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PubMed2006

Distinct induction patterns and functions of two closely related interferon-inducible human genes, ISG54 and ISG56.

Human P54 and P56 proteins are tetratricopeptide proteins that are encoded by two closely related genes, ISG54 and ISG56. These genes are induced strongly but transiently when cells are treated with interferons or double-stranded RNA or infected with a variety of viruses. We observed that, although double-stranded RNA or Sendai virus infection induced the two genes with similar kinetics, their induction kinetics in response to interferon-beta were quite different. The induction kinetics by virus infection were also different between two cell lines. Functionally the two proteins were similar. Like P56, P54 bound to the translation initiation factor eIF3 and inhibited translation. However, unlike P56, P54 bound to both the "e" and the "c" subunits of eIF3. Consequently, P54 inhibited two functions of eIF3. Like P56, it inhibited the ability of eIF3 to stabilize the eIF2 x GTP x Met-tRNA(i) ternary complex. But in addition, it also inhibited the formation of the 48 S pre-initiation complex between the 40 S ribosomal subunit and the 20 S complex composed of eIF3, ternary complex, eIF4F, and mRNA. Thus, although similar in structure, the human P54 and P56 proteins are induced differently and function differently.

Adaptor Proteins, Signal Transducing↗

PubMed2004

The tetraspan protein EMP2 modulates the surface expression of caveolins and glycosylphosphatidyl inositol-linked proteins.

Caveolae are a subset of lipid rafts enriched in glycosphingolipids and cholesterol-rich domains, but selectively lacking glycosylphosphatidyl inositol-anchored proteins (GPI-APs). Caveolin proteins are the organizing component of caveolae, but the corresponding proteins for other classes of lipid rafts are poorly defined. Epithelial membrane protein-2 (EMP2), a member of the four-transmembrane superfamily, facilitates plasma membrane delivery of certain integrins. In this study, we found by laser confocal microscopy that EMP2 was associated with GPI-APs (detected by the GPI-AP binding bacterial toxin proaerolysin). Biochemical membrane fractionation and methyl-beta-cyclodextrin treatment demonstrated that this association occurred within lipid rafts. EMP2 did not associate with caveolin-bearing membrane structures, and recombinant overexpression of EMP2 in NIH3T3 cells decreased caveolin-1 and caveolin-2 protein levels while increasing the surface expression of GPI-APs. Conversely, a ribozyme construct that specifically cleaves the EMP2 transcript reduced surface GPI-APs and increased caveolin protein expression. These findings suggest that EMP2 facilitates the formation and surface trafficking of lipid rafts bearing GPI-APs, and reduces caveolin expression, resulting in impaired formation of caveolae.

Adaptor Protein Complex gamma Subunits↗

PubMed1999

A beta-turn endocytic code is required for optimal internalization of the growth hormone receptor but not for alpha-adaptin association.

Intracellular trafficking of GH and its receptor, more particularly the chicken GH receptor (cGHR), was examined in COS-7 cells using biochemical and structural studies. Internalization of radioactive GH by the cGHR is reduced as compared with the rat GHR. On the contrary, activation of gene transcription through Janus kinase-2 was similar for both species. Secondary structures of the cytoplasmic domain of chicken and rat GHR were compared, since beta-turns were reported as internalization signals. The substitution of Pro335-Asp335, present in mammalian GH receptors, with Thr307-Gln308 in the cGHR leads to the loss of a beta-turn within a conserved cytoplasmic region. Mutational analysis indicated that the lower rate of internalization of cGHR, as compared with mammalian GHR, was due to this motif. Our data further show that alpha-adaptin, a subunit of adaptor protein AP-2, associates with the GHR upon hormone stimulation. The clathrin-coated pit pathway therefore seems to be involved in the endocytosis of cGHR, as AP-2 is known to intervene in the recruitment of receptors to these pits. Interaction with alpha-adaptin may occur through a common epitope of the chicken and mammalian GHR, since receptors from both species bind similar amounts of alpha-adaptin; alternatively, two different epitopes with similar affinity may be involved. Therefore, not alpha-adaptin but an uncharacterized factor, presumably interacting with the identified beta-turn endocytic code, is responsible for the difference in internalization kinetics. Finally, the present study illustrates that functional amino acid motifs of receptors can be derived from comparative studies.

