Association of Caspr/paranodin with tumour suppressor schwannomin/merlin and beta1 integrin in the central nervous system

Caspr/paranodin is an essential neuronal component of paranodal axoglial junctions, associated with contactin/F3. Its short intracellular domain contains a conserved motif (GNP motif) capable of binding protein 4.1 domains [FERM domains (four point one, ezrin, radixin, moesin)]. Schwannomin/merlin is a tumour suppressor expressed in many cell types, including in neurons, the function and partners of which are still poorly characterized. We show that the FERM domain of schwannomin binds to the paranodin GNP motif in glutathione S-transferase (GST)-pull down assays and in transfected COS-7 cells. The two proteins co-immunoprecipitated in brain extracts. In addition, paranodin and schwannomin were associated with integrin beta1 in transfected cells and in brain homogenates. The presence of paranodin increased the association between integrin beta1 and schwannomin or its N-terminal domain, suggesting that the interactions between these proteins are interdependent. In jimpy mutant mice, which display a severe dysmyelination with deficient paranodal junctions, the interactions between paranodin, schwannomin and integrin beta1 were profoundly altered. Our results show that schwannomin and integrin beta1 can be associated with paranodin in the central nervous system. Since integrin beta1 and schwannomin do not appear to be enriched in paranodes they may be quantitatively minor partners of paranodin in these regions and/or be associated with paranodin at other locations.     

IGFBP-3 and IGFBP-5 associate with the cell binding domain (CBD) of fibronectin

We have used Surface Plasmon Resonance (SPR) - based biosensor technology to investigate the interaction of the six high affinity insulin-like growth factor binding proteins (IGFBP 1-6) with the cell binding domain (CBD) of fibronectin. Using a biotinylated derivative of the ninth and tenth TypeIII domains of FN (^9-10FNIII), we show that IGFBP-3 and -5 bind to FN-CBD. We show that this binding is inhibited by IGF-I and that, for IGFBP-5, binding occurs through the C-terminal heparin binding domain of the protein. Using site-directed mutagenesis of ^9-10FNIII, we show both the “synergy” and RGD sites within these FN domains are required for maximum binding of both IGFBPs. We discuss the possible biological consequences of our results.     

Mutations in the extracellular domains of glutamate-gated chloride channel alpha3 and beta subunits from ivermectin-resistant Cooperia oncophora affect agonist sensitivity

Two full-length glutamate-gated chloride channel (GluCl) cDNAs, encoding GluClalpha3 and GluClbeta subunits, were cloned from ivermectin-susceptible (IVS) and -resistant (IVR) Cooperia oncophora adult worms. The IVS and IVR GluClalpha3 subunits differ at three amino acid positions, while the IVS and IVR GluClbeta subunits differ at two amino acid positions. The aim of this study was to determine whether mutations in the IVR subunits affect agonist sensitivity. The subunits were expressed singly and in combination in Xenopus laevis oocytes. Electrophysiological whole-cell voltage-clamp recordings showed that mutations in the IVR GluClalpha3 caused a modest but significant threefold loss of sensitivity to glutamate, the natural ligand for GluCl receptors. As well, a significant decrease in sensitivity to the anthelmintics ivermectin and moxidectin was observed in the IVR GluClalpha3 receptor. Mutations in the IVR GluClbeta subunit abolished glutamate sensitivity. Co-expressing the IVS GluClalpha3 and GluClbeta subunits resulted in heteromeric channels that were more sensitive to glutamate than the respective homomeric channels, demonstrating co-assembly of the subunits. In contrast, the heteromeric IVR channels were less sensitive to glutamate than the homomeric IVR GluClalpha3 channels. The heteromeric IVS channels were significantly more sensitive to glutamate than the heteromeric IVR channels. Of the three amino acids distinguishing the IVS and IVR GluClalpha3 subunits, only one of them, L256F, accounted for the differences in response between the IVS and IVR GluClalpha3 homomeric channels.     

