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PubMed2000

Electrophoretic mobility shift assay for the detection of specific DNA-protein complex in nuclear extracts from the cultured cells and frozen autopsy human brain tissue.

The electrophoretic mobility shift assay (EMSA) is generally used to study the interaction of transcription factors to specific DNA sequences. The preparation of high quality nuclear extracts is an important step before performing the assay. Here we describe a rapid method for the isolation of good-quality DNA-binding proteins from cultured cell lines and autopsy tissue samples from the human brain. The 'rapid method' (RM) utilizes the low salt/detergent lysis steps followed by high salt extraction of nuclei. To test and compare the activity of nuclear extracts prepared by the standard and 'rapid' methods for its ability to form the specific DNA-protein complex, EMSA was carried out with three different oligonucleotide probes: AP1, NF-kappaB and URE. A comparative study indicates that the capacity to form the specific DNA-protein complex with these oligonucleotide probes by standard and RM nuclear extracts was very similar. Each nuclear extract formed the corresponding DNA-protein complex, the specificity of which was checked by the competition experiment. In some cases unspecific bands were observed and which were present in nuclear extracts from both preparations. Thus the simplicity of the 'rapid method' permits the preparation of nuclear extracts from several cell lines and tissue samples at the same time at much shorter time than the 'standard' method without compromising the DNA-binding activity. The RM can be applied to determine the cell type or tissue specificity of transcription factors in an efficient, economical and consistent manner.

5' Untranslated Regions↗

PubMed2003

Raft disorganization leads to reduced plasmin activity in Alzheimer's disease brains.

The serine protease plasmin can efficiently degrade amyloid peptide in vitro, and is found at low levels in the hippocampus of patients with Alzheimer's disease (AD). The cause of such paucity remains unknown. We show here that the levels of total brain plasminogen and plasminogen-binding molecules are normal in these brain samples, yet plasminogen membrane binding is greatly reduced. Biochemical analysis reveals that the membranes of these brains have a mild, still significant, cholesterol reduction compared to age-matched controls, and anomalous raft microdomains. This was reflected by the loss of raft-enriched proteins, including plasminogen-binding and -activating molecules. Using hippocampal neurons in culture, we demonstrate that removal of a similar amount of membrane cholesterol is sufficient to induce raft disorganization, leading to reduced plasminogen membrane binding and low plasmin activity. These results suggest that brain raft alterations may contribute to AD by rendering the plasminogen system inefficient.

Adaptor Protein Complex gamma Subunits↗

PubMed1995

Different domains of the AP-1 adaptor complex are required for Golgi membrane binding and clathrin recruitment.

The assembly of clathrin-coated buds on the Golgi requires the recruitment of the heterotetrameric AP-1 adaptor complex, which is dependent on both guanine nucleotides and the small GTP-binding protein ADP-ribosylation factor (ARF). Here, we have investigated the structural domains of the AP-1 complex necessary for ARF-mediated translocation of the adaptor complex onto Golgi membranes and the subsequent recruitment of clathrin onto the membrane. Controlled proteolysis of purified AP-1, derived from bovine adrenal coated vesicles, was used to generate AP-1 core fragments composed of the amino-terminal trunk regions of the beta 1 and gamma subunits and associated mu 1 and sigma 1 subunits, and lacking either the beta 1 subunit carboxyl-terminal appendage or both beta 1 and gamma subunit appendages. On addition of these truncated fragments to AP-1-depleted adrenal cytosol, both types of core fragments were efficiently recruited onto Golgi membranes in the presence of GTP gamma S. Recruitment of both core fragments was inhibited by the fungal metabolite brefeldin A, indicative of an ARF-dependent process. Limited tryptic digestion of recruited, intact cytosolic AP-1 resulted in the quantitative release of the globular carboxyl-terminal appendage domains of the beta 1 and gamma subunits. The adaptor core complex remained associated with the Golgi membranes. Recruitment of cytosolic clathrin onto the Golgi membranes was strictly dependent on the presence of intact AP-1. Tryptic removal of the beta 1 subunit appendage prevented subsequent clathrin recruitment. We conclude that the structural determinants required for the ARF-mediated binding of cytosolic AP-1 onto Golgi membranes are contained within the adaptor core, and that the carboxyl-terminal appendage domains of the beta 1 and gamma subunits do not play any role in this process. Subsequent recruitment of cytosolic clathrin, however, requires an intact beta 1 subunit.

3T3 Cells↗

PubMed1999

Cloning, expression, and vesicular localization of zinc transporter Dri 27/ZnT4 in intestinal tissue and cells.