Adaptor Protein Complex alpha Subunits↗

PubMed2003

Endocytosis of epithelial apical junctional proteins by a clathrin-mediated pathway into a unique storage compartment.

The adherens junction (AJ) and tight junction (TJ) are key regulators of epithelial polarity and barrier function. Loss of epithelial phenotype is accompanied by endocytosis of AJs and TJs via unknown mechanisms. Using a model of calcium depletion, we defined the pathway of internalization of AJ and TJ proteins (E-cadherin, p120 and beta-catenins, occludin, JAM-1, claudins 1 and 4, and ZO-1) in T84 epithelial cells. Proteinase protection assay and immunocytochemistry revealed orchestrated internalization of AJs and TJs into a subapical cytoplasmic compartment. Disruption of caveolae/lipid rafts did not prevent endocytosis, nor did caveolin-1 colocalize with internalized junctional proteins. Furthermore, AJ and TJ proteins did not colocalize with the macropinocytosis marker dextran. Inhibitors of clathrin-mediated endocytosis blocked internalization of AJs and TJs, and junctional proteins colocalized with clathrin and alpha-adaptin. AJ and TJ proteins were observed to enter early endosomes followed by movement to organelles that stained with syntaxin-4 but not with markers of late and recycling endosomes, lysosomes, or Golgi. These results indicate that endocytosis of junctional proteins is a clathrin-mediated process leading into a unique storage compartment. Such mechanisms may mediate the disruption of intercellular contacts during normal tissue remodeling and in pathology.

Adaptor Protein Complex alpha Subunits↗

PubMed1998

Cloning and characterization of hSRP1 gamma, a tissue-specific nuclear transport factor.

Nuclear import of proteins containing a nuclear localization signal (NLS) is dependent on the presence of a cytoplasmic NLS receptor, the GTPase Ran, and p10/ NTF2. The NLS receptor is a heterodimeric proteins consisting of subunits of approximately 60 and 97 kDa, which have been termed importin alpha/beta, karyopherin alpha/beta, or PTAC 58/ 97. Members of the 60-kDa/importin alpha subunit family directly bind to the NLS motif and have been shown to function as adaptors that tether NLS-containing proteins to the p97/ importin beta subunit and to the downstream transport machinery. Herein we report the identification and characterization of hSRP1 gamma, a human importin alpha homologue. The hSRP1 gamma protein is around 45% identical to the previously identified human importin alpha homologues hSRP1 alpha/Rch1 and NPI/ hSRP1. hSRP1 gamma can form a complex with importin beta and is able to mediate import of a BSA-NLS substrate in an in vitro nuclear import system. Interestingly, hSRP1 gamma shows a very selective expression pattern and is most abundantly expressed in skeletal muscle, representing more than 1% of the total protein in this tissue. A potential role for hSRP1 gamma in tissue-specific transport events is discussed.

Amino Acid Sequence↗

PubMed1995

Comparative localization of mannose-6-phosphate receptor with 2,6sialyltransferase in HepG2 cells: an analysis by confocal double immunofluorescence microscopy.

