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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↗

PubMed1994

Role of p85 subunit of phosphatidylinositol-3-kinase as an adaptor molecule linking the insulin receptor, p62, and GTPase-activating protein.

After insulin stimulation of rat HTC hepatoma cells overexpressing normal human insulin receptors (IR), an antiserum to the p85 subunit of phosphatidylinositol-3-kinase (PIK) (alpha-p85) immunoprecipitated three major tyrosine-phosphorylated proteins: IR, insulin receptor substrate-1 (IRS-1), and a new 62-kDa protein (p62). Studies with antibodies to GTPase activating protein (alpha-GAP) and p62 GAP-associated protein suggested that p62 was the same as (or closely related to) p62 GAP-associated protein. In order to understand how p62 interacts with p85, we employed: 1) antibodies to the p110 subunit of PIK (alpha-p110); and 2) antiserum to IRS-1. To determine which subunit of PIK (p110 or p85) p62 associates with, we first immunoprecipitated insulin-treated cell lysates with alpha-p110 and subsequently immunoprecipitated with alpha-p85 followed by Western blotting analysis with anti-phosphotyrosine antibody (alpha-PY). In response to insulin, most of the tyrosine-phosphorylated p62 was complexed to p85 alone rather than with the PIK heterodimer. Moreover, p62 was absent in alpha-IRS-1 immunoprecipitates. These data suggest that: 1) p62 GAP-associated protein is tyrosine phosphorylated after insulin stimulation of cells; 2) p62 and IRS-1 form separate complexes with p85; 3) p62-GAP complex may be linked to p85 that is not bound to p110; 4) p85 may serve as an adaptor molecule in insulin receptor signaling, interacting with and regulating other intracellular proteins via SH2 domains.

Animals↗

PubMed2003

The B cell coreceptor CD22 associates with AP50, a clathrin-coated pit adapter protein, via tyrosine-dependent interaction.

The B cell coreceptor CD22 plays an important role in regulating signal transduction via the B cell Ag receptor. Studies have shown that surface expression of CD22 can be modulated in response to binding of ligand (i.e., mAb). Thus, it is possible that alterations in the level of CD22 expression following binding of natural ligand(s) may affect its ability to modulate the Ag receptor signaling threshold at specific points during B cell development and differentiation. Therefore, it is important to delineate the physiologic mechanism by which CD22 expression is controlled. In the current study, yeast two-hybrid analysis was used to demonstrate that CD22 interacts with AP50, the medium chain subunit of the AP-2 complex, via tyrosine-based internalization motifs in its cytoplasmic domain. This interaction was further characterized using yeast two-hybrid analysis revealing that Tyr(843) and surrounding amino acids in the cytoplasmic tail of CD22 comprise the primary binding site for AP50. Subsequent studies using transfectant Jurkat cell lines expressing wild-type or mutant forms of CD22 demonstrated that either Tyr(843) or Tyr(863) is sufficient for mAb-mediated internalization of CD22 and that these motifs are involved in its interaction with the AP-2 complex, as determined by coprecipitation of alpha-adaptin. Finally, experiments were performed demonstrating that treatment of B cells with either intact anti-Ig Ab or F(ab')(2) blocks ligand-mediated internalization of CD22. In conclusion, these studies demonstrate that internalization of CD22 is dependent on its association with the AP-2 complex via tyrosine-based internalization motifs.

Adaptor Protein Complex 2↗

PubMed2003

Human myometrial quiescence and activation during gestation and parturition involve dramatic changes in expression and activity of particulate type II (RII alpha) protein kinase A holoenzyme.

