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PubMed1997

Transactivation through Ets and Ap1 transcription sites determines the expression of the tumor-suppressing gene maspin.

Tumor invasion and metastasis are processes poorly understood at the molecular level. Maspin is a serine protease inhibitor (serpin) with tumor-suppressing function in the mammary gland. Maspin gene expression is decreased with malignancy and is lost in metastatic cells. We show in this report that differential expression of maspin in normal and carcinoma-derived mammary epithelial cells is regulated at the transcriptional level. We have identified the Ets and Ap1 sites in the maspin promoter that are active in regulating maspin expression in normal mammary epithelial cells but inactive in tumor cells. The Ets site alone is sufficient to activate transcription in a heterologous promoter, whereas the Ap1 site cooperates with Ets in activation. The enhancing function by Ets and Ap1 elements is decreased in primary tumor cells (21NT) and is abolished in invasive tumor cells (MDA-231). Thus, loss of maspin expression during tumor progression results at least in part from the absence of transactivation through the Ets and Ap1 sites.

Adaptor Protein Complex 1↗
PubMed1997

Transcription factor activation during signal-induced apoptosis of immature CD4(+)CD8(+) thymocytes. A protective role of c-Fos.

Many signals that cause apoptotic cell death operate by inducing transcription and translation of other (presumably death effector) mediators, and it is well established that stimulus-induced apoptosis can often be blocked by inhibiting transcription and translation. Transcriptional regulation of apoptosis, however, is incompletely understood. To gain insight into nuclear events associated with signal-induced apoptosis during T cell development, we studied signal-induced apoptosis of ex vivo isolated immature CD8(+)4(+) double-positive (DP) thymocytes. Stimuli utilizing the T cell receptor (TCR) signaling pathway or its parts (an alphaCD3/TCR monoclonal antibody, a Ca2+ ionophore, or a protein kinase C-activating phorbol ester) or a stimulus that antagonizes TCR signaling and apoptosis in T cell hybridoma (forskolin, a cyclic AMP-signaling activator) resulted in massive apoptosis of DP thymocytes. At the same time, these stimuli induced qualitatively similar but quantitatively unique patterns of inducible transcription factors (TFs) NF-kappaB/RelA-p50, AP-1 (Fos-Jun), and NUR-77. We focused our attention on the role of AP-1 (Fos-Jun) complex, which was strongly induced by all of the above stimuli and thus was a candidate for a proapoptotic TF. However, we found that AP-1/c-Fos induction was vital in prolonging DP thymocyte life, as judged by increased spontaneous and induced death of DP cells in Fos-/- mice. In direct support of this hypothesis, experiments with antisense oligonucleotides demonstrated that c-Fos plays an essential role in protecting normal DP thymocytes from Ca2+- and cAMP-induced apoptosis but not from TCR-mediated death. Together, these results demonstrate a physiological role for c-Fos in maintaining longevity of DP thymocytes.

Adaptor Protein Complex alpha Subunits↗
PubMed1998

Regulation of adipocyte gene expression by polyunsaturated fatty acids.

A wide number of adipocyte genes are regulated by exogenous polyunsaturated fatty acids (PUFA) through the actions of the peroxisome proliferator activated receptor. Such genes include the adipocyte lipid-binding protein (ALBP or aP2) which plays a central role in facilitating the trafficking of fatty acids within adipocytes. Work from a number of laboratories has suggested the key elements of the lipid signal transduction pathway include: (1) the transport of exogenous PUFAs across the plasma membrane, (2) metabolism of polyunsaturated fatty acids to second messengers including 15-deoxy delta 12,14 prostaglandin J2 (15dPGJ2), (3) trafficking of 15dPGJ2 and other second messengers from the smooth ER to the nucleus for association with peroxisome proliferator activated receptor gamma (PPAR gamma), and (4) dimerization of PPAR gamma with retinoid X receptor (RXR) permitting regulation of transcription via association with any of several nuclear co-activators or repressors. In addition to the aP2 gene being a target of activation by fatty acids, at the protein level ALBP/aP2 plays a role in trafficking of fatty acids and/or their metabolises. We report here that in a heterologous system using CV-1 cells transiently transfected with PPAR gamma 2, co-expression of ALBP/aP2 enhances the PPAR-dependent activation of gene transcription. These results suggest that ALBP/aP2 functions as a positive factor in fatty acid signalling by directly targetting and delivering fatty acids metabolites to the lipid signal transduction pathway.