The integrins alpha3beta1 and alpha6beta1 physically and functionally associate with CD36 in human melanoma cells. Requirement for the extracellular domain OF CD36

Lateral association between different transmembrane glycoproteins can serve to modulate integrin function. Here we characterize a physical association between the integrins alpha(3)beta(1) and alpha(6)beta(1) and CD36 on the surface of melanoma cells and show that ectopic expression of CD36 by CD36-negative MV3 melanoma cells increases their haptotactic migration on extracellular matrix components. The association was demonstrated by co-immunoprecipitation, reimmunoprecipitation, and immunoblotting of surface-labeled cells lysed in Brij 96 detergent. Confocal microscopy illustrated the co-association of alpha(3) and CD36 in cell membrane projections and ruffles. A requirement for the extracellular domain of CD36 in this association was shown by co-immunoprecipitation experiments using surface-labeled MV3 melanoma or COS-7 cells that had been transiently transfected with chimeric constructs between CD36 and intercellular adhesion molecule 1 (ICAM-1) or with a truncation mutant of CD36. CD36 is known to engage in signal transduction and to localize to membrane microdomains or rafts in several cell types. Toward a mechanistic explanation for the functional effects of CD36 expression, we demonstrate that in fractionated Triton X-100 lysates of the MV3 cells stably transfected with CD36, CD36 was greatly enriched with the detergent-insoluble fractions that represent plasma membrane rafts. Significantly, when these fractionated lysates were reprobed for endogenous beta(1) integrin, it was found that a 4-fold increase in the proportion of the mature protein was contained within the detergent-insoluble fractions when extracted from the CD36-transfected cells compared with MV3 cells transfected with vector only. These results suggest that in melanoma cells CD36 expression may induce the sequestration of certain integrins into membrane microdomains and promote cell migration.     

The cysteine-rich domain of human ADAM 12 supports cell adhesion through syndecans and triggers signaling events that lead to beta1 integrin-dependent cell spreading

The ADAMs (a disintegrin and metalloprotease) family of proteins is involved in a variety of cellular interactions, including cell adhesion and ecto- domain shedding. Here we show that ADAM 12 binds to cell surface syndecans. Three forms of recombinant ADAM 12 were used in these experiments: the cys-teine-rich domain made in Escherichia coli (rADAM 12-cys), the disintegrin-like and cysteine-rich domain made in insect cells (rADAM 12-DC), and full-length human ADAM 12-S tagged with green fluorescent protein made in mammalian cells (rADAM 12-GFP). Mesenchymal cells specifically and in a dose-dependent manner attach to ADAM 12 via members of the syndecan family. After binding to syndecans, mesenchymal cells spread and form focal adhesions and actin stress fibers. Integrin beta1 was responsible for cell spreading because function-blocking monoclonal antibodies completely inhibited cell spreading, and chondroblasts lacking beta1 integrin attached but did not spread. These data suggest that mesenchymal cells use syndecans as the initial receptor for the ADAM 12 cysteine-rich domain-mediated cell adhesion, and then the beta1 integrin to induce cell spreading. Interestingly, carcinoma cells attached but did not spread on ADAM 12. However, spreading could be efficiently induced by the addition of either 1 mM Mn(2+) or the beta1 integrin-activating monoclonal antibody 12G10, suggesting that in these carcinoma cells, the ADAM 12-syndecan complex fails to modulate the function of beta1 integrin.     

Role of auxiliary beta1-, beta2-, and beta3-subunits and their interaction with Na(v)1.8 voltage-gated sodium channel

The nociceptive C-fibers of the dorsal root ganglion express several sodium channel isoforms that associate with one or more regulatory beta-subunits (beta1-beta4). To determine the effects of individual and combinations of the beta-subunit isoforms, we co-expressed Nav1.8 in combination with these beta-subunits in Xenopus oocytes. Whole-cell inward sodium currents were recorded using the two-microelectrode voltage clamp method. Our studies revealed that the co-expression beta1 alone or in combination with other beta-subunits enhanced current amplitudes, accelerated current decay kinetics, and negatively shifted the steady-state curves. In contrast, beta2 alone and in combination with beta1 altered steady-state inactivation of Nav1.8 to more depolarized potentials. Co-expression of beta3 shifted steady-state inactivation to more depolarized potentials; however, combined beta1beta3 expression caused no shift in channel availability. The results in this study suggest that the functional behavior of Nav1.8 will vary depending on the type of beta-subunit that expressed under normal and disease states.     

