SEARCH · PubMed Health

Results for “Adaptor Protein Complex mu Subunits”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 73 records · Page 4Linked to original sources

PubMed2002

A phosphatidylinositol (4,5)-bisphosphate binding site within mu2-adaptin regulates clathrin-mediated endocytosis.

The clathrin adaptor complex AP-2 serves to coordinate clathrin-coated pit assembly with the sorting of transmembrane cargo proteins at the plasmalemma. How precisely AP-2 assembly and cargo protein recognition at sites of endocytosis are regulated has remained unclear, but recent evidence implicates phosphoinositides, in particular phosphatidylinositol (4,5)-bisphosphate (PI[4,5]P2), in these processes. Here we have identified and functionally characterized a conserved binding site for PI(4,5)P2 within mu2-adaptin, the medium chain of the clathrin adaptor complex AP-2. Mutant mu2 lacking a cluster of conserved lysine residues fails to bind PI(4,5)P2 and to compete the recruitment of native clathrin/AP-2 to PI(4,5)P2-containing liposomes or to presynaptic membranes. Moreover, we show that expression of mutant mu2 inhibits receptor-mediated endocytosis in living cells. We suggest that PI(4,5)P2 binding to mu2-adaptin regulates clathrin-mediated endocytosis and thereby may contribute to structurally linking cargo recognition to coat formation.

Adaptor Protein Complex 1↗

PubMed1988

Molecular cloning and complete amino acid sequence of AP50, an assembly protein associated with clathrin-coated vesicles.

AP50 is the 50,000-dalton protein component found in clathrin-coated vesicles as part of the coat assembly protein (AP) complex, AP-2. AP50 cDNA clones were isolated from rat brain cDNA libraries, and their nucleotide sequence was determined. The isolated cDNA clones represent the entire coding sequence for the rat brain AP50. They encode a polypeptide containing 435 amino acids with a molecular weight of 49,612 daltons. Comparison with the partially sequenced bovine brain AP50 shows a primary structure that is highly conserved. AP50 does not have detectable sequence similarity with other known kinases or with other proteins of known sequence.

Adaptor Protein Complex 2↗

PubMed1994

unc-101, a gene required for many aspects of Caenorhabditis elegans development and behavior, encodes a clathrin-associated protein.

Our genetic analysis indicates that the unc-101 gene of Caenorhabditis elegans is required for many aspects of development and behavior, including negative regulation of vulval differentiation. We have cloned unc-101 and found that it encodes a homolog of the mammalian medium chains of clathrin-associated protein complexes located at the trans-Golgi and the plasma membrane, AP47 and AP50, respectively. Therefore, clathrin-mediated events might contribute to the negative regulation of vulval differentiation. Comparison of sequences, including a full-length sequence of a C. elegans AP50 homolog, reveals that UNC-101 is most closely related to AP47. Mouse AP47 and nematode UNC-101 proteins are functionally equivalent as assayed in transgenic nematodes. We have sequenced the mutant alleles of unc-101 identified in various genetic screens and shown that all but one are deletions or nonsense mutations, suggesting that these alleles severely reduce unc-101 function.

Adaptor Protein Complex 2↗

PubMed2001

Mu 1A deficiency induces a profound increase in MPR300/IGF-II receptor internalization rate.

The mannose-6-phosphate/IGF-II receptor MPR300 mediates sorting of lysosomal enzymes from the trans-Golgi network to endosomes and endocytosis of hormones, for example, of IGF-II. We analyzed transport of MPR300 in mu1A-adaptin-deficient fibroblasts, which lack a functional AP-1 clathrin adaptor complex. In mu1A-adaptin-deficient fibroblasts, the homologous MPR46 accumulates in endosomes due to a block in retrograde transport to the trans-Golgi network. The MPR300-mediated endocytosis is markedly enhanced. We demonstrate that the seven-fold increase in endocytosis is not associated with an increased steady-state concentration of receptors at the plasma membrane, but with an increased internalization rate of MPR300. Internalization of other receptors that are also endocytosed by AP-2 is not affected. More MPR300 receptors are found in clathrin-coated pits of the plasma membrane, whereas outside coated-areas, more MPR300 are concentrated in clusters and all intracellular receptors reside in endosomes, which are in equilibrium with the plasma membrane. Thus AP-1-mediated transport of MPR300 from endosomes to the TGN controls indirectly the recycling rate of the receptor between the plasma membrane and endosomes.