Adaptor Protein Complex 2↗
PubMed2003

Identification of a family of endocytic proteins that define a new alpha-adaptin ear-binding motif.

Endocytosis by clathrin-coated vesicles (CCVs) is an important mechanism mediating protein internalization. Here, we show that two proteins identified through a proteomics analysis of CCVs are new components of the endocytic machinery. The proteins, named NECAP (adaptin-ear-binding coat-associated protein) 1 and 2, are paralogues that display no sequence similarity or common domains with any known protein. Both are enriched in CCV coats, and further analysis of the brain-enriched isoform, NECAP 1, shows its partial localization to clathrin-coated pits and direct binding to the globular ear domain of the alpha-adaptin subunit (alpha-ear) of the adaptor protein 2 (AP-2) complex. Intriguingly, this interaction is mediated by a new motif, WVQF, that uses a distinct alpha-ear interface relative to known alpha-ear-binding partners. Disruption of this interaction blocks clathrin-mediated endocytosis. Together, our studies identify a new family of endocytic proteins that define a unique AP-2-binding motif.

Adaptor Protein Complex alpha Subunits↗
PubMed2002

Regulating the clathrin-coated vesicle cycle by AP2 subunit phosphorylation.

Clathrin-coated pits form from the concerted assembly of many different protein components that interact with each other and with the lipid bilayer. When the coated pit pinches off to form a coated vesicle, the coat is removed to allow fusion of the endocytic vesicle with an early endosome, and the disassembled coat components can then recycle through the cytosol to participate in new rounds of coated pit formation. One potential mechanism to regulate the cycles of coat protein assembly and disassembly is reversible phosphorylation, particularly given that many of the coat components, such as the AP2 adaptor complex, are phosphorylated on multiple sites both in vitro and in vivo. Recent studies have provided particular insight into the role of phosphorylation of the cargo-binding micro2 subunit of AP2.

Adaptor Protein Complex 1↗
PubMed2002

Rab11-FIP2, an adaptor protein connecting cellular components involved in internalization and recycling of epidermal growth factor receptors.

Rab11-FIP2 is a member of a newly identified family of Rab11-binding proteins that have been implicated in the function of recycling endosomes. Here we show that Rab11-FIP2 may also be involved with the process of receptor-mediated endocytosis. First we demonstrate that Rab11-FIP2 contains an NPF motif that allows it to bind Reps1, a member of a family of EH domain proteins involved in endocytosis. We also show that Rab11-FIP2 associates with the alpha-adaptin subunit of AP-2 complexes, which are known to recruit receptors into clathrin-coated vesicles. Finally, we find that overexpression of Rab11-FIP2 suppresses the internalization of epidermal growth factor receptors, but not transferrin receptors, through binding sites that promote complex formation with Rab11, Reps1, and alpha-adaptin. These findings suggest that Rab11-FIP2 may participate in the coupling of receptor-mediated endocytosis to the subsequent sorting of receptor-containing vesicles in endosomes.

3T3 Cells↗
PubMed2001

The role of endocytic l1 trafficking in polarized adhesion and migration of nerve growth cones.