Recent advances in confocal immunofluorescent microscopy have led to significant improvements in delineating membrane-bounded organelles. In this study using HepG2 cells we focused on two functionally distinct but closely apposed organelles that have been difficult to distinguish by conventional immunofluorescent microscopy, namely the Golgi apparatus, the trans Golgi network (TGN) and late endosomes. The following markers were used: for the Golgi apparatus beta 1,4galactosyltransferase (gal-T), for the TGN, 2, 6(N)sialytransferase (sia-T) and for late endosomes/TGN, the mannose-6-phosphate/insulin growth factor II receptor (CIMPR). In addition, that part of the TGN previously shown to contain CIMPR was also identified using antibodies to the gamma-chain of the HA-1 adaptor (Klumperman et al. J. Cell Biol. 121, 997-1010 (1993)). True colocalization of intracellular antigens was ascertained by double staining of gal-T using both monoclonal and polyclonal antibodies. As previously reported, our results revealed essentially complete colocalization of gal-T and sia-T in this cell line. While the compartments containing CIMPR appeared to overlap with those containing sia-T by conventional immunofluorescence, both compartments were clearly distinct by double-label confocal microscopy. Differences between these organelles became more evident following treatment with brefeldin A. Finally, HA-1 gamma-chain was also localized to structures that were close to but clearly different from the sia-T-containing compartment. Absence of colocalization of CIMPR or HA-1 gamma-chain with sia-T indicates that these markers are enriched in distinct domains of the trans Golgi network.

Adaptor Protein Complex gamma Subunits↗

PubMed2003

Plasma membrane-cytoskeleton-endoplasmic reticulum complexes in neurons and astrocytes.

The possibility that certain integral plasma membrane (PM) proteins involved in Ca(2+) homeostasis form junctional units with adjacent endoplasmic reticulum (ER) in neurons and glia was explored using immunoprecipitation and immunocytochemistry. Rat brain membranes were solubilized with the mild, non-ionic detergent, IGEPAL CA-630. Na(+)/Ca(2+) exchanger type 1 (NCX1), a key PM Ca(2+) transporter, was immunoprecipitated from the detergent-soluble fraction. Several abundant PM proteins co-immunoprecipitated with NCX1, including the alpha2 and alpha3 isoforms of the Na(+) pump catalytic (alpha) subunit, and the alpha2 subunit of the dihydropyridine receptor. The adaptor protein, ankyrin 2 (Ank 2), and the cytoskeletal proteins, alpha-fodrin and beta-spectrin, also selectively co-immunoprecipitated with NCX1, as did the ER proteins, Ca(2+) pump type 2 (SERCA 2), and inositol-trisphosphate receptor type 1 (IP(3)R-1). In contrast, a number of other abundant PMs, adaptors, and cytoskeletal proteins did not co-immunoprecipitate with NCX1, including the Na(+) pump alpha1 isoform, PM Ca(2+) pump type 1 (PMCA1), beta-fodrin, and Ank 3. In reciprocal experiments, immunoprecipitation with antibodies to the Na(+) pump alpha2 and alpha3 isoforms, but not alpha1, co-immunoprecipitated NCX1; the antibodies to alpha1 did, however, co-immunoprecipitate PMCA1. Antibodies to Ank 2, alpha-fodrin, beta-spectrin and IP(3)R-1 all co-immunoprecipitated NCX1. Immunocytochemistry revealed partial co-localization of beta-spectrin with NCX1, Na(+) pump alpha3, and IP(3)R-1 in neurons and of alpha-fodrin with NCX1 and SERCA2 in astrocytes. The data support the idea that in neurons and glia PM microdomains containing NCX1 and Na(+) pumps with alpha2 or alpha3 subunits form Ca(2+) signaling complexes with underlying ER containing SERCA2 and IP(3)R-1. These PM and ER components appear to be linked through the cytoskeletal spectrin network, to which they are probably tethered by Ank 2.

Animals↗

PubMed2000

Trafficking of major histocompatibility complex class II molecules in human B-lymphoblasts deficient in the AP-3 adaptor complex.