Adaptor Protein Complex 1↗

PubMed1987

The clathrin coat assembly polypeptide complex. Autophosphorylation and assembly activities.

A 50-kDa polypeptide that is rapidly phosphorylated on addition of [gamma-32P]ATP to isolated clathrin-coated vesicles is shown here to be identical to the 50-kDa component (AP50) of the clathrin assembly protein (AP), a complex that promotes the assembly of clathrin coat structures under physiological conditions of pH and ionic strength. Phosphorylation of the AP50 occurred readily at 0 degrees C, almost exclusively on a threonyl residue(s). This reaction is attributable to autophosphorylation, since the AP50 was able to covalently incorporate 32P from [gamma-32P]ATP after separation by either one- or two-dimensional sodium dodecyl sulfate gel electrophoresis. Kinetic studies in solution were consistent with an intramolecular phosphorylation event; in addition, a concentration-dependent increase in AP50 phosphorylation was observed that may reflect intermolecular AP-AP activation of autophosphorylation. The phosphorylated AP50 was resistant to several inorganic phosphatases tested but was a substrate for protein phosphatases 1 and 2A, suggesting that a physiological phosphorylation-dephosphorylation cycle may exist. The phosphorylation state of the AP50 did not affect the ability of the AP to promote in vitro clathrin coat assembly. These and other data suggest that unique structural domains of the assembly protein are responsible for assembly (the 100-kDa components) and autophosphorylation (the AP50) and that the latter may be active as a protein kinase in the intact cell.

Adaptor Protein Complex 2↗

PubMed2006

Functional analysis of AP-2 alpha and mu2 subunits.

The AP-2 adaptor complex plays a key role in cargo recognition and clathrin-coated vesicle formation at the plasma membrane. To investigate the functions of individual binding sites and domains of the AP-2 complex in vivo, we have stably transfected HeLa cells with wild-type and mutant small interfering RNA-resistant alpha and mu2 subunits and then used siRNA knockdowns to deplete the endogenous proteins. Mutating the PtdIns(4,5)P2 binding site of alpha, the phosphorylation site of mu2, or the YXXPhi binding site of mu2 impairs AP-2 function, as assayed by transferrin uptake. In contrast, removing the C-terminal appendage domain of alpha, or mutating the PtdIns(4,5)P2 binding site of mu2, has no apparent effect. However, adding a C-terminal GFP tag to alpha renders it completely nonfunctional. These findings demonstrate that there is some functional redundancy in the binding sites of the various AP-2 subunits, because no single mutation totally abolishes function. They also help to explain why GFP-tagged AP-2 never appears to leave the plasma membrane in some live cell imaging studies. Finally, they establish a new model system that can be used both for additional structure-function analyses, and as a way of testing tagged constructs for function in vivo.

Adaptor Protein Complex alpha Subunits↗

PubMed2003

The adaptor complex 2 directly interacts with the alpha 1b-adrenergic receptor and plays a role in receptor endocytosis.

Using the yeast two-hybrid system, we identified the mu 2 subunit of the clathrin adaptor complex 2 as a protein interacting with the C-tail of the alpha 1b-adrenergic receptor (AR). Direct association between the alpha 1b-AR and mu 2 was demonstrated using a solid phase overlay assay. The alpha 1b-AR/mu 2 interaction occurred inside the cells, as shown by the finding that the transfected alpha 1b-AR and the endogenous mu 2 could be coimmunoprecipitated from HEK-293 cell extracts. Mutational analysis of the alpha 1b-AR revealed that the binding site for mu 2 does not involve canonical YXX Phi or dileucine motifs but a stretch of eight arginines on the receptor C-tail. The binding domain of mu 2 for the receptor C-tail involves both its N terminus and the subdomain B of its C-terminal portion. The alpha 1b-AR specifically interacted with mu 2, but not with the mu 1, mu 3, or mu 4 subunits belonging to other AP complexes. The deletion of the mu 2 binding site in the C-tail markedly decreased agonist-induced receptor internalization as demonstrated by confocal microscopy as well as by the results of a surface receptor biotinylation assay. The direct association of the adaptor complex 2 with a G protein-coupled receptor has not been reported so far and might represent a common mechanism underlying clathrin-mediated receptor endocytosis.

Adaptor Protein Complex 2↗

PubMed1997

Identification of two new mu-adaptin-related proteins, mu-ARP1 and mu-ARP2.