Motility of the nerve growth cone is highly dependent on its dynamic interactions with the microenvironment mediated by cell adhesion molecules (CAMs). These adhesive interactions can be spatially regulated by changing the density and avidity of CAMs on the growth cone. Previous studies have shown that L1, a member of the immunoglobulin superfamily of CAMs, is endocytosed at the central domain of the growth cone followed by centrifugal vesicular transport and reinsertion into the plasma membrane of the leading edge. The present paper focuses on the functional significance of endocytic L1 trafficking in dorsal root ganglia neurons in vitro. We demonstrate that the rate of L1-based neurite growth has a positive correlation with the amount of endocytosed L1 in the growth cone, whereas stimulation of neurite growth via an N-cadherin-dependent mechanism does not increase L1 endocytosis. A growth cone that migrates on an L1 substrate exhibits a steep gradient of L1-mediated adhesion (strong adhesion at the growth cone's leading edge and weak adhesion at the central domain). This gradient of L1 adhesion is attenuated after inhibition of L1 endocytosis in the growth cone by intracellular loading of a function-blocking antibody against alpha-adaptin, a subunit of the clathrin-associated AP-2 adaptor. Inhibition of L1 endocytosis by this antibody also decreased the rate of L1-dependent growth cone migration. These results indicate that the growth cone actively translocates CAMs to create spatial asymmetry in adhesive interactions with its environment and that this spatial asymmetry is important for growth cone migration.

Adaptor Protein Complex alpha Subunits↗
PubMed2003

Effect of clathrin heavy chain- and alpha-adaptin-specific small inhibitory RNAs on endocytic accessory proteins and receptor trafficking in HeLa cells.

To assess the contribution of individual endocytic proteins to the assembly of clathrin coated pits, we depleted the clathrin heavy chain and the alpha-adaptin subunit of AP-2 in HeLa-cells using RNA interference. 48 h after transfection with clathrin heavy chain-specific short interfering RNA both, the heavy and light chains were depleted by more than 80%. Residual clathrin was mainly membrane-associated, and an increase in shallow pits was noted. The membrane-association of adaptors, clathrin assembly lymphoid myeloid leukemia protein (CALM), epsin, dynamin, and Eps15 was only moderately affected by the knockdown and all proteins still displayed a punctate staining distribution. Clathrin depletion inhibited the uptake of transferrin but not that of the epidermal growth factor. However, efficient sorting of the epidermal growth factor into hepatocyte growth factor-regulated tyrosine kinase substrate-positive endosomes was impaired. Depletion of alpha-adaptin abolished almost completely the plasma membrane association of clathrin. Binding of Eps15 to membranes was strongly and that of CALM moderately reduced. Whereas the uptake of transferrin was efficiently blocked in alpha-adaptin knockdown cells, the internalization and sorting of the epidermal growth factor was not significantly impaired. Since neither clathrin nor AP-2 is essential for the internalization of EGF, we conclude that it is taken up by an alternative mechanism.

Adaptor Protein Complex alpha Subunits↗
PubMed2003

Tensile stress induces alpha-adaptin C production in mouse calvariae in an organ culture: possible involvement of endocytosis in mechanical stress-stimulated osteoblast differentiation.

We previously demonstrated that tensile stress (TS)-induced osteoblast differentiation eventually led to osteogenesis in an organ culture of mouse calvarial sutures. In the present study, we employed RNA-fingerprinting using an arbitrarily primed polymerase chain reaction (RAP-PCR) to identify alpha-adaptin C, a component of the endocytosis machinery AP2, as a TS-inducible gene. Protein production, as well as the gene expression of alpha-adaptin C, was induced by TS as early as 3 h following the initiation of loading. In situ hybridization and immunohistochemical analysis revealed that the induction of alpha-adaptin C mostly occurred in fibroblastic cells in the sutures, suggesting that it precedes TS-induced osteoblast differentiation. Consistent with this result, TS significantly increased the number of coated pits (CPs) and coated vesicles (CVs) in the undifferentiated fibroblastic cells but not in the osteoblastic cells around calvarial bones. Further, TS-induced osteoblast differentiation was suppressed when endocytosis was inhibited by potassium depletion. These results, taken together, suggest that TS accelerates osteoblast differentiation and osteogenesis, possibly through the induction of the alpha-adaptin C expression and consequent activation of receptor-mediated endocytosis.

Adaptor Protein Complex 2↗
PubMed2006

Clathrin and adaptin accumulation in drusen, Bruch's membrane and choroid in AMD and non-AMD donor eyes.

