Phosphorylation of the medium chain subunit of the AP-2 adaptor complex does not influence its interaction with the tyrosine based internalisation motif of TGN38.
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Tyrosine based motifs conforming to the consensus YXXphi (where phi represents a bulky hydrophobic residue) have been shown to interact with the medium chain subunit of clathrin adaptor complexes. These medium chains are targets for phosphorylation by a kinase activity associated with clathrin coated vesicles. We have used the clathrin coated vesicle associated kinase activity to specifically phosphorylate a soluble recombinant fusion protein of mu2, the medium chain subunit of the plasma membrane associated adaptor protein complex AP-2. We have tested whether this phosphorylation has any effect on the interaction of mu2 with the tyrosine based motif containing protein, TGN38, that has previously been shown to interact with mu2. Phosphorylation of mu2 was shown to have no significant effect on the in vitro interaction of mu2 with the cytosolic domain of TGN38, indicating that reversible phosphorylation of mu2 does not play a role in regulating its direct interaction with tyrosine based internalisation motifs. In addition, although a casein kinase II-like activity has been shown to be associated with clathrin coated vesicles, we show that mu2 is not phosphorylated by casein kinase II implying that another kinase activity is present in clathrin coated vesicles. Furthermore the kinase activity associated with clathrin coated vesicles was shown to be capable of phosphorylating dynamin 1. Phosphorylation of dynamin 1 has previously been shown to regulate its interaction with other proteins involved in clathrin mediated endocytosis. (+info)
Inhibition of the receptor-binding function of clathrin adaptor protein AP-2 by dominant-negative mutant mu2 subunit and its effects on endocytosis.
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Although interactions between the mu2 subunit of the clathrin adaptor protein complex AP-2 and tyrosine-based internalization motifs have been implicated in the selective recruitment of cargo molecules into coated pits, the functional significance of this interaction for endocytosis of many types of membrane proteins remains unclear. To analyze the function of mu2-receptor interactions, we constructed an epitope-tagged mu2 that incorporates into AP-2 and is targeted to coated pits. Mutational analysis revealed that Asp176 and Trp421 of mu2 are involved in the interaction with internalization motifs of TGN38 and epidermal growth factor (EGF) receptor. Inducible overexpression of mutant mu2, in which these two residues were changed to alanines, resulted in metabolic replacement of endogenous mu2 in AP-2 complexes and complete abrogation of AP-2 interaction with the tyrosine-based internalization motifs. As a consequence, endocytosis of the transferrin receptor was severely impaired. In contrast, internalization of the EGF receptor was not affected. These results demonstrate the potential usefulness of the dominant-interfering approach for functional analysis of the adaptor protein family, and indicate that clathrin-mediated endocytosis may proceed in both a mu2-dependent and -independent manner. (+info)
Mu1B, a novel adaptor medium chain expressed in polarized epithelial cells.
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The apical and basolateral plasma membrane domains of polarized epithelial cells contain distinct sets of integral membrane proteins. Biosynthetic targeting of proteins to the basolateral plasma membrane is mediated by cytosolic tail determinants, many of which resemble signals involved in the rapid endocytosis or lysosomal targeting. Since these signals are recognized by adaptor proteins, we hypothesized that there could be epithelial-specific adaptors involved in polarized sorting. Here, we report the identification of a novel member of the adaptor medium chain family, named mu1B, which is closely related to the previously described mu1A (79% amino acid sequence identity). Northern blotting and in situ hybridization analyses reveal the specific expression of mu1B mRNA in a subset of polarized epithelial and exocrine cells. Yeast two-hybrid analyses show that mu1B is capable of interacting with generic tyrosine-based sorting signals. These observations suggest that mu1B may be involved in protein sorting events specific to polarized cells. (+info)
Early endosomes are required for major histocompatiblity complex class II transport to peptide-loading compartments.
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Antigen presentation to CD4(+) T lymphocytes requires transport of newly synthesized major histocompatibility complex (MHC) class II molecules to the endocytic pathway, where peptide loading occurs. This step is mediated by a signal located in the cytoplasmic tail of the MHC class II-associated Ii chain, which directs the MHC class II-Ii complexes from the trans-Golgi network (TGN) to endosomes. The subcellular machinery responsible for the specific targeting of MHC class II molecules to the endocytic pathway, as well as the first compartments these molecules enter after exit from the TGN, remain unclear. We have designed an original experimental approach to selectively analyze this step of MHC class II transport. Newly synthesized MHC class II molecules were caused to accumulate in the Golgi apparatus and TGN by incubating the cells at 19 degrees C, and early endosomes were functionally inactivated by in vivo cross-linking of transferrin (Tf) receptor-containing endosomes using Tf-HRP complexes and the HRP-insoluble substrate diaminobenzidine. Inactivation of Tf-containing endosomes caused a marked delay in Ii chain degradation, peptide loading, and MHC class II transport to the cell surface. Thus, early endosomes appear to be required for delivery of MHC class II molecules to the endocytic pathway. Under cross-linking conditions, most alphabetaIi complexes accumulated in tubules and vesicles devoid of gamma-adaptin and/or mannose-6-phosphate receptor, suggesting an AP1-independent pathway for the delivery of newly synthesized MHC class II molecules from the TGN to endosomes. (+info)
Gamma-synergin: an EH domain-containing protein that interacts with gamma-adaptin.
