para a super expressão PLD2 nas formas catalítica ativa (CA) e inativa (CI). O papel da PLD2 foi examinado nestas células com o objetivo de elucidar sua atuação no processo de secreção incluindo o aparelho de Golgi e os grânulos secretores. As células CA e CI possuem maior atividavidade de -hexosaminidase total, porém quando estimuladas mostram uma deficiência na liberação desta enzima, quando comparadas com as células selvagens. A PLD2 nas células CA, CI, VET e RBL-2H3 está localizada no citosol, sendo abundante na região justanuclear, principalmente nas células CI, sugerindo uma associação com o aparelho de Golgi. A dupla marcação com o mAb AA4, que imunomarca gangliosídeos derivados do GD1b da membrana plasmática e com anti-PLD2, mostrou que esta enzima não se localiza na membrana plasmática. A dupla marcação com anti-PLD2 e anti-GM130 mostrou que as áreas de maior concentração da PLD2 se co-localizam com o aparelho de Golgi, especialmente nas células CI. A marcação com anti-GM130 e os experimentos com microscopia eletrônica de transmissão mostraram que o aparelho de Golgi está organizado nas células CA e desorganizado nas células CI, onde se encontra disperso no citoplasma. Ainda, as células CI expressam menos GM130 em comparação com as demais linhagens celulares. Quando a produção de PA pela PLD está inibida pelo 1-Butanol, as células CA apresentam as mesmas características fenotípicas das células CI. A incubação das CI com PA resulta na reestruturação do aparelho de Golgi. A manutenção estrutural do aparelho de Golgi, também está relacionada com os microtúbulos. Nas células CI o centro organizador de microtúbulos é dificilmente identificado. Os microtúbulos nas células CI são desordenados em comparação com as demais linhagens celulares. Estes resultados mostram que a produção de PA pela PLD2 é importante na organização de microtúbulos e na manutenção da estrutura do aparelho de Golgi. As alterações celulares relacionadas com os microtúbulos e o aparelho de Golgi afetam o processo secretor nestas células e, provavelmente, em outros tipos de células secretoras. Estes achados poderão levar a novas estratégias terapêuticas para controlar a liberação de mediadores durante processos alérgicos e inflamatórios. / Mast cells are components of the immune system that liberate a wide variety of pharmacologically active mediators. The principle method of activating mast cells is through the high affinity receptor for IgE (FcRI). This activation then culminates with the release of mediators. Phospholipase D (PLD) acts on phospholipids, hydrolyzing phosphatidylcholine to phosphatidic acid (PA) and choline. PLD is activated following stimulation via FcRI and plays an important role in signal transduction in mast cells. PLD has two isoforms, PLD1 and PLD2, which are differentially expressed depending on the cell type where none, one or both may be expressed. RBL-2H3 cells, a mast cell line, transfected to super express catalytically active (CA) and inactive (CI) forms of PLD2 were used in the present study. The role of PLD2 was examined in these cells in order to clarify the action of PLD2 in the secretory process. Although the CA and CI cells posses a greater total -hexosaminidase activity, when stimulated these cells release less -hexosaminidase than cells transfected with empty vector or wild type RBL-2H3 cells. In all cell lines, PLD2 was dispersed throughout the cytoplasm with a concentration in the juxtanuclear region suggesting an association of PLD2 with the Golgi apparatus. Double labeling with anti-PLD2 and mAb AA4, which recognizes gangliosides derived from GD1b on the plasma membrane, showed that PLD2 was not associated with the plasma membrane. When the cells were double labeled with anti-PLD2 and anti-GM130, which labels the cis-Golgi saccules, PLD2 does colocalize with the Golgi apparatus, especially in CI cells. Labeling with anti-GM130 alone as well as experiments employing transmission electron microscopy revealed that the Golgi apparatus is well organized in the CA cells, but is disorganized and dispersed in the cytoplasm in the CI cells. By Western Blotting, the CI cells also expressed less GM130 than the other cell lines. When the production of PA by PLD2 was inhibited by 1-Butanol, the Golgi apparatus of the CA cells presented the same phenotypic characteristics as that of the CI cells. Conversely, incubation of the CI cells with PA resulted in the reorganization of the Golgi apparatus. The structural maintenance of the Golgi apparatus is also related to microtubules. In the CI cells, the microtubule organizing center was difficult to identify and the microtubules were disorganized in the cytoplasm as compared to the other cell lines. These results show that the production of PA by PLD2 is important in the arrangement of the microtubules and in maintaining the structure of the Golgi apparatus. Alterations in the distribution of the microtubules and the structure of the Golgi apparatus in the CI cells affect the secretory process in these cells, and such alterations may affect the secretory process in other cell types as well. The findings presented here may lead to new therapeutic strategies to control the production and release of mediators during allergic and inflammatory processes.