We have identified the Dri 27 cDNA on the basis of its upregulated expression during rat intestinal development. It encodes a hydrophobic protein of 430 amino acids that shares significant homology with members of the mammalian zinc transporter family ZnT. The murine homologue of Dri 27 (named ZnT4) was recently associated with the mouse mutation "lethal milk." The primary sequence of Dri 27/ZnT4 displays features characteristic of polytopic membrane proteins. In this paper, we show that Dri 27/ZnT4 is localized in the membrane of intracellular vesicles, the majority of which concentrate in the basal cytoplasmic region of polarized enterocytes. A Dri 27/ZnT4 myc-tagged construct, transiently transfected in intestinal Caco-2 cells, partially colocalizes with the transferrin receptor and with the beta-subunits of the clathrin adaptor complexes AP-1 and AP-2 in a subpopulation of endosomal vesicles. By subcloning distinct portions of the protein in frame with glutathione-S-transferase, we also provide experimental evidence of their function as zinc-binding and protein-protein-interaction domains.

Amino Acid Sequence↗

PubMed2000

COP I domains required for coatomer integrity, and novel interactions with ARF and ARF-GAP.

We performed a systematic mapping of interaction domains on COP I subunits to gain novel insights into the architecture of coatomer. Using the two-hybrid system, we characterize the domain structure of the alpha-, beta'-, epsilon-COP and beta-, gamma-, delta-, zeta-COP coatomer subcomplexes and identify links between them that contribute to coatomer integrity. Our results demonstrate that the domain organization of the beta-, gamma-, delta-, zeta-COP subcomplex and AP adaptor complexes is related. Through in vivo analysis of alpha-COP truncation mutants, we characterize distinct functional domains on alpha-COP. Its N-terminal WD40 domain is dispensable for yeast cell viability and overall coatomer function, but is required for KKXX-dependent trafficking. The last approximately 170 amino acids of alpha-COP are also non-essential for cell viability, but required for epsilon-COP incorporation into coatomer and maintainance of normal epsilon-COP levels. Further, we demonstrate novel direct interactions of coatomer subunits with regulatory proteins: beta'- and gamma-COP interact with the ARF-GTP-activating protein (GAP) Glo3p, but not Gcs1p, and beta- and epsilon-COP interact with ARF-GTP. Glo3p also interacts with intact coatomer in vitro.

ADP-Ribosylation Factors↗

PubMed2002

Association of mumps virus V protein with RACK1 results in dissociation of STAT-1 from the alpha interferon receptor complex.

It has been reported that mumps virus protein V or the C-terminal Cys-rich region of protein V (Vsp) is associated with blocking of the interferon (IFN) signal transduction pathway through a decrease in STAT-1 production. The intracellular target of the V protein was investigated by using a two-hybrid screening system with Vsp as bait. Full-length V protein and Vsp were able to bind to RACK1, and the interaction did not require two WD domains, WD1 and WD2, in RACK1. A significant interaction between V protein and RACK1 was also demonstrated in cells persistently infected with mumps virus (FLMT cells), and the formation of the complex was not affected by treatment with IFN. On the other hand, in uninfected cells, STAT-1 was associated with the long form of the beta subunit of the alpha IFN receptor, and this association was mediated by the function of RACK1 as an adaptor protein. Immunoprecipitation and glutathione S-transferase pull-down experiments revealed that the association of RACK1 or mumps virus V protein with the IFN receptor was undetectable in mumps virus-infected cells. Furthermore, RACK1 interacted with mumps virus V protein with a higher affinity than STAT-1 did. Therefore, it is suggested that mumps virus V protein has the ability to interact strongly with RACK1 and consequently to bring about the disruption of the complex formed from STAT-1, RACK1, and the IFN receptor.

Cells, Cultured↗

PubMed1999

Altered trafficking of lysosomal proteins in Hermansky-Pudlak syndrome due to mutations in the beta 3A subunit of the AP-3 adaptor.

Hermansky-Pudlak syndrome (HPS) is a genetic disorder characterized by defective lysosome-related organelles. Here, we report the identification of two HPS patients with mutations in the beta 3A subunit of the heterotetrameric AP-3 complex. The patients' fibroblasts exhibit drastically reduced levels of AP-3 due to enhanced degradation of mutant beta 3A. The AP-3 deficiency results in increased surface expression of the lysosomal membrane proteins CD63, lamp-1, and lamp-2, but not of nonlysosomal proteins. These differential effects are consistent with the preferential interaction of the AP-3 mu 3A subunit with tyrosine-based signals involved in lysosomal targeting. Our results suggest that AP-3 functions in protein sorting to lysosomes and provide an example of a human disease in which altered trafficking of integral membrane proteins is due to mutations in a component of the sorting machinery.

