Heparan Sulfate Proteoglycans Are Receptors for the Cell-surface Trafficking and Biological Activity of Transglutaminase-2

Transglutaminase type 2 (TG2) is both a protein cross-linking enzyme and a cell adhesion molecule with an elusive unconventional secretion pathway. In normal conditions, TG2-mediated modification of the extracellular matrix modulates cell motility, proliferation and tissue repair, but under continuous cell insult, higher expression and elevated extracellular trafficking of TG2 contribute to the pathogenesis of tissue scarring. In search of TG2 ligands that could contribute to its regulation, we characterized the affinity of TG2 for heparan sulfate (HS) and heparin, an analogue of the chains of HS proteoglycans (HSPGs). By using heparin/HS solid-binding assays and surface plasmon resonance we showed that purified TG2 has high affinity for heparin/HS, comparable to that for fibronectin, and that cell-surface TG2 interacts with heparin/HS. We demonstrated that cell-surface TG2 directly associates with the HS chains of syndecan-4 without the mediation of fibronectin, which has affinity for both syndecan-4 and TG2. Functional inhibition of the cell-surface HS chains of wild-type and syndecan-4-null fibroblasts revealed that the extracellular cross-linking activity of TG2 depends on the HS of HSPG and that syndecan-4 plays a major but not exclusive role. We found that heparin binding did not alter TG2 activity per se. Conversely, fibroblasts deprived of syndecan-4 were unable to effectively externalize TG2, resulting in its cytosolic accumulation. We propose that the membrane trafficking of TG2, and hence its extracellular activity, is linked to TG2 binding to cell-surface HSPG.Transglutaminase type 2 (TG2,² EC 2.3.2.13) is the most widespread member of a large family of enzymes that catalyze the Ca²⁺-dependent post-translational modification of proteins leading to intra- or intermolecular Nϵ(γ-glutamyl)lysine bonds (1, 2). Unlike other family members, TG2 is uniquely exported through a yet to be elucidated non-conventional pathway. Once secreted, TG2 finds in the extracellular compartment the ideal conditions of high Ca²⁺ and low GTP concentration for the activation of its intrinsic transamidation activity (cross-linking) (2, 3). Intracellularly, GTP binding suppresses the Ca²⁺-dependent cross-linking activity and determines the additional GTPase activity of TG2 (4, 5), which is responsible for signal transduction (6). Once externalized, TG2 remains tightly bound to the cell surface and to the extracellular matrix (ECM) (7, 8), and it is rarely found free in the conditioned medium, unless overexpressed by cell transfection (9).Extracellular TG2 activity is involved in the cross-linking of the ECM, conferring resistance to matrix metalloproteinase and promoting cell-matrix interactions via cross-linking of fibronectin (FN) and collagen (1, 7, 11, 12). TG2 has an additional non-enzymatic role in the matrix as an integrin-β₁ co-receptor (8) by supporting RGD-independent cell adhesion to FN (8, 13, 14).Extracellular cross-linking and TG2-mediated adhesion facilitate the repair process in many tissue compartments (1, 2, 15, 16). On the other hand, uncontrolled cross-linking as a consequence of chronic cell insult and secretion of TG2 has been implicated in a number of pathological conditions, including kidney, liver, and pulmonary fibrosis (17–20).Understanding how TG2 is exported and targeted to the cell surface is critical for limiting its cellular secretion and extracellular action. Although a key trigger for TG2 export is cell stress (2, 21, 22), TG2 is not unspecifically released, because extracellular trafficking occurs in the absence of leakage of intracellular components and cells remain viable (23). We know that TG2 requires the tertiary structure of its active site region to be secreted (9); moreover, TG2 is acetylated on the N terminus (24), a process reported to affect membrane targeting of non-conventional secreted proteins (25). Two main binding partners for TG2, FN and integrin-β1, have both been attributed a possible role in the transport of TG2 to the cell surface (8, 26). FN was shown to co-localize with TG2 once released (26), and integrin-β1 to co-associate with TG2 in cells induced to differentiate (8).TG2 has also long been known to have some affinity for heparin (27, 28), a highly sulfated analogue of heparan sulfate (HS) glycosaminoglycan chains, which are abundant constituents of the cell surface/ECM. HS chains are linear polysaccharides consisting of alternating N-acetylated or N-sulfated glucosamine units (GlcNAc or GlcNS), and uronic acids (glucuronic acid GlcA or iduronic acid IdoA residues) (29), which only exist covalently bound to the core protein of cell-surface proteoglycans (syndecans and glypicans) and secreted proteoglycans (29). Heparin binding is a property common to many ECM proteins (29), but the level of affinity has never been established for TG2, which makes it difficult to estimate the real biological significance of this interaction. Heparan sulfate proteoglycans (HSPG) bind ECM ligands through the HS chains, influencing their biological activity, trafficking, and secretion. Among the HSPG subfamilies, the syndecans act as co-receptors for both ECM components and soluble ligands (30), and syndecan-4 has overlapping roles with extracellular TG2 in wound healing and fibrosis (31, 32). In this study, we show that TG2 has a surprisingly high affinity for heparin and HS, raising the hypothesis that HSPG are involved in its biological activity. We demonstrate that HSPGs are essential for the transamidating activity of TG2 at the cell surface and that syndecan-4 acts as a receptor for TG2, which is involved in the trafficking and cell-surface localization, and thus activity of TG2.     