The major histocompatibility complex class II subunits (MHC-II) alpha and beta assemble with the invariant chain (Ii) in the endoplasmic reticulum and are transported to endosomal-lysosomal organelles known as MHC class II compartments (MIICs). Although it has been shown that two dileucine-based signals in the cytosolic tail of Ii, as well as a dileucine-based signal in the tail of the beta chain mediate sorting to MIICs, the molecular mechanisms by which alphabetaIi complexes are sorted have yet to be resolved fully. The AP-3 adaptor complex stands out as a particularly good candidate for mediating this targeting because: (i) it has a proven role in the trafficking of membrane proteins to lysosome-related organelles; and (ii) it has the ability to interact with dileucine-based signals in vitro. To investigate the potential role of AP-3 in transport of MHC-II to MIICs, we have examined MHC-II trafficking in human B-lymphoblast lines from patients with Hermansky-Pudlak syndrome type 2 (HPS-2), which are deficient in the AP-3 complex. Pulse-chase analyses revealed no significant alteration in the kinetics of synthesis and degradation of either MHC-II subunits or Ii. Moreover, we observed neither impairment of the formation of compact SDS-resistant alphabeta dimers, nor delay in the appearance of a conformational epitope indicative of a mature, Ii-free alphabeta dimer. Finally, we demonstrated that in HPS-2 patients' cells, there was no delay in the expression of the alphabeta dimers on the cell surface. Thus, AP-3 does not seem to be essential for normal trafficking of MHC-II. These findings have important implications for HPS-2 patients, because they suggest that the recurrent bacterial infections suffered by these patients are not likely due to impaired antigen processing and presentation by MHC-II.

Adaptor Protein Complex alpha Subunits↗

PubMed2004

GADD34-PP1c recruited by Smad7 dephosphorylates TGFbeta type I receptor.

The cascade of phosphorylation is a pivotal event in transforming growth factor beta (TGFbeta) signaling. Reversible phosphorylation regulates fundamental aspects of cell activity. TGFbeta-induced Smad7 binds to type I receptor (TGFbeta type I receptor; TbetaRI) functioning as a receptor kinase antagonist. We found Smad7 interacts with growth arrest and DNA damage protein, GADD34, a regulatory subunit of the protein phosphatase 1 (PP1) holoenzyme, which subsequently recruits catalytic subunit of PP1 (PP1c) to dephosphorylate TbetaRI. Blocking Smad7 expression by RNA interference inhibits association of GADD34-PP1c complex with TbetaRI, indicating Smad7 acts as an adaptor protein in the formation of the PP1 holoenzyme that targets TbetaRI for dephosphorylation. SARA (Smad anchor for receptor activation) enhances the recruitment PP1c to the Smad7-GADD34 complex by controlling the specific subcellular localization of PP1c. Importantly, GADD34-PP1c recruited by Smad7 inhibits TGFbeta-induced cell cycle arrest and mediates TGFbeta resistance in responding to UV light irradiation. The dephosphorylation of TbetaRI mediated by Smad7 is an effective mechanism for governing negative feedback in TGFbeta signaling.

Activin Receptors, Type I↗

PubMed1992

100-kD proteins of Golgi- and trans-Golgi network-associated coated vesicles have related but distinct membrane binding properties.

The 100-110-kD proteins (alpha-, beta-, beta'-, and gamma-adaptins) of clathrin-coated vesicles and the 110-kD protein (beta-COP) of the nonclathrin-coated vesicles that mediate constitutive transport through the Golgi have homologous protein sequences. To determine whether homologous processes are involved in assembly of the two types of coated vesicles, the membrane binding properties of their coat proteins were compared. After treatment of MDBK cells with the fungal metabolite Brefeldin A (BFA), beta-COP was redistributed to the cytoplasm within 15 s, gamma-adaptin and clathrin in the trans-Golgi network (TGN) dispersed within 30 s, but the alpha-adaptin and clathrin present on coated pits and vesicles derived from the plasma membrane remained membrane associated even after a 15-min exposure to BFA. In PtK1 cells and MDCK cells, BFA did not affect beta-COP binding or Golgi morphology but still induced redistribution of gamma-adaptin and clathrin from TGN membranes to the cytoplasm. Thus BFA affects the binding of coat proteins to membranes in the Golgi region (Golgi apparatus and TGN) but not plasma membranes. However, the Golgi binding interactions of beta-COP and gamma-adaptin are distinct and differentially sensitive to BFA. BFA treatment did not release gamma-adaptin or clathrin from purified clathrin-coated vesicles, suggesting that their distribution to the cytoplasm after BFA treatment of cells was due to interference with their rebinding to TGN membranes after a normal cycle of disassembly. This was confirmed using an in vitro assay in which gamma-adaptin binding to TGN membranes was blocked by BFA and enhanced by GTP gamma S, similar to the binding of beta-COP to Golgi membranes. These results suggest the involvement of GTP-dependent proteins in the association of the 100-kD coat proteins with membranes in the Golgi region of the cell.