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The AP-1 adaptor complex is associated with the TGN, where it links selected membrane proteins to the clathrin lattice, enabling these proteins to be concentrated in clathrin-coated vesicles. To identify other proteins that participate in the clathrin-coated vesicle cycle at the TGN, we have carried out a yeast two- hybrid library screen using the gamma-adaptin subunit of the AP-1 complex as bait. Two novel, ubiquitously expressed proteins were found: p34, which interacts with both gamma-adaptin and alpha-adaptin, and gamma-synergin, an alternatively spliced protein with an apparent molecular mass of approximately 110-190 kD, which only interacts with gamma-adaptin. gamma-Synergin is associated with AP-1 both in the cytosol and on TGN membranes, and it is strongly enriched in clathrin-coated vesicles. It binds directly to the ear domain of gamma-adaptin and it contains an Eps15 homology (EH) domain, although the EH domain is not part of the gamma-adaptin binding site. In cells expressing alpha-adaptin with the gamma-adaptin ear, a construct that goes mainly to the plasma membrane, much of the gamma-synergin is also rerouted to the plasma membrane, indicating that it follows AP-1 onto membranes rather than leading it there. The presence of an EH domain suggests that gamma-synergin links the AP-1 complex to another protein or proteins. (+info)
Duplications on human chromosome 22 reveal a novel Ret Finger Protein-like gene family with sense and endogenous antisense transcripts.
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Analysis of 600 kb of sequence encompassing the beta-prime adaptin (BAM22) gene on human chromosome 22 revealed intrachromosomal duplications within 22q12-13 resulting in three active RFPL genes, two RFPL pseudogenes, and two pseudogenes of BAM22. The genomic sequence of BAM22vartheta1 shows a remarkable similarity to that of BAM22. The cDNA sequence comparison of RFPL1, RFPL2, and RFPL3 showed 95%-96% identity between the genes, which were most similar to the Ret Finger Protein gene from human chromosome 6. The sense RFPL transcripts encode proteins with the tripartite structure, composed of RING finger, coiled-coil, and B30-2 domains, which are characteristic of the RING-B30 family. Each of these domains are thought to mediate protein-protein interactions by promoting homo- or heterodimerization. The MID1 gene on Xp22 is also a member of the RING-B30 family and is mutated in Opitz syndrome (OS). The autosomal dominant form of OS shows linkage to 22q11-q12. We detected a polymorphic protein-truncating allele of RFPL1 in 8% of the population, which was not associated with the OS phenotype. We identified 6-kb and 1.2-kb noncoding antisense mRNAs of RFPL1S and RFPL3S antisense genes, respectively. The RFPL1S and RFPL3S genes cover substantial portions of their sense counterparts, which suggests that the function of RFPL1S and RFPL3S is a post-transcriptional regulation of the sense RFPL genes. We illustrate the role of intrachromosomal duplications in the generation of RFPL genes, which were created by a series of duplications and share an ancestor with the RING-B30 domain containing genes from the major histocompatibility complex region on human chromosome 6. (+info)
A novel clathrin adaptor complex mediates basolateral targeting in polarized epithelial cells.
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Although polarized epithelial cells are well known to maintain distinct apical and basolateral plasma membrane domains, the mechanisms responsible for targeting membrane proteins to the apical or basolateral surfaces have remained elusive. We have identified a novel form of the AP-1 clathrin adaptor complex that contains as one of its subunits mu1B, an epithelial cell-specific homolog of the ubiquitously expressed mu1A. LLC-PK1 kidney epithelial cells do not express mu1B and missort many basolateral proteins to the apical surface. Stable expression of mu1B selectively restored basolateral targeting, improved the overall organization of LLC-PK1 monolayers, and had no effect on apical targeting. We conclude that basolateral sorting is mediated by an epithelial cell-specific version of the AP-1 complex containing mu1B. (+info)
Association of AP1 adaptor complexes with GLUT4 vesicles.
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Nycodenz gradients have been used to examine the in vitro effects of GTP-(gamma)-S on adaptor complex association with GLUT4 vesicles. On addition of GTP-(gamma)-S, GLUT4 fractionates as a heavier population of vesicles, which we suggest is due to a budding or coating reaction. Under these conditions there is an increase in co-sedimentation of GLUT4 with AP1, but not with AP3. Western blotting of proteins associated with isolated GLUT4 vesicles shows the presence of high levels of AP1 and some AP3 but very little AP2 adaptor complexes. Cell free, in vitro association of the AP1 complex with GLUT4 vesicles is increased approximately 4-fold by the addition of GTP-(gamma)-S and an ATP regenerating system. Following GTP-(gamma)-S treatment in vitro, ARF is also recruited to GLUT4 vesicles, and the temperature dependence of ARF recruitment closely parallels that of AP1. The recruitment of both AP1 and ARF are partially blocked by brefeldin A. These data demonstrate that the coating of GLUT4 vesicles can be studied in isolated cell-free fractions. Furthermore, at least two distinct adaptor complexes can associate with the GLUT4 vesicles and it is likely that these adaptors are involved in mediating distinct intracellular sorting events at the level of TGN and endosomes. (+info)