Multiple Heparan Sulfate Binding Site Engagements Are Required for the Infectious Entry of Human Papillomavirus Type 16

Human papillomavirus (HPV) entry is accompanied by multiple receptor-induced conformational changes (CCs) affecting both the major and minor capsid proteins, L1 and L2. Interaction of heparan sulfate (HS) with L1 is essential for successful HPV16 entry. Recently, cocrystallization of HPV16 with heparin revealed four distinct binding sites. Here we characterize mutant HPV16 to delineate the role of engagement with HS binding sites during infectious internalization. Site 1 (Lys278, Lys361), which mediates primary binding, is sufficient to trigger an L2 CC, exposing the amino terminus. Site 2 (Lys54, Lys356) and site 3 (Asn57, Lys59, Lys442, Lys443) are engaged following primary attachment and are required for infectious entry. Site 2 mutant particles are efficiently internalized but fail to undergo an L1 CC on the cell surface and subsequent uncoating in the endocytic compartment. After initial attachment to the cell, site 3 mutants undergo L1 and L2 CCs and then accumulate on the extracellular matrix (ECM). We conclude that the induction of CCs following site 1 and site 2 interactions results in reduced affinity for the primary HS binding site(s) on the cell surface, which allows engagement with site 3. Taken together, our findings suggest that HS binding site engagement induces CCs that prepare the virus for downstream events, such as the exposure of secondary binding sites, CCs, transfer to the uptake receptor, and uncoating.     

Heparan Sulfate Proteoglycans Are Important for Islet Amyloid Formation and Islet Amyloid Polypeptide-induced Apoptosis

Deposition of β cell toxic islet amyloid is a cardinal finding in type 2 diabetes. In addition to the main amyloid component islet amyloid polypeptide (IAPP), heparan sulfate proteoglycan is constantly present in the amyloid deposit. Heparan sulfate (HS) side chains bind to IAPP, inducing conformational changes of the IAPP structure and an acceleration of fibril formation. We generated a double-transgenic mouse strain (hpa-hIAPP) that overexpresses human heparanase and human IAPP but is deficient of endogenous mouse IAPP. Culture of hpa-hIAPP islets in 20 mm glucose resulted in less amyloid formation compared with the amyloid load developed in cultured islets isolated from littermates expressing human IAPP only. A similar reduction of amyloid was achieved when human islets were cultured in the presence of heparin fragments. Furthermore, we used CHO cells and the mutant CHO pgsD-677 cell line (deficient in HS synthesis) to explore the effect of cellular HS on IAPP-induced cytotoxicity. Seeding of IAPP aggregation on CHO cells resulted in caspase-3 activation and apoptosis that could be prevented by inhibition of caspase-8. No IAPP-induced apoptosis was seen in HS-deficient CHO pgsD-677 cells. These results suggest that β cell death caused by extracellular IAPP requires membrane-bound HS. The interaction between HS and IAPP or the subsequent effects represent a possible therapeutic target whose blockage can lead to a prolonged survival of β cells.     