There are substantial data indicating that components of the cAMP-signaling pathway are differentially expressed in the human myometrium during pregnancy. The effects of cAMP in most tissues and cell types are mainly modulated via protein kinase A, a heterotetrameric protein complex consisting of two regulatory (R) and two catalytic (C) subunits. In the studies presented here, we used specific antibodies in Western blotting/immunoprecipitation, RT-PCR, and functional protein kinase A (PKA) phosphorylation assays to determine the PKA holoenzymes that are expressed in the human myometrium throughout pregnancy and labor. We report that as early as the second trimester of pregnancy, there is a significant increase in expression of the regulatory RII alpha protein subunit of PKA in the myometrium. This increase in protein expression is also mirrored at the mRNA level, indicating transcriptional control throughout pregnancy, whereas during parturition both transcript and protein are significantly decreased. This increase in RII alpha protein also resulted in increased particulate PKA activity in the myometrium during gestation, which was subsequently decreased during labor. Two specific A kinase anchoring proteins, AKAP95 and AKAP79, which have high binding affinities for RII alpha subunits, were found to form complexes with myometrial RII alpha species employing immunoprecipitation assays, but their levels of expression remained uniform in all myometrial tissue samples investigated. Our findings indicate that increased particulate type II PKA activity occurs throughout pregnancy, therefore directing the cAMP quiescence signal to specific subcellular loci within myometrial smooth muscle cells including the contractile machinery at the cytoskeleton; this effect is then removed during parturition.

A Kinase Anchor Proteins↗

PubMed2006

Nck in a complex containing the catalytic subunit of protein phosphatase 1 regulates eukaryotic initiation factor 2alpha signaling and cell survival to endoplasmic reticulum stress.

Stress imposed on the endoplasmic reticulum (ER) induces the phosphorylation of the alpha-subunit of the eukaryotic initiation factor 2 (eIF2) on Ser51. This results in transient inhibition of general translation initiation while concomitantly activating a signaling pathway that promotes the expression of genes whose products improve ER function. Conversely, dephosphorylation of eIF2alphaSer51 is accomplished by protein phosphatase 1 (PP1c) complexes containing either the protein CReP or GADD34, which target PP1c to eIF2. Here, we demonstrate that the Src homology (SH) domain-containing adaptor Nck is a key component of a molecular complex that controls eIF2alpha phosphorylation and signaling in response to ER stress. We show that overexpression of Nck decreases basal and ER stress-induced eIF2alpha phosphorylation and the attendant induction of ATF4 and CHOP. In contrast, we demonstrate that the mouse embryonic fibroblasts lacking both isoforms of Nck (Nck1-/-Nck2-/-) show higher levels of eIF2alpha phosphorylation and premature induction of ATF4, CHOP, and GADD34 in response to ER stress and finally, are more resistant to cell death induced by prolonged ER stress conditions. We establish that a significant amount of Nck protein localizes at the ER and is in a complex with eIF2 subunits. Further analysis of this complex revealed that it also contains the Ser/Thr phosphatase PP1c, its regulatory subunit CReP, and dephosphorylates eIF2alpha on Ser51 in vitro. Overall, we demonstrate that Nck as a component of the CReP/PP1c holophosphatase complex contributes to maintain eIF2alpha in a hypophosphorylated state. In this manner, Nck modulates translation and eIF2alpha signaling in response to ER stress.

Activating Transcription Factor 4↗

PubMed2003

Hyaluronan-mediated CD44 interaction with RhoGEF and Rho kinase promotes Grb2-associated binder-1 phosphorylation and phosphatidylinositol 3-kinase signaling leading to cytokine (macrophage-colony stimulating factor) production and breast tumor progression.