Adaptor Proteins, Vesicular Transport↗

PubMed2006

Dopamine D3 receptors regulate GABAA receptor function through a phospho-dependent endocytosis mechanism in nucleus accumbens.

The dopamine D3 receptor, which is highly enriched in nucleus accumbens (NAc), has been suggested to play an important role in reinforcement and reward. To understand the potential cellular mechanism underlying D3 receptor functions, we examined the effect of D3 receptor activation on GABAA receptor (GABAAR)-mediated current and inhibitory synaptic transmission in medium spiny neurons of NAc. Application of PD128907 [(4aR,10bR)-3,4a,4,10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano-[4,3-b]-1,4-oxazin-9-ol hydrochloride], a specific D3 receptor agonist, caused a significant reduction of GABAAR current in acutely dissociated NAc neurons and miniature IPSC amplitude in NAc slices. This effect was blocked by dialysis with a dynamin inhibitory peptide, which prevents the clathrin/activator protein 2 (AP2)-mediated GABAA receptor endocytosis. In addition, the D3 effect on GABAAR current was prevented by agents that manipulate protein kinase A (PKA) activity. Infusion of a peptide derived from GABAAR beta subunits, which contains an atypical binding motif for the clathrin AP2 adaptor complex and the major PKA phosphorylation sites and binds with high affinity to AP2 only when dephosphorylated, diminished the D3 regulation of IPSC amplitude. The phosphorylated equivalent of the peptide was without effect. Moreover, PD128907 increased GABAAR internalization and reduced the surface expression of GABAA receptor beta subunits in NAc slices, which was prevented by dynamin inhibitory peptide or cAMP treatment. Together, our results suggest that D3 receptor activation suppresses the efficacy of inhibitory synaptic transmission in NAc by increasing the phospho-dependent endocytosis of GABAA receptors.

Analysis of Variance↗

PubMed2001

Similar subunit interactions contribute to assembly of clathrin adaptor complexes and COPI complex: analysis using yeast three-hybrid system.

Clathrin adaptor protein (AP) complexes are heterotetramers composed of two large, one medium, and one small subunits. By exploiting the yeast three-hybrid system, we have found that an interaction between the two large subunits of the AP-1 complex, gamma-adaptin and beta1-adaptin, is markedly enhanced in the presence of the small subunit, sigma1. Similarly, two large subunits of the AP-4 complex, epsilon-adaptin and beta4-adaptin, are found to interact with each other only in the presence of the small subunit, sigma4. Furthermore, we have found that an interaction between two large subunits of the COPI F subcomplex, gamma-COP and beta-COP, is detectable only in the presence of zeta-COP. Because these COPI subunits have common ancestral origins to the corresponding AP subunits, these three-hybrid data, taken together with the previous two-hybrid data, suggest that the AP complexes and the COPI F subcomplex assemble by virtue of similar subunit interactions.

Adaptor Protein Complex 1↗

PubMed1994

Purification of Golgi adaptor protein 1 from bovine adrenal gland and characterization of its beta 1 (beta') subunit by microsequencing.

A method for the purification of the Golgi adaptor protein 1 from bovine adrenal gland tissue was devised to investigate the relationship of its beta 1 (formerly referred to as beta') subunit to known beta-type sequences. Adrenal gland tissue was chosen for this study because it yielded 2-3 times more adaptor protein 1 than a comparable preparation from bovine brain. Like its neuronal isoform, the beta 1 subunit from adrenal gland adaptor protein 1 is readily cleaved by trypsin into a 63-kDa N-terminal fragment and a 40-kDa C-terminal fragment, while the gamma subunit is largely refractory to digestion. Based on microsequencing of 167 residues from the 63-kDa fragment, we noted 11 differences to the corresponding region of the beta 2 (formerly beta) subunit of the plasma membrane adaptor protein 2, but only one difference to the corresponding region of a beta-type protein encoded by the rat cDNA clone AP105a which is supposed to be a variant of the beta 2 subunit of the plasma membrane adaptor protein 2 [Kirchhausen, T., Nathanson, K. L., Matsui, W., Vaisberg, A., Chow, E. P., Burne, C., Keen, J. H. & Davis, A. E. (1989) Proc. Natl Acad. Sci. USA 84, 8805-8809]. Alignment of 187 residues from the 40-kDa beta 1 C-terminal fragment revealed differences in 77 positions to the corresponding region of the beta 2 subunit and differences in 23 positions compared to the supposed beta 2-like protein. These findings suggest that the protein encoded by the rat cDNA clone AP105a is more closely related to the beta 1 subunit of the bovine adrenal Golgi adaptor protein 1 than to the beta 2 subunit of the rat plasma membrane adaptor protein 2.