Heparan Sulfate 2-O-Sulfotransferase Is Required for Triglyceride-rich Lipoprotein Clearance

Hepatic clearance of triglyceride-rich lipoproteins depends on heparan sulfate and low density lipoprotein receptors expressed on the basal membrane of hepatocytes. Binding and uptake of the lipoproteins by way of heparan sulfate depends on the degree of sulfation of the chains based on accumulation of plasma triglycerides and delayed clearance of triglyceride-rich lipoproteins in mice bearing a hepatocyte-specific alteration of N-acetylglucosamine (GlcNAc) N-deacetylase-N-sulfotransferase 1 (Ndst1) (MacArthur, J. M., Bishop, J. R., Stanford, K. I., Wang, L., Bensadoun, A., Witztum, J. L., and Esko, J. D. (2007) J. Clin. Invest. 117, 153–164). Inactivation of Ndst1 led to decreased overall sulfation of heparan sulfate due to coupling of uronyl 2-O-sulfation and glucosaminyl 6-O-sulfation to initial N-deacetylation and N-sulfation of GlcNAc residues. To determine whether lipoprotein clearance depends on 2-O-and 6-O-sulfation, we evaluated plasma triglyceride levels in mice containing loxP-flanked conditional alleles of uronyl 2-O-sulfotransferase (Hs2st^f/f) and glucosaminyl 6-O-sulfotransferase-1 (Hs6st1^f/f) and the bacterial Cre recombinase expressed in hepatocytes from the rat albumin (Alb) promoter. We show that Hs2st^f/fAlbCre⁺ mice accumulated plasma triglycerides and exhibited delayed clearance of intestinally derived chylomicrons and injected human very low density lipoproteins to the same extent as observed in Ndst1^f/fAlbCre⁺ mice. In contrast, Hs6st1^f/fAlbCre⁺ mice did not exhibit any changes in plasma triglycerides. Chemically modified heparins lacking N-sulfate and 2-O-sulfate groups did not block very low density lipoprotein binding and uptake in isolated hepatocytes, whereas heparin lacking 6-O-sulfate groups was as active as unaltered heparin. Our findings show that plasma lipoprotein clearance depends on specific subclasses of sulfate groups and not on overall charge of the chains.     

Small Oligomers of Ribulose-bisphosphate Carboxylase/Oxygenase (Rubisco) Activase Are Required for Biological Activity

Ribulose-bisphosphate carboxylase/oxygenase (Rubisco) activase uses the energy from ATP hydrolysis to remove tight binding inhibitors from Rubisco, thus playing a key role in regulating photosynthesis in plants. Although several structures have recently added much needed structural information for different Rubisco activase enzymes, the arrangement of these subunits in solution remains unclear. In this study, we use a variety of techniques to show that Rubisco activase forms a wide range of structures in solution, ranging from monomers to much higher order species, and that the distribution of these species is highly dependent on protein concentration. The data support a model in which Rubisco activase forms an open spiraling structure rather than a closed hexameric structure. At protein concentrations of 1 μm, corresponding to the maximal activity of the enzyme, Rubisco activase has an oligomeric state of 2–4 subunits. We propose a model in which Rubisco activase requires at least 1 neighboring subunit for hydrolysis of ATP.     

Heparan Sulfate Is Required for Embryonic Stem Cells to Exit from Self-renewal

Pluripotent embryonic stem cells (ESCs) must select between alternative fates of self-renewal and lineage commitment at each division during continuous proliferation. Heparan sulfate (HS) is a highly sulfated polysaccharide and is present abundantly on the ESC surface. In this study, we investigated the role of HS in ESC self-renewal by examining Ext1^−/− ESCs that are deficient in HS. We found that Ext1^−/− ESCs retained their self-renewal potential but failed to transit from self-renewal to differentiation upon removal of leukemia inhibitory factor. Furthermore, we found that the aberrant cell fate commitment is caused by defects in fibroblast growth factor signaling, which directly retained high expression of the pluripotency gene Nanog in Ext1^−/− ESCs. Therefore, our studies identified and defined HS as a novel factor that controls ESC fate commitment and also delineates that HS facilitates fibroblast growth factor signaling, which, in turn, inhibits Nanog expression and commits ESCs to lineage differentiation.     