Background: Light-adaptation is a multifaceted process in the retina that helps adjust the visual system to changing illumination levels. Many studies are focused on the photochemical mechanism of light-adaptation. Neural network adaptation mechanisms at the photoreceptor synapse are largely unknown. We find that large, spontaneous Excitatory Amino Acid Transporter (EAATs) activity in cone terminals may contribute to cone synaptic adaptation, specifically with respect to how these signals change in differing conditions of light. EAATs in neurons quickly transport glutamate from the synaptic cleft, and also elicit large thermodynamically uncoupled Cl- currents when activated. We recorded synaptic EAAT currents from cones to study glutamate-uptake events elicited by glutamate release from the local cone, and from adjacent photoreceptors. We find that cones are synaptically connected via EAATs in dark ; this synaptic connection is diminished in light-adapted cones. Methods: Whole-cell patch-clamp was performed on dark- and transiently light-adapted tiger salamander cones. Endogenous EAAT currents were recorded in cones with a short depolarization to -10mV/2ms, while spontaneous transporter currents from network cones were observed while a local cone holding at -70mV constantly. DHKA, a specific transporter inhibitor, was used to identify EAAT2 currents in the cone terminals, while TBOA identified other EAAT subtypes. GABAergic and glycinergic network inputs were always blocked with picrotoxin and strychnine. Results: Spontaneous EAAT currents were observed in cones held constantly at -70mV in dark, indicating that the cones received glutamate inputs from adjacent photoreceptors. These spontaneous EAAT currents disappeared in presence of a strong light, possibly because the light suppressed glutamate releases from the adjacent photoreceptors. The spontaneous EAAT currents were blocked with TBOA, but not DHKA, an inhibitor for EAAT2 subtype, suggesting that a / non-EAAT 2 subtype may reside in a basal or perisynaptic area of cones, with a specialized ability to bind exocytosed glutamate from adjacent cones in dark. Furthermore, these results could be artificially replicated by dual-electrode recordings from two adjacent cones. When glutamate release was elicited from one cone, the TBOA-sensitive EAAT currents were observed from the other cone. Conclusions: Cones appear to act like a meshwork, synaptically connected via glutamate transporters. Light attenuates glutamate release and diminishes the cone-cone synaptic connections. This process may act as an important network mechanism for cone light adaptation. / by Matthew JM Rowan. / Thesis (Ph.D.)--Florida Atlantic University, 2011. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2011. Mode of access: World Wide Web.

Crotalfina é um peptídeo sintetizado a partir da sequência de um composto analgésico purificado do veneno de serpentes Crotalus durissus terrificus. Apesar da atividade opióide, ensaios moleculares indicam que esse peptídeo não se liga diretamente aos receptores opióides, sugerindo que a liberação de opióides endógenos sejam os responsáveis pela atividade analgésica. Baseado em dados da literatura que demonstram a estreita relação entre o sistema canabinóide e o sistema opióide, o objetivo deste projeto foi avaliar o possível efeito da crotalfina sobre o sistema canabinóide. Os resultados indicam que receptores canabinóides CB2 estão envolvidos na antinocicepção acarretada pela crotalfina, na vigência de hiperalgesia induzida por PGE2. Ainda, este efeito é dependente de liberação de opióides endógenos, particularmente dinorfina A, agonista endógeno de receptores opióides do tipo kappa, sendo essa liberação dependente da ativação de receptores CB2. / Crotalphine is a peptide synthesized based on the sequence of the analgesic compound purified from the Crotalus durissus terrificus snake venom. Despite the opioid activity, molecular assays indicate that this peptide does not directly bind to opioid receptors, suggesting that the endogenous opioid release could be responsible for analgesic activity. Based on data from literature demonstrating the close relationship between the cannabinoid and the opioid systems, the goal of this project was to evaluate the possible effect of crotalphine on the cannabinoid system. Results demonstrated that CB2 cannabinoid receptors are involved in the antinociception induced by crotalphine during PGE2-induced hyperalgesia. In agreement, this effect is dependent of the release of endogenous opioids, particularly dynorphin A, the endogenous agonist of kappa opioid receptors. This release is dependent of the CB2 receptor activation.