Adaptor Protein Complex 1↗

PubMed1990

Cloning and expression of gamma-adaptin, a component of clathrin-coated vesicles associated with the Golgi apparatus.

Adaptins are the major components of adaptors, the protein complexes that link clathrin to transmembrane proteins (e.g., receptors) in coated pits and vesicles. The plasma membrane adaptor contains an alpha-adaptin subunit and a beta-adaptin subunit, while the Golgi adaptor contains a gamma-adaptin subunit and a beta'-adaptin subunit. A partial cDNA clone encoding gamma-adaptin was isolated from a bovine brain expression library by screening with antibodies, and was used to obtain a cDNA clone from a mouse brain library containing the full coding sequence. The identity of the clones was confirmed by protein sequencing. The deduced amino acid sequence of gamma-adaptin was found to be homologous to that of alpha-adaptin, with several stretches of identical amino acids or conservative substitutions in the first approximately 70 kD, and 25% identity overall. Weaker homology was seen between gamma- and beta-adaptins. Like both alpha- and beta-adaptins, gamma-adaptin has a proline and glycine-rich hinge region, dividing it into NH2- and COOH-terminal domains. A chimeric gamma-adaptin was constructed from the mouse and bovine cDNAs and transfected into Rat 1 fibroblasts. Immunofluorescence microscopy was carried out using an mAb which recognizes an epitope present on the chimera but not found on the rodent protein. The construct was found to have a distribution typical of endogenous gamma-adaptin. Using this transfection system, it should now be possible to exchange domains between alpha- and gamma-adaptins, to try to find out how adaptors are targeted to the appropriate membrane compartment of the cell, and how they recruit the appropriate receptors into the coated vesicle.

Adaptor Protein Complex gamma Subunits↗

PubMed2000

Peptide-in-groove interactions link target proteins to the beta-propeller of clathrin.

The "WD40" domain is a widespread recognition module for linking partner proteins in intracellular networks of signaling and sorting. The clathrin amino-terminal domain, which directs incorporation of cargo into coated pits, is a beta-propeller closely related in structure to WD40 modules. The crystallographically determined structures of complexes of the clathrin-terminal domain with peptides derived from two different cargo adaptors, beta-arrestin 2 and the beta-subunit of the AP-3 complex, reveal strikingly similar peptide-in-groove interactions. The two peptides in our structures contain related, five-residue motifs, which form the core of their contact with clathrin. A number of other proteins involved in endocytosis have similar "clathrin-box" motifs, and it therefore is likely that they all bind the terminal domain in the same way. We propose that a peptide-in-groove interaction is an important general mode by which beta-propellers recognize specific target proteins.

Adaptor Proteins, Vesicular Transport↗

PubMed2004

Sorting of H,K-ATPase beta-subunit in MDCK and LLC-PK cells is independent of mu 1B adaptin expression.

The cytoplasmic tail of the H,K-ATPase beta-subunit contains a putative tyrosine-based motif that directs the beta-subunit's basolateral sorting when it is expressed in Madin-Darby Canine Kidney (MDCK) cells. When expressed in LLC-PK(1) cells, however, the beta-subunit is localized to the apical membrane. Several proteins that contain tyrosine-based motifs, including the low-density lipoprotein and transferrin receptors, show a similar sorting 'defect' when expressed in LLC-PK(1) cells. For low-density lipoprotein and transferrin receptors, this behavior is due to the differential expression of the mu 1B subunit of the AP-1B clathrin adaptor complex. mu 1B is expressed by MDCK cells, but not LLC-PK(1) cells, and transfection of mu 1B into LLC-PK(1) cells restores basolateral localization of low-density lipoprotein and transferrin receptors. For the beta-subunit, however, mu B expression in LLC-PK(1) cells does not induce its basolateral expression. We found that the beta-subunit interacts with both mu 1B and mu 1A in vitro and in vivo. The capacity to participate in a mu 1B interaction therefore is not sufficient to program the beta-subunit's basolateral localization in MDCK cells. Our data suggest that the H,K-ATPase beta-subunit's basolateral sorting signal is either masked in certain epithelial cells, or requires an interaction with sorting machinery other than AP-1B for delivery to the basolateral plasma membrane.