We report the cDNA cloning, primary structure and tissue distribution of two new proteins homologous to mu-adaptins, the medium chains of the clathrin coat adaptor complexes. Both predicted proteins share 60% amino acid sequence identity with each other and 27-31%, identity with mu1-adaptin (ap47) and mu2-adaptin (ap50). Lower similarity (23-25% identity) is found with two other mu-adaptin-related proteins, p47A/B, and there is similarity over the N-terminal 150 amino acids with the adaptin small chains and deltaCOP. The mRNAs of both molecules are expressed in all tissues analyzed, but with different profiles of relative abundance. mu-ARP1 is most abundant in brain, ovary and lung, whereas mu-ARP2 is prominently expressed in testis. These proteins suggest the existence of as yet uncharacterized types of clathrin- or non-clathrin-associated protein coats in cellular membrane traffic, of which they are probably prototype subunits, and provide molecular markers and probes for their characterization.

Adaptor Protein Complex 1↗

PubMed2004

Recognition of the tryptophan-based endocytosis signal in the neonatal Fc Receptor by the mu subunit of adaptor protein-2.

Endocytosis of membrane proteins is typically mediated by signals present in their cytoplasmic domains. These signals usually contain an essential tyrosine or pair of leucine residues. Both tyrosine- and dileucine-based endocytosis signals are recognized by the adaptor complex AP-2. The best understood of these interactions occurs between the tyrosine-based motif, YXXPhi, and the mu2 subunit of AP-2. We recently reported a tryptophan-based endocytosis signal, WLSL, contained within the cytoplasmic domain of the neonatal Fc receptor. This signal resembles YXXPhi. We have investigated the mechanism by which the tryptophan-based signal is recognized. Both interaction assays in vitro and endocytosis assays in vivo show that mu2 binds the tryptophan-based signal. Furthermore, the WLSL sequence binds the same site as YXXPhi. Unlike the WXXF motif, contained in stonin 2 and other endocytic proteins, WLSL does not bind the alpha subunit of AP-2. These observations reveal a functional similarity between the tryptophan-based endocytosis signal and the YXXPhi motif, and an unexpected versatility of mu2 function.

Adaptor Protein Complex 2↗

PubMed2005

Leucine-specific, functional interactions between human immunodeficiency virus type 1 Nef and adaptor protein complexes.

The human immunodeficiency virus type 1 virulence protein Nef interacts with the endosomal sorting machinery via a leucine-based motif. Similar sequences within the cytoplasmic domains of cellular transmembrane proteins bind to the adaptor protein (AP) complexes of coated vesicles to modulate protein traffic, but the molecular basis of the interactions between these motifs and the heterotetrameric complexes is controversial. To identify the target of the Nef leucine motif, the native sequence was replaced with either leucine- or tyrosine-based AP-binding sequences from cellular proteins, and the interactions with AP subunits were correlated with function. Tyrosine motifs predictably modulated the interactions between Nef and the mu subunits of AP-1, AP-2, and AP-3; heterologous leucine motifs caused little change in these interactions. Conversely, leucine motifs mediated a ternary interaction between Nef and hemicomplexes containing the sigma1 plus gamma subunits of AP-1 or the sigma3 plus delta subunits of AP-3, whereas tyrosine motifs did not. Similarly, only leucine motifs supported the Nef-mediated association of AP-1 and AP-3 with endosomal membranes in cells treated with brefeldin A. Functionally, Nef proteins containing leucine motifs down-regulated CD4 from the cell surface and enhanced viral replication, whereas those containing tyrosine motifs were inactive. Apparently, the interaction of Nef with the mu subunits of AP complexes is insufficient for function. A leucine-specific mode of interaction that likely involves AP hemicomplexes is further required for Nef activity. The mu and hemicomplex interactions may cooperate to yield high avidity binding of AP complexes to Nef. This binding likely underlies the unusual ability of Nef to induce the stabilization of these complexes on endosomal membranes, an activity that correlates with enhancement of viral replication.

Adaptor Protein Complex mu Subunits↗

PubMed2005

The mouse CD1d cytoplasmic tail mediates CD1d trafficking and antigen presentation by adaptor protein 3-dependent and -independent mechanisms.