Adaptor Protein Complex alpha Subunits↗

PubMed2002

Detecting and responding to hypoxia.

Adaptation to hypoxia is a topic of considerable clinical relevance, as it influences the pathophysiology of anaemia, polycythaemia, tissue ischaemia and cancer. A growing number of physiologically relevant genes are regulated in response to changes in intracellular oxygen tension. These include genes encoding erythropoietin, vascular endothelial growth factor and tyrosine hydroxylase. Studies on the regulation of the erythropoietin gene have provided insights into the common mechanism of oxygen sensing and signal transduction, leading to activation of the hypoxia-inducible transcription factor 1 (HIF-1). Activation of HIF-1 by hypoxia depends on rescue of its alpha-subunit from oxygen-dependent degradation in the proteasome, allowing it to form a heterodimer with HIF-1 beta. This then translocates to the nucleus. There, HIF-1 assembles with a highly conserved orphan nuclear receptor, HNF-4, and a critical transcriptional adaptor, p300. This complex binds to a 3' enhancer on the erythropoietin gene, enabling transcription of erythropoietin. HIF-1 also activates other genes, the cis-acting elements of which contain cognate hypoxia response elements. There is growing evidence that the oxygen sensor is a flavohaem protein and that the signal transduction pathway involves changes in the level of intracellular reactive oxygen intermediates. We have recently cloned a novel fusion protein called cytochrome b5/b5 reductase, which is a cyanide-insensitive NADPH oxidase and, therefore, a candidate to be the oxygen sensor. This flavohaem protein is widely expressed in cell lines and tissues, with localization in the perinuclear space. In the presence of oxygen and iron, it may induce oxidative modifications that target HIF-1 alpha for ubiquitination and degradation.

Adaptation, Physiological↗

PubMed2002

Phosphorylation of the AP2 mu subunit by AAK1 mediates high affinity binding to membrane protein sorting signals.

During receptor-mediated endocytosis, AP2 complexes act as a bridge between the cargo membrane proteins and the clathrin coat by binding to sorting signals via the mu 2 subunit and to clathrin via the beta subunit. Here we show that binding of AP2 to sorting signals in vitro is regulated by phosphorylation of the mu 2 subunit of AP2. Phosphorylation of mu 2 enhances the binding affinity of AP2 for sorting motifs as much as 25-fold compared with dephosphorylated AP2. The recognition of sorting signals was not affected by the phosphorylation status of the alpha or beta 2 subunit, suggesting that phosphorylation of mu 2 is critical for regulation of AP2 binding to sorting signals. Phosphorylation of mu 2 occurs at a single threonine residue (Thr-156) and is mediated by the newly discovered adaptor-associated kinase, AAK1, which copurifies with AP2. We propose that phosphorylation of the AP2 mu 2 subunit by AAK1 ensures high affinity binding of AP2 to sorting signals of cargo membrane proteins during the initial steps of receptor-mediated endocytosis.

Adaptor Protein Complex 2↗

PubMed1999

Nuclear import of Cdk/cyclin complexes: identification of distinct mechanisms for import of Cdk2/cyclin E and Cdc2/cyclin B1.