RBBP6 interacts with multifunctional protein YB-1 through its RING finger domain, leading to ubiquitination and proteosomal degradation of YB-1

RBBP6 (retinoblastoma binding protein 6) is a 250-kDa multifunctional protein that interacts with both p53 and pRb and has been implicated in mRNA processing. It has also been identified as a putative E3 ubiquitin ligase due to the presence of a RING finger domain, although no substrate has been identified up to now. Using the RING finger domain as bait in a yeast two-hybrid screen, we identified YB-1 (Y-box binding protein 1) as a binding partner of RBBP6, localising the interaction to the last 62 residues of YB-1. We showed, furthermore, that both full-length RBBP6 and the isolated RING finger domain were able to ubiquitinate YB-1, resulting in its degradation in the proteosome. As a result, RBBP6 was able to suppress the levels of YB-1 in vivo and to reduce its transactivational ability. In the light of the important role that YB-1 appears to play in tumourigenesis, our results suggest that RBBP6 may be a relevant target for therapeutic drugs aimed at modifying the activity of YB-1.     

Down Regulated Expression of the β1 Subunit of the Big-conductance Ca^2＋ Sensitive K＋ Channel in Sphincter of Oddi Cells from Rabbits Fed with a High Cholesterol Diet

Hypercholesterolemia,which is closely related to gallbladder bile stasis,can cause sphincter ofOddi dysfunction (SOD) by increasing the tension of sphincter of Oddi (SO).Intracellular calcium ionconcentration ([Ca~(2 )]_i) could influence the tension of SO.The β1 subunit of the big-conductance Ca~(2 )sensitive K~ channel (BK_(Ca) can enhance the sensitivity of the BK_(Ca) channel to [Ca~(2 )]_i.Absence and decline ofthe BK_(Ca) channel subunit β1 could lead to many diseases.However,the relationship betweenhypercholesterolemia and the expression of β1 subunit is not well understood.In this study,we successfullyexpressed and purified the rabbit BK_(Ca) β1 subunit protein and prepared its polyclonal antibody.The specificityof the prepared antibody was determined by western blotting.A SOD rabbit model induced by a high cholesteroldiet was established and the expression of the β1 subunit of SO was determined by immunohistochemicalstaining and western blotting.Compared with the controls,our results demonstrated that hypercholesterolemiacould decrease the expression of the β1 subunit in the SO cells from rabbits.This indicates that lowerexpression of BK_(Ca) channel β1 subunit might induce SOD.     

The integrin alpha9beta1 mediates adhesion to activated endothelial cells and transendothelial neutrophil migration through interaction with vascular cell adhesion molecule-1

The integrin alpha9beta1 has been shown to be widely expressed on smooth muscle and epithelial cells, and to mediate adhesion to the extracellular matrix proteins osteopontin and tenascin-C. We have found that the peptide sequence this integrin recognizes in tenascin-C is highly homologous to the sequence recognized by the closely related integrin alpha4beta1, in the inducible endothelial ligand, vascular cell adhesion mole-cule-1 (VCAM-1). We therefore sought to determine whether alpha9beta1 also recognizes VCAM-1, and whether any such interaction would be biologically significant. In this report, we demonstrate that alpha9beta1 mediates stable cell adhesion to recombinant VCAM-1 and to VCAM-1 induced on human umbilical vein endothelial cells by tumor necrosis factor-alpha. Furthermore, we show that alpha9beta1 is highly and selectively expressed on neutrophils and is critical for neutrophil migration on VCAM-1 and tenascin-C. Finally, alpha9beta1 and alpha4 integrins contribute to neutrophil chemotaxis across activated endothelial monolayers. These observations suggest a possible role for alpha9beta1/VCAM-1 interactions in extravasation of neutrophils at sites of acute inflammation.     

Extracellular matrix protein 1 interacts with the domain III of fibulin-1C and 1D variants through its central tandem repeat 2

Extracellular matrix protein 1 (ECM1), a widely expressed glycoprotein, has been shown to harbor mutations in lipoid proteinosis (LP), an autosomal recessive disorder characterized by profound alterations in the extracellular matrix of connective tissue. The biological function of ECM1 and its role in the pathomechanisms of LP are unknown. Fibulins comprise a family of extracellular matrix components, and the prototype of this family, fibulin-1, is expressed in various connective tissues and plays a role in developmental and pathologic processes. In this study, we demonstrate that ECM1, and specifically the second tandem repeat domain which is alternatively spliced, interacts with the C-terminal segments of fibulins 1C and 1D splice variants which differ in their C-terminal domain III. The interactions were detected by yeast two-hybrid genetic system and confirmed by co-immunoprecipitations. Kinetics of the binding between ECM1 and fibulin-1D, measured by biosensor assay, revealed a K(d) of 5.71 x 10(-8) M, indicating a strong protein-protein interaction. Since distinct splice variants of ECM1 and fibulin-1 have been shown to be co-expressed in tissues affected in LP, we propose that altered ECM1/fibulin-1 interactions may play a role in the pathogenesis of this disease as well as in a number of processes involving the extracellular matrix of connective tissues.     