An Emerging Role of Sonic Hedgehog Shedding as a Modulator of Heparan Sulfate Interactions

Major developmental morphogens of the Hedgehog (Hh) family act at short range and long range to direct cell fate decisions in vertebrate and invertebrate tissues. To this end, Hhs are released from local sources and act at a distance on target cells that express the Hh receptor Patched. However, morphogen secretion and spreading are not passive processes because all Hhs are synthesized as dually (N- and C-terminal) lipidated proteins that firmly tether to the surface of producing cells. On the cell surface, Hhs associate with each other and with heparan sulfate (HS) proteoglycans. This raises the question of how Hh solubilization and spreading is achieved. We recently discovered that Sonic hedgehog (Shh) is solubilized by proteolytic processing (shedding) of lipidated peptide termini in vitro. Because unprocessed N termini block Patched receptor binding sites in the cluster, we further suggested that their proteolytic removal is required for simultaneous Shh activation. In this work we confirm inactivity of unprocessed protein clusters and demonstrate restored biological Shh function upon distortion or removal of N-terminal amino acids and peptides. We further show that N-terminal Shh processing targets and inactivates the HS binding Cardin-Weintraub (CW) motif, resulting in soluble Shh clusters with their HS binding capacities strongly reduced. This may explain the ability of Shh to diffuse through the HS-containing extracellular matrix, whereas other HS-binding proteins are quickly immobilized. Our in vitro findings are supported by the presence of CW-processed Shh in murine brain samples, providing the first in vivo evidence for Shh shedding and subsequent solubilization of N-terminal-truncated proteins.     

Sulfated Polysaccharides Are Required for Collagen-Induced Vascular Tube Formation

We have previously shown that soluble type I collagen can induce vascular tube formation when it contacts the apical side of a confluent endothelial monolayer. In this study we have examined which soluble agent(s) are required for collagen-induced tube formation. Human neo-natal foreskin microvascular endothelial cells, maintained in basal medium, were preincubated with each test agent for 2 h prior to the addition of solubilised type I collagen (100 μg/ml). After 6 h, tube formation was quantitated using image analysis and expressed as the mean area of tube formation (mm²) per microscopic field of view. Collagen-induced tube formation did not occur in the presence of endothelial cell growth supplement, basic fibroblast growth factor, or normal pooled human serum. In contrast, the addition of heparin at 5 or 50 μg/ml caused extensive tube formation (0.22 ± 0.07 and 0.30 ± 0.12 mm², respectively) whereas at 500 μg/ml little tube formation occurred (0.03 ± 0.02 mm²). Protamine sulfate, an antagonist of heparin, inhibited collagen-induced tube formation in a dose-dependent manner. Pentosan polysulfate, dextran sulfate, heparan sulfate, and chondroitin sulfate mimicked the action of heparin. Partially sulfated heparin (de-N-sulfated heparin) stimulated less tube formation compared to heparin (0.15 ± 0.06 mm² at 50 μg/ml). The nonsulfated polysaccharides, xylan and dextran, had no effect on tube formation. In summary, sulfated polysaccharides are required for collagen-induced vascular tube formation in vitro. The sulfation of these molecules appears to be vital for collagen-induced tube formation.     

Characterization of the Host Factors Required for Hepadnavirus Covalently Closed Circular (ccc) DNA Formation

Synthesis of the covalently closed circular (ccc) DNA is a critical, but not well-understood step in the life cycle of hepadnaviruses. Our previous studies favor a model that removal of genome-linked viral DNA polymerase occurs in the cytoplasm and the resulting deproteinized relaxed circular DNA (DP-rcDNA) is subsequently transported into the nucleus and converted into cccDNA. In support of this model, our current study showed that deproteinization of viral double-stranded linear (dsl) DNA also took place in the cytoplasm. Furthermore, we demonstrated that Ku80, a component of non-homologous end joining DNA repair pathway, was essential for synthesis of cccDNA from dslDNA, but not rcDNA. In an attempt to identify additional host factors regulating cccDNA biosynthesis, we found that the DP-rcDNA was produced in all tested cell lines that supported DHBV DNA replication, but cccDNA was only synthesized in the cell lines that accumulated high levels of DP-rcDNA, except for NCI-H322M and MDBK cells, which failed to synthesize cccDNA despite of the existence of nuclear DP-rcDNA. The results thus imply that while removal of the genome-linked viral DNA polymerase is most likely catalyzed by viral or ubiquitous host function(s), nuclear factors required for the conversion of DP-rcDNA into cccDNA and/or its maintenance are deficient in the above two cell lines, which could be useful tools for identification of the elusive host factors essential for cccDNA biosynthesis or maintenance.     