A enzima Na+, K+-ATPase é uma proteína de membrana altamente conservada em eucariotos, capaz de gerar um gradiente eletroquímico, fundamental para o balanço osmótico das células, o potencial de repouso das membranas e a propriedade excitatória das células musculares e nervosas. Além de seu papel regulatório na homeostasia iônica, desempenha um papel na transdução de sinal e na ativação de transcrição gênica, modulando na presença de ouabaína o crescimento celular, migração e morte celular programada. A Ouabaína (OUA) é um esteróide cardiotônico, produzido no córtex da adrenal e no hipotálamo. Em linhas gerais, a sinalização da Na+, K+-ATPase promovida pela OUA parece ativar vias associadas à modulação de fatores de transcrição como a via da Src, MAPK, Ca2+ e NF-<font face=\"Symbol\">kB. Evidências indicam que o NF-<font face=\"Symbol\">kB exerça algum tipo de modulação na via canônica do WNT, no entanto os mecanismos moleculares ainda são desconhecidos. A via de sinalização WNT desempenha função importante na embriogênese e na homeostase de tecidos adultos. Assim, o objetivo do presente projeto é verificar se a administração intrahipocampal de OUA é capaz de modular a atividade das vias canônicas do NF-<font face=\"Symbol\">kB e da WNT. Estas vias foram estudadas em um decurso temporal imediato (1 -2 horas) e tardio (10, 24 e 48 horas) utilizando técnicas como Western Blotting, RT-PCR e EMSA. Os resultados encontrados mostram que a OUA (10 nM) foi capaz de ativar a via de sinalização NF-<font face=\"Symbol\">kB, após 1 hora, 10, 24 e 48 horas. A OUA também foi capaz de ativar a via canônica do WNT, sendo que após 10 horas ocorreu aumento da proteína pGSK-3<font face=\"Symbol\">b, enquanto que em 24 horas, observamos aumento da translocação nuclear da <font face=\"Symbol\">b-CATENINA. Além disso, pode-se verificar aumento de BDNF ao longo de todo o decurso temporal. / The enzyme Na+,K+-ATPase is an integral membrane protein, highly conserved in eukaryotes, that establishes the electrochemical gradient across the plasma membrane, which is essential to maintain the osmotic balance of cells, the resting membrane potential and the excitatory property of nerve and muscle cells. Besides its role in ion homeostasis, several recent studies suggest that this pump may also act as a signal transducer and transcription activator involved in cell growth, differentiation and programmed cell death. Ouabain (OUA), the ligand of Na+,K+-ATPase, is a steroid derivative that is produced by the adrenal cortex and hypothalamus. After OUA binding, the Na+,K+-ATPase signaling seems to activate pathways such as Src, MAPK, NF-<font face=\"Symbol\">kB and Ca2+. Some evidences indicate a possible crosstalk between the NF-<font face=\"Symbol\">kB signaling pathway and the canonical WNT pathway, however the molecular mechanisms are still unknown. The canonical WNT play important roles during embryogenesis and in adult tissue homeostasis. The aim of this project is to verify if the intrahipocampal administration of OUA is able to modulate the activity of the canonical pathways of NF-<font face=\"Symbol\">kB and WNT. Both pathways were studied after 1 and 2 hours, and after 10, 24 and 48 hours by methods such Western blot, RT-PCR and Electrophoretic mobility shift assays. The results show that the OUA (10 nM) was able to activate the signaling pathway NF-<font face=\"Symbol\">kB after 1, 10, 24 and 48 hours. The OUA was also able to activate the canonical WNT pathway, since after 10 hours there was an increased in pGSK-3<font face=\"Symbol\">b protein, whereas in 24 hours, we observed increased nuclear translocation of <font face=\"Symbol\">b-CATENIN. Moreover, we found increased levels of BDNF throughout the time course.