The short cytoplasmic tail of mouse CD1d (mCD1d) is required for its endosomal localization, for the presentation of some glycolipid Ags, and for the development of Valpha14i NKT cells. This tail has a four-amino acid Tyr-containing motif, Tyr-Gln-Asp-Ile (YQDI), similar to those sequences known to be important for the interaction with adaptor protein complexes (AP) that mediate the endosomal localization of many different proteins. In fact, mCD1d has been shown previously to interact with the AP-3 adaptor complex. In the present study, we mutated each amino acid in the YQDI motif to determine the importance of the entire motif sequence in influencing mCD1d trafficking, its interaction with adaptors, and its intracellular localization. The results indicate that the Y, D, and I amino acids are significant functionally because mutations at each of these positions altered the intracellular distribution of mCD1d and reduced its ability to present glycosphingolipids to NKT cells. However, the three amino acids are not all acting in the same way because they differ with regard to how they influence the intracellular distribution of CD1d, its rate of internalization, and its ability to interact with the mu subunit of AP-3. Our results emphasize that multiple steps, including interactions with the adaptors AP-2 and AP-3, are required for normal trafficking of mCD1d and that these different steps are mediated by only a few cytoplasmic amino acids.

Adaptor Protein Complex 2↗

PubMed2002

The polarized epithelia-specific mu 1B-adaptin complements mu 1A-deficiency in fibroblasts.

The heterotetrameric AP-1A adaptor complex of clathrin-coated vesicles is ubiquitously expressed. The mu 1-adaptin subunit of the complex exists as the ubiquitous mu 1A and the polarized epithelia-specific mu 1B, which are 80% identical. In polarized epithelia, mu 1B is incorporated into the AP-1B complex, which is required for basolateral plasma membrane sorting of the low-density lipoprotein receptor. Binding of AP-1B to subdomains of the trans-Golgi network (TGN) appears to be part of the mechanism by which protein sorting is mediated. We expressed mu 1B in mu 1A-deficient fibroblasts to test for mu 1B function in non-polarized cells. AP-1B complexes were formed and bound to the TGN and to endosomes. Moreover, AP-1B restored the AP-1A-dependent sorting of mannose 6-phosphate receptors between endosomes and the TGN. This demonstrates that mu 1A and mu 1B do have overlapping sorting functions and indicates that AP-1A and AP-1B mediate protein sorting along parallel pathways between the TGN and endosomes in polarized epithelia.

Adaptor Protein Complex mu Subunits↗

PubMed2006

Trafficking of the human transferrin receptor in plant cells: effects of tyrphostin A23 and brefeldin A.

Plant cells possess much of the molecular machinery necessary for receptor-mediated endocytosis (RME), but this process still awaits detailed characterization. In order to identify a reliable and well-characterized marker to investigate RME in plant cells, we have expressed the human transferrin receptor (hTfR) in Arabidopsis protoplasts. We have found that hTfR is mainly found in endosomal (Ara7- and FM4-64-positive) compartments, but also at the plasma membrane, where it mediates binding and internalization of its natural ligand transferrin (Tfn). Cell surface expression of hTfR increases upon treatment with tyrphostin A23, which inhibits the interaction between the YTRF endocytosis signal in the hTfR cytosolic tail and the mu2-subunit of the AP2 complex. Indeed, tyrphostin A23 inhibits Tfn internalization and redistributes most of hTfR to the plasma membrane, suggesting that the endocytosis signal of hTfR is functional in Arabidopsis protoplasts. Co-immunoprecipitation experiments show that hTfR is able to interact with a mu-adaptin subunit from Arabidopsis cytosol, a process that is blocked by tyrphostin A23. In contrast, treatment with brefeldin A, which inhibits recycling from endosomes back to the plasma membrane in plant cells, leads to the accumulation of Tfn and hTfR in larger patches inside the cell, reminiscent of BFA compartments. Therefore, hTfR has the same trafficking properties in Arabidopsis protoplasts as in animal cells, and cycles between the plasma membrane and endosomal compartments. The specific inhibition of Tfn/hTfR internalization and recycling by tyrphostin A23 and BFA, respectively, thus provide valuable molecular tools to characterize RME and the recycling pathway in plant cells.

Adaptor Protein Complex mu Subunits↗

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↗

PubMed2006

Stimulation of phosphatidylinositol kinase type I-mediated phosphatidylinositol (4,5)-bisphosphate synthesis by AP-2mu-cargo complexes.

Phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P(2)] is an important factor for a variety of cellular functions ranging from cell signaling to actin cytoskeletal dynamics and endocytic membrane traffic. Here, we have identified the clathrin adaptor complex AP-2 as a regulator of phosphatidylinositol 4-phosphate 5-kinase (PIPK)-mediated PI(4,5)P(2) synthesis. AP-2 directly interacts with the kinase core domain of type I PIPK isozymes via its mu2-subunit in vitro and in native protein extracts. Endocytic cargo protein binding to mu2 leads to a potent stimulation of PIPK activity. These data thus identify a positive feedback loop consisting of endocytic cargo proteins, AP-2mu, and PIPK type I which may provide a specific pool of PI(4,5)P(2) dedicated to clathrin/AP-2-dependent receptor internalization.

Adaptor Protein Complex 2↗

PubMed2003

The AP-1A and AP-1B clathrin adaptor complexes define biochemically and functionally distinct membrane domains.

Most epithelial cells contain two AP-1 clathrin adaptor complexes. AP-1A is ubiquitously expressed and involved in transport between the TGN and endosomes. AP-1B is expressed only in epithelia and mediates the polarized targeting of membrane proteins to the basolateral surface. Both AP-1 complexes are heterotetramers and differ only in their 50-kD mu1A or mu1B subunits. Here, we show that AP-1A and AP-1B, together with their respective cargoes, define physically and functionally distinct membrane domains in the perinuclear region. Expression of AP-1B (but not AP-1A) enhanced the recruitment of at least two subunits of the exocyst complex (Sec8 and Exo70) required for basolateral transport. By immunofluorescence and cell fractionation, the exocyst subunits were found to selectively associate with AP-1B-containing membranes that were both distinct from AP-1A-positive TGN elements and more closely apposed to transferrin receptor-positive recycling endosomes. Thus, despite the similarity of the two AP-1 complexes, AP-1A and AP-1B exhibit great specificity for endosomal transport versus cell polarity.

Adaptor Protein Complex 1↗

PubMed2004

Hypertension-linked mutation in the adducin alpha-subunit leads to higher AP2-mu2 phosphorylation and impaired Na+,K+-ATPase trafficking in response to GPCR signals and intracellular sodium.

Alpha-adducin polymorphism in humans is associated with abnormal renal sodium handling and high blood pressure. The mechanisms by which mutations in adducin affect the renal set point for sodium excretion are not known. Decreases in Na+,K+-ATPase activity attributable to endocytosis of active units in renal tubule cells by dopamine regulates sodium excretion during high-salt diet. Milan rats carrying the hypertensive adducin phenotype have a higher renal tubule Na+,K+-ATPase activity, and their Na+,K+-ATPase molecules do not undergo endocytosis in response to dopamine as do those of the normotensive strain. Dopamine fails to promote the interaction between adaptins and the Na+,K+-ATPase because of adaptin-mu2 subunit hyperphosphorylation. Expression of the hypertensive rat or human variant of adducin into normal renal epithelial cells recreates the hypertensive phenotype with higher Na+,K+-ATPase activity, mu2-subunit hyperphosphorylation, and impaired Na+,K+-ATPase endocytosis. Thus, increased renal Na+,K+-ATPase activity and altered sodium reabsorption in certain forms of hypertension could be attributed to a mutant form of adducin that impairs the dynamic regulation of renal Na+,K+-ATPase endocytosis in response to natriuretic signals.

Adaptor Protein Complex 2↗

PubMed2004

HIV-1 Nef disrupts MHC-I trafficking by recruiting AP-1 to the MHC-I cytoplasmic tail.

To avoid immune recognition by cytotoxic T lymphocytes (CTLs), human immunodeficiency virus (HIV)-1 Nef disrupts the transport of major histocompatibility complex class I molecules (MHC-I) to the cell surface in HIV-infected T cells. However, the mechanism by which Nef does this is unknown. We report that Nef disrupts MHC-I trafficking by rerouting newly synthesized MHC-I from the trans-Golgi network (TGN) to lysosomal compartments for degradation. The ability of Nef to target MHC-I from the TGN to lysosomes is dependent on expression of the mu1 subunit of adaptor protein (AP) AP-1A, a cellular protein complex implicated in TGN to endolysosomal pathways. We demonstrate that in HIV-infected primary T cells, Nef promotes a physical interaction between endogenous AP-1 and MHC-I. Moreover, we present data that this interaction uses a novel AP-1 binding site that requires amino acids in the MHC-I cytoplasmic tail. In sum, our evidence suggests that binding of AP-1 to the Nef-MHC-I complex is an important step required for inhibition of antigen presentation by HIV.

Adaptor Protein Complex 1↗