In this study we have examined CD44 (a hyaluronan (HA) receptor) interaction with a RhoA-specific guanine nucleotide exchange factor (p115RhoGEF) in human metastatic breast tumor cells (MDA-MB-231 cell line). Immunoprecipitation and immunoblot analyses indicate that both CD44 and p115RhoGEF are expressed in MDA-MB-231 cells and that these two proteins are physically associated as a complex in vivo. The binding of HA to MDA-MB-231 cells stimulates p115RhoGEF-mediated RhoA signaling and Rho kinase (ROK) activity, which, in turn, increases serine/threonine phosphorylation of the adaptor protein, Gab-1 (Grb2-associated binder-1). Phosphorylated Gab-1 promotes PI 3-kinase recruitment to CD44v3. Subsequently, PI 3-kinase is activated (in particular, alpha, beta, gamma forms but not the delta form of the p110 catalytic subunit), AKT signaling occurs, the cytokine (macrophage-colony stimulating factor (M-CSF)) is produced, and tumor cell-specific phenotypes (e.g. tumor cell growth, survival and invasion) are up-regulated. Our results also demonstrate that HA/CD44-mediated oncogenic events (e.g. AKT activation, M-CSF production and breast tumor cell-specific phenotypes) can be effectively blocked by a PI 3-kinase inhibitor (LY294002). Finally, we have found that overexpression of a dominant-negative form of ROK (by transfection of MBA-MD-231 cells with the Rho-binding domain cDNA of ROK) not only inhibits HA/CD44-mediated RhoA-ROK activation and Gab-1 phosphorylation but also down-regulates oncogenic signaling events (e.g. Gab-1.PI 3-kinase-CD44v3 association, PI 3-kinase-mediated AKT activation, and M-CSF production) and tumor cell behaviors (e.g. cell growth, survival, and invasion). Taken together, these findings strongly suggest that CD44 interaction with p115RhoGEF and ROK plays a pivotal role in promoting Gab-1 phosphorylation leading to Gab-1.PI 3-kinase membrane localization, AKT signaling, and cytokine (M-CSF) production during HA-mediated breast cancer progression.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Interaction of SAP97 with minus-end-directed actin motor myosin VI. Implications for AMPA receptor trafficking.

SAP97 is a modular protein composed of three PDZ domains, an SH3 domain, and a guanylate kinase-like domain. It has been implicated functionally in the assembly and structural stability of synaptic junctions as well as in the trafficking, recruitment, and localization of specific ion channels and neurotransmitter receptors. The N terminus of SAP97 (S97N) has been shown to play a key role in the selection of binding partners and the localization of SAP97 at adhesion sites, as well as the clustering of ion channels in heterologous cells. Using the S97N domain as bait in a yeast two-hybrid screen, we identified the minus-end-directed actin-based motor, myosin VI, as an S97N binding partner. Moreover, in light membrane fractions prepared from rat brain, we found that myosin VI and SAP97 form a trimeric complex with the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subunit, GluR1. These data suggest that SAP97 may serve as a molecular link between GluR1 and the actin-dependent motor protein myosin VI during the dynamic translocation of AMPA receptors to and from the postsynaptic plasma membrane.

Adaptor Proteins, Signal Transducing↗

PubMed1995

Involvement of stress-activated protein kinase in the cellular response to 1-beta-D-arabinofuranosylcytosine and other DNA-damaging agents.

The cellular response to 1-beta-D-arabinofuranosylcytosine (ara-C) includes activation of Jun/AP-1, induction of c-jun transcription, and programmed cell death. The stress-activated protein (SAP) kinases stimulate the transactivation function of c-jun by amino terminal phosphorylation. The present work demonstrates that ara-C activates p54 SAP kinase. The finding that SAP kinase is also activated by alkylating agents (mitomycin C and cisplatinum) and the topoisomerase I inhibitor 9-amino-camptothecin supports DNA damage as an initial signal in this cascade. The results demonstrate that ara-C also induces binding of SAP kinase to the SH2/SH3-containing adapter protein Grb2. SAP kinase binds to the SH3 domains of Grb2, while interaction of the p85 alpha-subunit of phosphatidylinositol 3-kinase complex. The results also demonstrate that ara-C treatment is associated with inhibition of lipid and serine kinase activities of PI 3-kinase. The potential significance of the ara-C-induced interaction between SAP kinase and PI 3-kinase is further supported by the demonstration that Wortmannin, an inhibitor of PI 3-kinase, stimulates SAP kinase activity. The finding that Wortmannin treatment is also associated with internucleosomal DNA fragmentation may support a potential link between PI 3-kinase and regulation of both SAP kinase and programmed cell death.

Adaptor Proteins, Signal Transducing↗

PubMed1998

SLP-65: a new signaling component in B lymphocytes which requires expression of the antigen receptor for phosphorylation.