START-GAP1/DLC1 is localized in focal adhesions through interaction with the PTB domain of tensin2

START-GAP1, also termed as DLC1, is a negative-regulator for RhoA and Cdc42. START-GAP1 is localized in focal adhesions via the FAT (focal adhesion targeting) domain located in its N-terminal half and interacts with tensin family proteins, that constitutes focal adhesion components. This study has provided evidence that the interaction between START-GAP1 and tensin2 occurs in a PTB domain-dependent manner. It was revealed that FAT3, the third subdomain of the FAT domain divided into five that consists of 39 amino acids, binds directly to the PTB domain of tensin2. This interaction does not require protein phosphorylation, since the interaction was detected with proteins expressed in bacterial expression system. In mammalian genome, there are three genes encoding START domain containing RhoGAPs. START-GAP2/DLC2 and START-GAP3/DLC3, as well as STRT-GAP1/DLC1, bind to the PTB domain of tensin2, presumably due to the presence of highly conserved residues in the center of FAT3. Deletion of this sub-region abrogates the interaction with the tensin PTB domain. Furthermore, D368, H369, G372, F374, P375 and L378 in the highly conserved region of START-GAP1 have been revealed to be essential for the interaction. The tensin2-PTB domain seems to determine the subcellular localization of FAT3. Nevertheless, our study with deletion mutants revealed that FAT3 is essential but not sufficient for the focal adhesion localization of START-GAP1. These results suggest that the interaction between the tensin PTB domain and FAT3 contributes to START-GAP1 localization but only partially. Other factors could affect the START-GAP1 localization.     

The second transmembrane domain of the large conductance, voltage- and calcium-gated potassium channel beta(1) subunit is a lithocholate sensor

Bile acids and other steroids modify large conductance, calcium- and voltage-gated potassium (BK) channel activity contributing to non-genomic modulation of myogenic tone. Accessory BK beta(1) subunits are necessary for lithocholate (LC) to activate BK channels and vasodilate. The protein regions that sense steroid action, however, remain unknown. Using recombinant channels in 1-palmitoyl-2-oleoyl-phosphatidylethanolamine/1-palmitoyl-2-oleoyl-phosphatidylserine bilayers we now demonstrate that complex proteolipid domains and cytoarchitecture are unnecessary for beta(1) to mediate LC action; beta(1) and a simple phospholipid microenvironment suffice. Since beta(1) senses LC but beta(4) does not, we made chimeras swapping regions between these subunits and, following channel heterologous expression, demonstrate that beta(1) TM2 is a bile acid-recognizing sensor.     

CT domain of CCN2/CTGF directly interacts with fibronectin and enhances cell adhesion of chondrocytes through integrin alpha5beta1

Searching for CCN family protein 2/connective tissue growth factor (CCN2/CTGF) interactive proteins by yeast-two-hybrid screening, we identified fibronectin 1 gene product as a major binding partner of CCN2/CTGF in the chondrosarcoma-derived chondrocytic cell line HCS-2/8. Only the CT domain of CCN2/CTGF bound directly to fibronectin (FN). CCN2/CTGF and its CT domain enhanced the adhesion of HCS-2/8 cells to FN in a dose-dependent manner. The CCN2/CTGF-enhancing effect on cell adhesion to FN was abolished by a blocking antibody against alpha5beta1 integrin (alpha5beta1), but not by one against anti-alphavbeta3 integrin. These findings suggest for the first time that CCN2/CTGF enhances chondrocyte adhesion to FN through direct interaction of its C-terminal CT domain with FN, and that alpha5beta1 is involved in this adhesion.     