<p>Prokaryotes are continually exposed to variations in their environment. Survival in unstable milieu requires a wide range of transcriptional regulators (TRs) that respond to specific environmental and cellular signals by modulating gene expression and provide an appropriate physiological response to external stimuli. These adaptive responses to environmental signals are mostly mediated by TRs from one of two families: the single or the two component signal transduction systems (1CSTS; 2CSTS). In this thesis the structural analysis of two 1CSTS – DntR and NikR − are presented. One study was carried out to try to develop a bacterial biosensor for synthetic dinitrotulenes compounds, the other to characterise the Ni-sensing mechanism that contributes to the acid adaptation of the human pathogen<i> Helicobacter pylori.</i> DntR belongs to the LysR family and the crystal structures obtained have allowed the proposal a model of the interaction of DntR with salicylate inducer as well as giving insights into the signal propagation mechanism in LysR-type transcription factors (<b>paper I</b>). DntR mutant crystal structures combined with the modelling of DntR-2,4-dnt interactions led to the design of a DntR mutant that has a limited response to 2,4-dnt in a whole cell biosensor system (<b>paper 2</b>). Crystal structures of apo-NikR from <i>H. pylori </i>(HpNikR) and of Ni-bound intermediary states of the protein were obtained. The latter have helped in unravelling the Ni incorporation and selectivity mechanisms of NikRs and have shown a strong cooperativity between conformational changes in the Ni binding domain with movements of the DNA binding domain (<b>paper 3</b>). Biochemical studies and comparisons of the HpNikR crystal structures with those of NikR homologues strongly suggest that HpNikR has evolved different surface properties (<b>paper 4</b>) and a new mode of DNA binding. </p>

<p>Basement membranes are thin extracellular sheets that surround muscle, fat and peripheral nerve cells and underlay epithelial and endothelial cells. Laminins are one of the main protein families of these matrices. Integrins and dystroglycan are receptors for laminins, connecting cells to basement membranes. Each laminin consists of three different chains, (α, β, γ). Laminin-1 (α1β1γ1) was the first laminin to be found and is the most frequently studied. Despite this, it was unclear where its α1 chain was expressed. A restricted distribution of the α1 chain in the adult epithelial basement membranes was demonstrated in the present study. In contrast, dystroglycan was found to have a much broader distribution. Dystroglycan is an important receptor for α2-laminins in muscle, but binds also α1-laminins. The more ubiquitous α5-laminins were also shown to bind dystroglycan, but with distinctly lower affinity than α1- and α2- laminins. </p><p>The biological roles of different laminin isoforms have been investigated. Differences were found in the capacity of various tested laminins to promote epithelial cell adhesion. The α5-laminins were potent adhesive substrates, a property shown to be dependent on α3 and α6 integrins. Each receptor alone could promote efficient epithelial cell adhesion to α5-laminins. Yet, only α6 integrin and in particular the α6A cytoplasmic splice variant could be linked to laminin-mediated activation of a mitogen-activated protein kinase (MAP kinase) pathway. Attachment to either α1- or α5-laminins activated extracellular-signal regulated kinase (ERK) in cells expressing the integrin α6A variant, but not in cells expressing α6B. A new role for dystroglycan as a suppressor of this activation was demonstrated. Dystroglycan antibodies, or recombinant fragments with high affinity for dystroglycan, decreased ERK activation induced by integrin α6 antibodies. Integrin αvβ3 was identified as a novel co-receptor for α5-laminin trimers. Cell attachment to α5-laminins was found to facilitate growth factor induced cell proliferation. This proliferation could be blocked by antibodies against integrin αvβ3 or by an inhibitor of the MEK/ERK pathway. Therefore, integrin αvβ3 binding to α5-laminins could be of biological significance.</p>