The B cell antigen receptor (BCR) consists of the membrane-bound immunoglobulin (Ig) molecule as antigen-binding subunit and the Ig-alpha/Ig-beta heterodimer as signaling subunit. BCR signal transduction involves activation of protein tyrosine kinases (PTKs) and phosphorylation of several proteins, only some of which have been identified. The phosphorylation of these proteins can be induced by exposure of B cells either to antigen or to the tyrosine phosphatase inhibitor pervanadate/H2O2. One of the earliest substrates in B cells is a 65-kD protein, which we identify here as a B cell adaptor protein. This protein, named SLP-65, is part of a signaling complex involving Grb-2 and Vav and shows homology to SLP-76, a signaling element of the T cell receptor. In pervanadate/H2O2-stimulated cells, SLP-65 becomes phosphorylated only upon expression of the BCR. These data suggest that SLP-65 is part of a BCR transducer complex.

Adaptor Proteins, Signal Transducing↗

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↗

PubMed2002

Transforming growth factor-beta receptors interact with AP2 by direct binding to beta2 subunit.

Transforming growth factor-beta (TGF-beta) superfamily members regulate a wide range of biological processes by binding to two transmembrane serine/threonine kinase receptors, type I and type II. We have previously shown that the internalization of these receptors is inhibited by K(+) depletion, cytosol acidification, or hypertonic medium, suggesting the involvement of clathrin-coated pits. However, the involvement of the clathrin-associated adaptor complex AP2 and the identity of the AP2 subunit that binds the receptors were not known. Herein, we have studied these issues by combining studies on intact cells with in vitro assays. Using fluorescence photobleaching recovery to measure the lateral mobility of the receptors on live cells (untreated or treated to alter their coated pit structure), we demonstrated that their mobility is restricted by interactions with coated pits. These interactions were transient and mediated through the receptors' cytoplasmic tails. To measure direct binding of the receptors to specific AP2 subunits, we used yeast two-hybrid screens and in vitro biochemical assays. In contrast to most other plasma membrane receptors that bind to AP2 via the mu2 subunit, AP2/TGF-beta receptor binding was mediated by a direct interaction between the beta2-adaptin N-terminal trunk domain and the cytoplasmic tails of the receptors; no binding was observed to the mu2, alpha, or sigma2 subunits of AP2 or to mu1 of AP1. The data uniquely demonstrate both in vivo and in vitro the ability of beta2-adaptin to directly couple TGF-beta receptors to AP2 and to clathrin-coated pits, providing the first in vivo evidence for interactions of a transmembrane receptor with beta2-adaptin.

Adaptor Protein Complex 2↗

PubMed1999

Anchorage-dependent regulation of the mitogen-activated protein kinase cascade by growth factors is supported by a variety of integrin alpha chains.

Integrin cooperation with growth factor receptors to enable permissive signaling to the mitogen-activated protein (MAP) kinase pathway has important implications for cell proliferation, differentiation, and survival. Here we have sought to determine whether anchorage regulation of the MAP kinase pathway is specific to the alpha chain subunit of the integrins employed during adhesion. Human umbilical vein endothelial cells (HUVECs) anchored via endogenous alpha(2), alpha(3), or alpha(5) integrin subunits or NIH3T3 fibroblast cells lines anchored via ectopically expressed human integrin alpha(2) or alpha(5) subunits displayed comparable MAP kinase activation upon growth factor stimulation, regardless of the integrin alpha chain employed. In contrast, when either cell type was maintained in suspension, growth factor treatment inefficiently activated the MAP kinase pathway. The integrin-mediated enhancement of MAP kinase activation by growth factor correlated with the tyrosine phosphorylation of focal adhesion kinase but was independent of Shc. These data indicate that integrin modulation of the MAP kinase pathway is supported by a variety of integrin complexes and imply that other pathways may be required for the previously reported alpha chain-specific effects on cell cycle regulation and cell differentiation.

3T3 Cells↗

PubMed2006

Central role for MyD88 in the responses of microglia to pathogen-associated molecular patterns.