Clathrin was identified in a recent proteomic analysis of Bruch's membrane from age-related macular degeneration (AMD) donor eyes. The present study was conducted to determine the localization of clathrin in AMD tissues and to compare this distribution and relative content with that in non-AMD control tissues. The distribution of adaptin, which is functionally linked to clathrin, was also evaluated. Human eyes were from donors between 66 and 94 years of age; 13 eyes were from donors with AMD and 13 from non-AMD donors. Bruch's membrane and choroid from the macula of each donor eye were prepared for immunohistochemistry and Western blotting. Differences in immunoreactivity were quantitated. Drusen, Bruch's membrane and choroid from AMD tissues showed greater immunoreactivity for clathrin and adaptin than did non-AMD tissues. Western blots also showed more intense clathrin and adaptin immunoreactivity in AMD tissues than were present in non-AMD samples. This study suggests that accumulation of clathrin and adaptin in drusen, Bruch's membrane and choroid may reflect a higher rate of clathrin mediated endocytosis in AMD tissues. Alternatively, the accumulation of these proteins in these extracellular compartments may reflect a higher susceptibility to oxidative damage.

Adaptor Protein Complex alpha Subunits↗
PubMed2003

Age-related regulation of dendritic endocytosis associated with altered clathrin dynamics.

The protein composition of the neuronal plasma membrane is regulated by clathrin-mediated endocytosis and changes drastically over the neuronal lifespan. Here, we utilize the transition out of the period of early postnatal growth as a model system to study age-related changes in endocytosis. Previously, we have found that the dynamic behavior of endocytic clathrin coats in dendrites changes during this period, and that clathrin coat lifetime increases in older neurons. In this study, we examine endocytosis in neuronal dendrites by measuring transferrin (Tf) uptake, and find that it is markedly reduced in older neurons in culture. This decrease was not due to a reduction in transferrin receptor protein levels, nor to a decrease in the expression of endocytic proteins. However, imaging of endocytosis in living dendrites demonstrated that cargo transport through clathrin-coated pits was slower during internalization. Thus, endocytic function in dendrites is altered in older neurons, suggesting that as neurons age, protein trafficking mechanisms are controlled to complement maturational requirements.

Adaptor Protein Complex alpha Subunits↗
PubMed2006

Discrete residues in the c(2)b domain of synaptotagmin I independently specify endocytic rate and synaptic vesicle size.

It has been demonstrated that synapses lacking functional synaptotagmin I (Syt I) have a decreased rate of synaptic vesicle endocytosis. Beyond this, the function of Syt I during endocytosis remains undefined. Here, we demonstrate that a decreased rate of endocytosis in syt(null) mutants correlates with a stimulus-dependent perturbation of membrane internalization, assayed ultrastructurally. We then separate the mechanisms that control endocytic rate and vesicle size by mapping these processes to discrete residues in the Syt I C(2)B domain. Mutation of a poly-lysine motif alters vesicle size but not endocytic rate, whereas the mutation of calcium-coordinating aspartate residues (syt-D3,4N) alters endocytic rate but not vesicle size. Finally, slowed endocytic rate in the syt-D3,4N animals, but not syt(null) animals, can be rescued by elevating extracellular calcium concentration, supporting the conclusion that calcium coordination within the C(2)B domain contributes to the control of endocytic rate.

Adaptor Protein Complex alpha Subunits↗
PubMed2006

Drosophila homologue of Eps15 is essential for synaptic vesicle recycling.

The mammalian protein Eps15 is phosphorylated by EGF receptor tyrosine kinase and has been shown to interact with several components of the endocytic machinery. We have identified a hypomorphic Eps15 mutant in Drosophila which shows reversible paralysis and an altered physiology at restrictive temperatures. In addition, the temperature-sensitive paralytic defect of shibire mutant is enhanced by this mutant. Eps15 is enriched in the larval neuromuscular junction in endocytic 'hot spots' in a pattern similar to Dynamin. Eps15 mutants show a decrease in the alpha-Adaptin levels at the larval neuromuscular junction synapse. Genetic and biochemical studies of interactions with components of the endocytic machinery suggest that Eps15 has an important role in synaptic vesicle recycling and regulates recruitment of alpha-Adaptin.