Lateral Opening and Exit Pore Formation Are Required for BamA Function

1. Download : Download high-res image (415KB)
2. Download : Download full-size image

     

Multiple Domains Are Required for the Toxic Activity of Pseudomonas aeruginosa ExoU

Expression of ExoU by Pseudomonas aeruginosa is correlated with acute cytotoxicity in a number of epithelial and macrophage cell lines. In vivo, ExoU is responsible for epithelial injury. The absence of a known motif or significant homology with other proteins suggests that ExoU may possess a new mechanism of toxicity. To study the intracellular effects of ExoU, we developed a transient-transfection system in Chinese hamster ovary cells. Transfection with full-length but not truncated forms of ExoU inhibited reporter gene expression. Inhibition of reporter activity after cotransfection with ExoU-encoding constructs was correlated with cellular permeability and death. The toxicity of truncated versions of ExoU could be restored by coexpression of the remainder of the molecule from separate plasmids in trans. This strategy was used to map N- and C-terminal regions of ExoU that are necessary but not sufficient for toxicity. Disruption of a middle region of the protein reduces toxicity. This portion of the molecule is postulated to allow the N- and C-terminal regions to functionally complement one another. In contrast to ExoS and ExoT, native and recombinant ExoU molecules do not oligomerize or form aggregates. The complex domain structure of ExoU suggests that, like other P. aeruginosa-encoded type III effectors (ExoS and ExoT), ExoU toxicity may result from a molecule that possesses more than one activity.     

Functions of Chondroitin Sulfate and Heparan Sulfate in the Developing Brain

Chondroitin sulfate and heparan sulfate proteoglycans are major components of the cell surface and extracellular matrix in the brain. Both chondroitin sulfate and heparan sulfate are unbranched highly sulfated polysaccharides composed of repeating disaccharide units of glucuronic acid and N-acetylgalactosamine, and glucuronic acid and N-acetylglucosamine, respectively. During their biosynthesis in the Golgi apparatus, these glycosaminoglycans are highly modified by sulfation and C5 epimerization of glucuronic acid, leading to diverse heterogeneity in structure. Their structures are strictly regulated in a cell type-specific manner during development partly by the expression control of various glycosaminoglycan-modifying enzymes. It has been considered that specific combinations of glycosaminoglycan-modifying enzymes generate specific functional microdomains in the glycosaminoglycan chains, which bind selectively with various growth factors, morphogens, axon guidance molecules and extracellular matrix proteins. Recent studies have begun to reveal that the molecular interactions mediated by such glycosaminoglycan microdomains play critical roles in the various signaling pathways essential for the development of the brain.     

The Effect of Heparan Sulfate Application on Bone Formation during Distraction Osteogenesis

Bone morphogenetic proteins (BMPs) are recognized for their ability to induce bone formation in vivo and in vitro. Their osteogenic and osteoinductive properties are tightly regulated by the secretion of specific BMP antagonists, which have been shown to physically bind and sometimes be blocked by the extracellular proteoglycan heparan sulphate side chains (from hereon referred to as HS). The purpose of this study was to investigate if local application of 5 µg of HS proteoglycan to a bone regenerate site in a mouse model of distraction osteogenesis (DO) can accelerate bone healing and affect the expression of key members of the BMP signaling pathway. DO was performed on the right tibia of 115 adult male wild-type mice. At mid-distraction (day 11), half the group was injected locally with 5 µg of HS, while the other half was injected with saline. The mice were sacrificed at 2 time-points: mid-consolidation (34 days) and full consolidation (51 days). The distracted tibial zone was then collected for analysis by μCT, radiology, biomechanical testing, immunohistochemistry, and histology. While μCT data showed no statistically significant difference in bone formation, the results of biomechanical testing in stiffness and ultimate force were significantly lower in the HS-injected bones at 51 days, compared to controls. Immunohistochemistry results also suggested a decrease in expression of several key members of the BMP signaling pathway at 34 days. Furthermore, wound dehiscence and infection rates were significantly elevated in the HS group compared to the controls, which resulted in a higher rate of euthanasia in the treatment group. Our findings demonstrate that exogenous application of 5 µg of HS in the distracted gap of a murine model had a negative impact on bone and wound healing.     