Reversible phosphorylation of nuclear proteins is required for both DNA replication and entry into mitosis. Consequently, most cyclin-dependent kinase (Cdk)/cyclin complexes are localized to the nucleus when active. Although our understanding of nuclear transport processes has been greatly enhanced by the recent identification of nuclear targeting sequences and soluble nuclear import factors with which they interact, the mechanisms used to target Cdk/cyclin complexes to the nucleus remain obscure; this is in part because these proteins lack obvious nuclear localization sequences. To elucidate the molecular mechanisms responsible for Cdk/cyclin transport, we examined nuclear import of fluorescent Cdk2/cyclin E and Cdc2/cyclin B1 complexes in digitonin-permeabilized mammalian cells and also examined potential physical interactions between these Cdks, cyclins, and soluble import factors. We found that the nuclear import machinery recognizes these Cdk/cyclin complexes through direct interactions with the cyclin component. Surprisingly, cyclins E and B1 are imported into nuclei via distinct mechanisms. Cyclin E behaves like a classical basic nuclear localization sequence-containing protein, binding to the alpha adaptor subunit of the importin-alpha/beta heterodimer. In contrast, cyclin B1 is imported via a direct interaction with a site in the NH2 terminus of importin-beta that is distinct from that used to bind importin-alpha.

Animals↗

PubMed1999

Automated protein model building combined with iterative structure refinement.

In protein crystallography, much time and effort are often required to trace an initial model from an interpretable electron density map and to refine it until it best agrees with the crystallographic data. Here, we present a method to build and refine a protein model automatically and without user intervention, starting from diffraction data extending to resolution higher than 2.3 A and reasonable estimates of crystallographic phases. The method is based on an iterative procedure that describes the electron density map as a set of unconnected atoms and then searches for protein-like patterns. Automatic pattern recognition (model building) combined with refinement, allows a structural model to be obtained reliably within a few CPU hours. We demonstrate the power of the method with examples of a few recently solved structures.

Adaptor Protein Complex alpha Subunits↗

PubMed1997

Functional domain mapping of the clathrin-associated adaptor medium chains mu1 and mu2.

The clathrin-associated adaptors AP-1 and AP-2 are heterotetrameric complexes involved in the recognition of sorting signals present within the cytosolic domain of integral membrane proteins. The medium chains of these complexes, mu1 and mu2, have been implicated in two types of interaction: assembly with the beta1 and beta2 chains of the corresponding complexes and recognition of tyrosine-based sorting signals. In this study, we report the results of a structure-function analysis of the mu1 and mu2 chains aimed at identifying regions of the molecules that are responsible for each of the two interactions. Analyses using the yeast two-hybrid system and proteolytic digestion experiments suggest that mu1 and mu2 have a bipartite structure, with the amino-terminal one-third (residues 1-145 of mu1 and mu2) being involved in assembly with the beta chains and the carboxyl-terminal two-thirds (residues 147-423 of mu1 and 164-435 of mu2) binding tyrosine-based sorting signals. These observations support a model in which the amino-terminal one-third of mu2 is embedded within the core of the AP-2 complex, while the carboxyl-terminal two-thirds of the protein are exposed to the medium, placing this region in a position to interact with tyrosine-based sorting signals.

Adaptor Protein Complex 1↗

PubMed1999

Adaptor complex-independent clathrin function in yeast.