Adaptor Protein Complex alpha Subunits↗
PubMed2002

Cloning, physical mapping and structural characterization of the human alpha(A)-adaptin gene.

Adaptins are major structural components of heterotetrameric protein complexes called adaptors, which are involved in intracellular receptor transport via clathrin-coated vesicles. In mice, one of these adaptins has been shown to be encoded by two genes, alpha(A)-adaptin and alpha(C)-adaptin, the former of which is expressed as two alternatively spliced transcripts. Using positional cloning gene approaches, we were able to identify the human alpha(A)-adaptin gene, which consists of 24 exons spanning over 40 kb on chromosome 19q13.3 between the loci of the R-ras gene and the polynucleotide kinase phosphatase gene. The novel gene encodes a 977 amino acid, 107.6 kDa protein with 98% amino acid sequence identity to its murine ortholog. Human alpha(A)-adaptin is expressed as a full-length transcript in forebrain, skeletal muscle, spinal cord, cerebellum, salivary gland, heart and colon. It is also ubiquitously expressed in tissues and in ZR-75-1 breast cancer cells and LNCaP prostate carcinoma cells as a smaller variant generated by splicing out of an exon encoding 22 amino acids in the hinge region of the protein.

Adaptor Protein Complex 2↗
PubMed2002

The endocytic protein alpha-Adaptin is required for numb-mediated asymmetric cell division in Drosophila.

During asymmetric cell division in Drosophila sensory organ precursor cells, the Numb protein localizes asymmetrically and segregates into one daughter cell, where it influences cell fate by repressing signal transduction via the Notch receptor. We show here that Numb acts by polarizing the distribution of alpha-Adaptin, a protein involved in receptor-mediated endocytosis. alpha-Adaptin binds to Numb and localizes asymmetrically in a Numb-dependent fashion. Mutant forms of alpha-Adaptin that no longer bind to Numb fail to localize asymmetrically and cause numb-like defects in asymmetric cell division. Our results suggest a model in which Numb influences cell fate by downregulating Notch through polarized receptor-mediated endocytosis, since Numb also binds to the intracellular domain of Notch.

Adaptor Protein Complex alpha Subunits↗
PubMed2002

Numb: "Adapting" notch for endocytosis.

During sensory organ precursor divisions in Drosophila, the numb gene product segregates asymmetrically into one of the two daughter cells, to which it confers a specific fate by inhibiting Notch signaling. In this issue of Developmental Cell, Berdnik et al. show that Numb recruits alpha-Adaptin and that this physical interaction plays a role in downregulating Notch, presumably by stimulating endocytosis of Notch.

Animals↗
PubMed2001

AP-3 adaptor functions in targeting P-selectin to secretory granules in endothelial cells.

P-selectin, a cell adhesion protein participating in the early stages of inflammation, contains multiple sorting signals that regulate its cell surface expression. Targeting to secretory granules regulates delivery of P-selectin to the cell surface. Internalization followed by sorting from early to late endosomes mediates rapid removal of P-selectin from the surface. We show here that the P-selectin cytoplasmic domain bound AP-2 and AP-3 adaptor complexes in vitro. The amino acid substitution L768A, which abolishes endosomal sorting and impairs granule targeting of P-selectin, reduced binding of AP-3 adaptors but not AP-2 adaptors. Turnover of P-selectin was 2.4-fold faster than turnover of transferrin receptor in AP-3-deficient mocha fibroblasts, similar to turnover of these two proteins in AP-3-competent cells, demonstrating that AP-3 function is not required for endosomal sorting. However, sorting P-selectin to secretory granules was defective in endothelial cells from AP-3-deficient pearl mice, demonstrating a role for AP-3 adaptors in granule assembly in endothelial cells. P-selectin sorting to platelet alpha-granules was normal in pearl mice, consistent with earlier evidence that granule targeting of P-selectin is mechanistically distinct in endothelial cells and platelets. These observations establish that AP-3 adaptor functions in assembly of conventional secretory granules, in addition to lysosomes and the 'lysosome-like' secretory granules of platelets and melanocytes.

Adaptor Protein Complex alpha Subunits↗