Stearoyl-CoA Desaturase 1 Activity Is Required for Autophagosome Formation

Autophagy is one of the major degradation pathways for cytoplasmic components. The autophagic isolation membrane is a unique membrane whose content of unsaturated fatty acids is very high. However, the molecular mechanisms underlying formation of this membrane, including the roles of unsaturated fatty acids, remain to be elucidated. From a chemical library consisting of structurally diverse compounds, we screened for novel inhibitors of starvation-induced autophagy by measuring LC3 puncta formation in mouse embryonic fibroblasts stably expressing GFP-LC3. One of the inhibitors we identified, 2,5-pyridinedicarboxamide, N2,N5-bis[5-[(dimethylamino)carbonyl]-4-methyl-2-thiazolyl], has a molecular structure similar to that of a known stearoyl-CoA desaturase (SCD) 1 inhibitor. To determine whether SCD1 inhibition influences autophagy, we examined the effects of the SCD1 inhibitor 28c. This compound strongly inhibited starvation-induced autophagy, as determined by LC3 puncta formation, immunoblot analyses of LC3, electron microscopic observations, and p62/SQSTM1 accumulation. Overexpression of SCD1 or supplementation with oleic acid, which is a catalytic product of SCD1 abolished the inhibition of autophagy by 28c. Furthermore, 28c suppressed starvation-induced autophagy without affecting mammalian target of rapamycin activity, and also inhibited rapamycin-induced autophagy. In addition to inhibiting formation of LC3 puncta, 28c also inhibited formation of ULK1, WIPI1, Atg16L, and p62/SQSTM1 puncta. These results suggest that SCD1 activity is required for the earliest step of autophagosome formation.     

Heparanase 2 Interacts with Heparan Sulfate with High Affinity and Inhibits Heparanase Activity

Heparanase activity is highly implicated in cell dissemination associated with tumor metastasis, angiogenesis, and inflammation. Heparanase expression is induced in many hematological and solid tumors, associated with poor prognosis. Heparanase homolog, termed heparanase 2 (Hpa2), was cloned based on sequence homology. Detailed characterization of Hpa2 at the biochemical, cellular, and clinical levels has not been so far reported, and its role in normal physiology and pathological disorders is obscure. We provide evidence that unlike heparanase, Hpa2 is not subjected to proteolytic processing and exhibits no enzymatic activity typical of heparanase. Notably, the full-length Hpa2c protein inhibits heparanase enzymatic activity, likely due to its high affinity to heparin and heparan sulfate and its ability to associate physically with heparanase. Hpa2 expression was markedly elevated in head and neck carcinoma patients, correlating with prolonged time to disease recurrence (follow-up to failure; p = 0.006) and inversely correlating with tumor cell dissemination to regional lymph nodes (N-stage; p = 0.03). Hpa2 appears to restrain tumor metastasis, likely by attenuating heparanase enzymatic activity, conferring a favorable outcome of head and neck cancer patients.     

NHERF2/NHERF3 Protein Heterodimerization and Macrocomplex Formation Are Required for the Inhibition of NHE3 Activity by Carbachol

NHERF1, NHERF2, and NHERF3 belong to the NHERF (Na⁺/H⁺ exchanger regulatory factor) family of PSD-95/Discs-large/ZO-1 (PDZ) scaffolding proteins. Individually, each NHERF protein has been shown to be involved in the regulation of multiple receptors or transporters including Na⁺/H⁺ exchanger 3 (NHE3). Although NHERF dimerizations have been reported, results have been inconsistent, and the physiological function of NHERF dimerizations is still unknown. The current study semiquantitatively compared the interaction strength among all possible homodimerizations and heterodimerizations of these three NHERF proteins by pulldown and co-immunoprecipitation assays. Both methods showed that NHERF2 and NHERF3 heterodimerize as the strongest interaction among all NHERF dimerizations. In vivo NHERF2/NHERF3 heterodimerization was confirmed by FRET and FRAP (fluorescence recovery after photobleach). NHERF2/NHERF3 heterodimerization is mediated by PDZ domains of NHERF2 and the C-terminal PDZ domain recognition motif of NHERF3. The NHERF3-4A mutant is defective in heterodimerization with NHERF2 and does not support the inhibition of NHE3 by carbachol. This suggests a role for NHERF2/NHERF3 heterodimerization in the regulation of NHE3 activity. In addition, both PDZ domains of NHERF2 could be simultaneously occupied by NHERF3 and another ligand such as NHE3, α-actinin-4, and PKCα, promoting formation of NHE3 macrocomplexes. This study suggests that NHERF2/NHERF3 heterodimerization mediates the formation of NHE3 macrocomplexes, which are required for the inhibition of NHE3 activity by carbachol.     

Uclacyanin Proteins Are Required for Lignified Nanodomain Formation within Casparian Strips

1. Download : Download high-res image (206KB)
2. Download : Download full-size image