Microglia, the innate immune effector cells of the CNS parenchyma, express TLR that recognize conserved motifs of microorganisms referred to as pathogen-associated molecular patterns (PAMP). All TLRs identified to date, with the exception of TLR3, use a common adaptor protein, MyD88, to transduce activation signals. Recently, we reported that microglial activation in response to the Gram-positive bacterium Staphylococcus aureus was not completely attenuated following TLR2 ablation, suggesting the involvement of additional receptors. To assess the functional role of alternative TLRs in microglial responses to S. aureus and its cell wall product peptidoglycan as well as the Gram-negative PAMP LPS, we evaluated primary microglia from MyD88 knockout (KO) and wild-type mice. The induction of TNF-alpha, IL-12 p40, and MIP-2 (CXCL2) expression by S. aureus- and peptidoglycan-stimulated microglia was MyD88 dependent, as revealed by the complete inhibition of cytokine production in MyD88 KO cells. In addition, the expression of additional pattern recognition receptors, including TLR9, pentraxin-3, and lectin-like oxidized LDL receptor-1, was regulated, in part, via a MyD88-dependent manner as demonstrated by the attenuated expression of these receptors in MyD88 KO microglia. Microglial activation was only partially inhibited in LPS-stimulated MyD88 KO cells, suggesting the involvement of MyD88-independent pathways. Collectively, these findings reveal the complex mechanisms for microglia to respond to diverse bacterial pathogens, which occur via both MyD88-dependent and -independent pathways.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Characterization of the human ortholog of Mov34 reveals eight N-terminal residues important for MPN domain stability.

Eukaryotic MPN domain proteins are components of the complexes proteasome lid, COP9-signalosome (CSN), and translation initiation factor 3 (eIF3). The proteasome lid Rpn11 and COP9-signalosome Csn5 subunits, which contain the conserved JAMM motif involved in zinc ion coordination, show catalytic isopeptidase activity. Homology modeling indicates that the MPN domain of Mov34 cannot coordinate a zinc ion in the same manner as catalytically active MPN domains. In this work, we show that the MPN domain of Mov34 is highly resistant to proteolysis and the major product comprises residues 9-186, which includes the conserved MPN domain. Two clones containing the MPN domain region (MPN1-177 and MPN1-186) including the eight N-terminal residues show a less pronounced band in the 220 nm region of the CD, indicating lower alpha-helical content relative to the clones lacking these residues (MPN9-177 and MPN9-186). However, clones lacking residues 1-8 show lower expression levels and thermal stability, indicating that residues 1-8 are required for proper folding and stability of this particular MPN domain.

Adaptor Proteins, Signal Transducing↗

PubMed2004

A novel transcription regulatory complex containing death domain-associated protein and the ATR-X syndrome protein.

Death domain-associated protein (Daxx) is a multi-functional protein that modulates both apoptosis and transcription. Within the nucleus, Daxx is a component of the promyelocytic leukemia protein (PML) nuclear bodies (NBs) and interacts with a number of transcription factors, yet its precise role in transcription remains elusive. To further define the function of Daxx, we have isolated its interacting proteins in the nucleus using epitope-tagged affinity purification and identified X-linked mental retardation and alpha-thalassaemia syndrome protein (ATRX), a putative member of the SNF2 family of ATP-dependent chromatin remodeling proteins that is mutated in several X-linked mental retardation disorders. We show that substantial amounts of endogenous Daxx and ATRX exist in a nuclear complex. Daxx binds to ATRX through its paired amphipathic alpha helices domains. ATRX has ATPase activity that is stimulated by mononucleosomes, and patient mutations in the ATPase domain attenuate this activity. ATRX strongly represses transcription when tethered to a promoter. Daxx does not affect the ATPase activity of ATRX, however, it alleviates its transcription repression activity. In addition, ATRX is found in the PML-NBs, and this localization is mediated by Daxx. These results show that the ATRX.Daxx complex is a novel ATP-dependent chromatin-remodeling complex, with ATRX being the core ATPase subunit and Daxx being the targeting subunit. Moreover, the localization of ATRX to the PML-NBs supports the notion that these structures may play an important role in transcription regulation.