Tannins present in Cichorium intybus enhance glucose uptake and inhibit adipogenesis in 3T3-L1 adipocytes through PTP1B inhibition

Insulin resistance is a fundamental aspect for the etiology of non-insulin dependent diabetes mellitus (NIDDM) and has links with a wide array of secondary disorders including weight gain and obesity. The present study analyzes the effect of Cichorium intybus methanolic (CME) extract on glucose transport and adipocyte differentiation in 3T3-L1 cells by studying the radiolabelled glucose uptake and lipid accumulation assays, respectively. By performing detannification (CME/DT), the role of tannins present in CME on both the activities was evaluated. CME and CME/DT exhibited significant glucose uptake in 3T3-L1 adipocytes with a dose-dependent response. Glucose uptake profile in the presence of PI3K and IRTK inhibitors (Wortmannin and Genistein) substantiates the mechanism used by both the extracts. CME inhibited the differentiation of 3T3-L1 preadipocytes but failed to show glucose uptake in inhibitor treated cells. The activity exhibited by CME/DT is exactly vice versa to CME. Furthermore, the findings from PTP1B inhibition assay, mRNA and protein expression analysis revealed the unique behavior of CME and CME/DT. The duality exhibited by C. intybus through adipogenesis inhibition and PPARgamma up regulation is of interest. Current observation concludes that the activities possessed by C. intybus are highly desirable for the treatment of NIDDM because it reduces blood glucose levels without inducing adipogenesis in 3T3-L1 adipocytes.     

Abl-SH3 binding protein 2, 3BP2, interacts with CIN85 and HIP-55

The adapter 3BP2 is involved in leukocyte signaling downstream Src/Syk-kinases coupled immunoreceptors. Here, we show that 3BP2 directly interacts with the endocytic scaffold protein CIN85 and the actin-binding protein HIP-55. 3BP2 co-localized with CIN85 and HIP-55 in T cell rafts and at the T cell/APC synapse, an active zone of receptors and proteins recycling. A binding region of CIN85 SH3 domains on 3BP2 was mapped to a PVPTPR motif in the first proline-rich region of 3BP2, whereas the C-terminal SH3 domain of HIP-55 bound a more distal proline-rich domain of 3BP2. Together, our data suggest an unexpected role of 3BP2 in endocytic and cytoskeletal regulation through its interaction with CIN85 and HIP-55.     

Interactions of barley α-amylase isozymes with Ca2 , substrates and proteinaceous inhibitors

-Amylases are endo-acting retaining enzymes of glycoside hydrolase family 13 with a catalytic (β/)₈-domain containing an inserted loop referred to as domain B and a C-terminal anti-parallel β-sheet termed domain C. New insights integrate the roles of Ca^2 +, different substrates, and proteinaceous inhibitors for -amylases. Isozyme specific effects of Ca^2 + on the 80% sequence identical barley -amylases AMY1 and AMY2 are not obvious from the two crystal structures, containing three superimposable Ca^2 + with identical ligands. A fully hydrated fourth Ca^2 + at the interface of the AMY2/barley -amylase/subtilisin inhibitor (BASI) complex interacts with catalytic groups in AMY2, and Ca^2 + occupies an identical position in AMY1 with thiomaltotetraose bound at two surface sites. EDTA-treatment, DSC, and activity assays indicate that AMY1 has the highest affinity for Ca^2 +. Subsite mapping has revealed that AMY1 has ten functional subsites which can be modified by means protein engineering to modulate the substrate specificity. Other mutational analyses show that surface carbohydrate binding sites are critical for interaction with polysaccharides. The conserved Tyr380 in the newly discovered 'sugar tongs' site in domain C of AMY1 is thus critical for binding to starch granules. Furthermore, mutations of binding sites mostly reduced the degree of multiple attack in amylose hydrolysis. AMY1 has higher substrate affinity than AMY2, but isozyme chimeras with AMY2 domain C and other regions from AMY1 have higher substrate affinity than both parent isozymes. The latest revelations addressing various structural and functional aspects that govern the mode of action of barley -amylases are reported in this review.     

Swapping of the N-terminus of VDAC1 with VDAC3 restores full activity of the channel and confers anti-aging features to the cell

Voltage-dependent anion-selective channels (VDACs) are pore-forming proteins allowing the permeability of the mitochondrial outer membrane. The VDAC3 isoform is the least abundant and least active in a complementation assay performed in a yeast strain devoid of porin-1. We swapped the VDAC3 N-terminal 20 amino acids with homologous sequences from the other isoforms. The substitution of the VDAC3 N-terminus with the VDAC1 N-terminus caused the chimaera to become more active than VDAC1. The VDAC2 N-terminus improved VDAC3 activity, though to a lesser extent. The VDAC3 carrying the VDAC1 N-terminus was able to complement the lack of the yeast porin in mitochondrial respiration and in modulation of reactive oxygen species (ROS). This chimaera increased life span, indicating a more efficient bioenergetic metabolism and/or a better protection from ROS.