Clathrin-associated adaptor protein (AP) complexes are major structural components of clathrin-coated vesicles, functioning in clathrin coat assembly and cargo selection. We have carried out a systematic biochemical and genetic characterization of AP complexes in Saccharomyces cerevisiae. Using coimmunoprecipitation, the subunit composition of two complexes, AP-1 and AP-2R, has been defined. These results allow assignment of the 13 potential AP subunits encoded in the yeast genome to three AP complexes. As assessed by in vitro binding assays and coimmunoprecipitation, only AP-1 interacts with clathrin. Individual or combined disruption of AP-1 subunit genes in cells expressing a temperature-sensitive clathrin heavy chain results in accentuated growth and alpha-factor pheromone maturation defects, providing further evidence that AP-1 is a clathrin adaptor complex. However, in cells expressing wild-type clathrin, the same AP subunit deletions have no effect on growth or alpha-factor maturation. Furthermore, gel filtration chromatography revealed normal elution patterns of clathrin-coated vesicles in cells lacking AP-1. Similarly, combined deletion of genes encoding the beta subunits of the three AP complexes did not produce defects in clathrin-dependent sorting in the endocytic and vacuolar pathways or alterations in gel filtration profiles of clathrin-coated vesicles. We conclude that AP complexes are dispensable for clathrin function in S. cerevisiae under normal conditions. Our results suggest that alternative factors assume key roles in stimulating clathrin coat assembly and cargo selection during clathrin-mediated vesicle formation in yeast.

Adaptor Proteins, Vesicular Transport↗

PubMed2004

Runx2 deficiency in chondrocytes causes adipogenic changes in vitro.

Runx2 (runt-related transcription factor 2) is an important transcription factor for chondrocyte differentiation as well as for osteoblast differentiation. To investigate the function of Runx2 in chondrocytes, we isolated chondrocytes from the rib cartilage of Runx2-deficient (Runx2-/-) mice and examined the effect of Runx2 deficiency on chondrocyte function and behavior in culture for up to 12 days. At the beginning of the culture, Runx2-/- chondrocytes actively proliferated, had a polygonal shape and expressed type II collagen; these are all characteristics of chondrocytes. However, they gradually accumulated lipid droplets that stained with oil red O and resembled adipocytes. Northern blot analysis revealed that the expression of adipocyte-related differentiation marker genes including PPAR gamma (peroxisome proliferator-activated receptor gamma), aP2 and Glut4 increased over time in culture, whereas expression of type II collagen decreased. Furthermore, the expression of Pref-1, an important inhibitory gene of adipogenesis, was remarkably decreased. Adenoviral introduction of Runx2 or treatment with transforming growth factor-beta, retinoic acid, interleukin-1 beta, basic fibroblast growth factor, platelet-derived growth factor or parathyroid hormone inhibited the adipogenic changes in Runx2-/- chondrocytes. Runx2 and transforming growth factor-beta synergistically upregulated interleukin-11 expression, and the addition of interleukin-11 to the culture medium reduced adipogenesis in Runx2-/- chondrocytes. These findings indicate that depletion of Runx2 resulted in the loss of the differentiated phenotype in chondrocytes and induced adipogenic differentiation in vitro, and show that Runx2 plays important roles in maintaining the chondrocyte phenotype and in inhibiting adipogenesis. Our findings suggest that these Runx2-dependent functions are mediated, at least in part, by interleukin-11.

Adaptor Protein Complex 2↗

PubMed2003

Structural basis for binding of accessory proteins by the appendage domain of GGAs.

The Golgi-associated, gamma-adaptin-related, ADP-ribosylation-factor binding proteins (GGAs) and adaptor protein (AP)-1 are adaptors involved in clathrin-mediated transport between the trans-Golgi network and endosomal system. The appendage domains of GGAs and the AP-1 gamma-adaptin subunit are structurally homologous and have been proposed to bind to accessory proteins via interaction with short sequences containing phenylalanines and acidic residues. Here we present the structure of the human GGA1 appendage in complex with its cognate binding peptide from the p56 accessory protein (DDDDFGGFEAAETFD) as determined by X-ray crystallography. The interaction is governed predominantly by packing of the first two phenylalanine residues of the peptide with conserved basic and hydrophobic residues from GGA1. Additionally, several main chain hydrogen bonds cause the peptide to form an additional beta-strand on the edge of the preexisting beta-sheet of the protein. Isothermal titration calorimetry was used to assess the affinities of different peptides for the GGA and gamma-appendage domains.

ADP-Ribosylation Factors↗