Adaptor Proteins, Signal Transducing↗

PubMed2000

The association of CRKII with C3G can be regulated by integrins and defines a novel means to regulate the mitogen-activated protein kinases.

In studies to define mechanisms of ERK activation in Chinese hamster ovary cells, we have observed an inverse correlation between CRKII-C3G complex formation and ERK activity. That is, we were able to coprecipitate the guanine nucleotide exchange factor C3G with the adaptor protein CRKII in lysates from suspended cells that had low ERK activity, but we could not do so or could do so less efficiently in lysates of adherent cells with increased ERK activity. Consistent with the presence of a functional CRKII-C3G complex, we detected more GTP-loaded RAP1 in suspension than adherent lysates. Overexpression of cDNAs encoding B-RAF, CRKII W109L, and PTP1B C215S activated ERK in suspension cells, the latter two constructs also disrupting CRKII-C3G complex formation. Finally, we have also observed that certain integrin alpha subunit cytoplasmic splice variants differentially regulate ERK1/2 but also in a manner that correlated with levels of a CRKII-C3G complex. Thus, these data suggest the involvement of integrins in an ERK suppression pathway mediated by CRKII-C3G complex formation and downstream signaling from activated RAP1.

Alternative Splicing↗

PubMed2006

Phosphorylation of adaptor protein-2 mu2 is essential for Na+,K+-ATPase endocytosis in response to either G protein-coupled receptor or reactive oxygen species.

Activation of G protein-coupled receptor by dopamine and hypoxia-generated reactive oxygen species promote Na+,K+-ATPase endocytosis. This effect is clathrin dependent and involves the activation of protein kinase C (PKC)-zeta and phosphorylation of the Na+,K+-ATPase alpha-subunit. Because the incorporation of cargo into clathrin vesicles requires association with adaptor proteins, we studied whether phosphorylation of adaptor protein (AP)-2 plays a role in its binding to the Na+,K+-ATPase alpha-subunit and thereby in its endocytosis. Dopamine induces a time-dependent phosphorylation of the AP-2 mu2 subunit. Using specific inhibitors and dominant-negative mutants, we establish that this effect was mediated by activation of the adaptor associated kinase 1/PKC-zeta isoform. Expression of the AP-2 mu2 bearing a mutation in its phosphorylation site (T156A) prevented Na+,K+-ATPase endocytosis and changes in activity induced by dopamine. Similarly, in lung alveolar epithelial cells, hypoxia-induced endocytosis of Na+,K+-ATPase requires the binding of AP-2 to the tyrosine-based motif (Tyr-537) located in the Na+,K+-ATPase alpha-subunit, and this effect requires phosphorylation of the AP-2 mu2 subunit. We conclude that phosphorylation of AP-2 mu2 subunit is essential for Na+,K+-ATPase endocytosis in response to a variety of signals, such as dopamine or reactive oxygen species.

Adaptor Protein Complex 2↗

PubMed2004

Gamma-COP appendage domain - structure and function.

COPI-coated vesicles mediate retrograde transport from the Golgi back to the ER and intra-Golgi transport. The cytosolic precursor of the COPI coat, the heptameric coatomer complex, can be thought of as composed of two subcomplexes. The first consists of the beta-, gamma-, delta- and zeta-COP subunits which are distantly homologous to AP clathrin adaptor subunits. The second consists of the alpha-, beta'- and epsilon-COP subunits. Here, we present the structure of the appendage domain of gamma-COP and show that it has a similar overall fold as the alpha-appendage of AP2. Again, like the alpha-appendage the gamma-COP appendage possesses a single protein/protein interaction site on its platform subdomain. We show that in yeast this site binds to the ARFGAP Glo3p, and in mammalian gamma-COP this site binds to a Glo3p orthologue, ARFGAP2. On the basis of mutations in the yeast homologue of gamma-COP, Sec21p, a second binding site is proposed to exist on the gamma-COP appendage that interacts with the alpha,beta',epsilon COPI subcomplex.

ADP-Ribosylation Factors↗