Adaptor Protein Complex mu Subunits↗

PubMed1995

Interaction of the transcriptional activator Stat-2 with the type I interferon receptor.

Binding of interferon-alpha (IFN alpha) to the multisubunit type I IFN receptor (IFNR) induces activation of the Tyk-2 and Jak-1 kinases and tyrosine phosphorylation of multiple signaling elements, including the Stat proteins that form the ISGF3 alpha complex. Although Jak kinases are required for IFN alpha-dependent activation of Stats, the mechanisms by which Stats interact with these kinases are not known. We report that Stat-2 associates with beta s subunit of the type I IFN receptor in an interferon-dependent manner. This association is rapid, occurring within 1 min of interferon treatment of cells, and is inducible by various type I (alpha, beta, omega) but not type II (gamma) IFNs. The kinetics of Stat-2-IFNR association are similar to the kinetics of phosphorylation of Stat-2, suggesting that during its binding to the type I IFNR, Stat 2 acts as a substrate for interferon-dependent tyrosine kinase activity. These findings support the hypothesis that the type I IFNR acts as an adaptor, linking Stat proteins to Jak kinases. Interaction of Stat-2 with the beta s subunit of the type I IFNR may be a critical signaling event, required for the formation of the ISGF3 alpha complex and downstream transcription of interferon-stimulated genes.

Amino Acid Sequence↗

PubMed1993

Redistribution of clathrin-coated vesicle adaptor complexes during adipocytic differentiation of 3T3-L1 cells.

Mechanisms for intracellular retention of proteins are induced during adipocytic differentiation of 3T3-L1 cells. To investigate the potential role of clathrin lattices in these retention processes, we performed a morphological and biochemical analysis of coated vesicle components in 3T3-L1 cells. Optical sectioning and image restoration revealed a marked increase in the staining of clathrin and beta adaptins in the perinuclear region of cells with differentiation. In addition, predominance of beta (subunit of the AP-2, plasma membrane adaptor) over beta' (subunit of the AP-1, Golgi adaptor) adaptin was observed in immunoblots of clathrin-coated vesicles purified from nondifferentiated fibroblasts, and this ratio was reversed in coated vesicles purified from differentiated adipocytes. These results indicate that the relative abundance of TGN-derived clathrin lattices increases markedly during adipocytic differentiation. Subcellular fractionation indicated that cytosolic AP-1 and AP-2 adaptors comprised approximately 70% of the total cellular adaptor pool. Interestingly, neither the concentration nor the relative ratio of cytosolic AP-1 to AP-2 adaptors increased significantly during differentiation. These data suggest that the increase in TGN-derived lattices results from differentiation-induced mechanisms for enhanced assembly or stabilization of adaptors on Golgi membranes. Interestingly, double-immunofluorescence microscopy also revealed that whereas extensive colocalization between clathrin and beta adaptins occurred both in fibroblasts and adipocytes, structures stained only with anti-adaptin antibody could be detected. Taken together these results suggest that membranes coated with adaptors, but not clathrin, can exist in these cells.

3T3 Cells↗

PubMed2000

The interaction of beta-arrestin with the AP-2 adaptor is required for the clustering of beta 2-adrenergic receptor into clathrin-coated pits.

Beta-arrestins are cytosolic proteins that regulate the signaling and the internalization of G protein-coupled receptors (GPCRs). Although termination of receptor coupling requires beta-arrestin binding to agonist-activated receptors, GPCR endocytosis involves the coordinate interactions between receptor-beta-arrestin complexes and other endocytic proteins such as adaptor protein 2 (AP-2) and clathrin. Clathrin interacts with a conserved motif in the beta-arrestin C-terminal tail; however, the specific molecular determinants in beta-arrestin that bind AP-2 have not been identified. Moreover, the respective contributions of the interactions of beta-arrestin with AP-2 and clathrin toward the targeting of GPCRs to clathrin-coated vesicles have not been established. Here, we identify specific arginine residues (Arg(394) and Arg(396)) in the beta-arrestin 2 C terminus that mediate beta-arrestin binding to AP-2 and show, in vitro, that these domains in beta-arrestin 1 and 2 interact equally well with AP-2 independently of clathrin binding. We demonstrate in HEK 293 cells by fluorescence microscopy that beta(2)-adrenergic receptor-beta-arrestin complexes lacking the beta-arrestin-clathrin binding motif are still targeted to clathrin-coated pits. In marked contrast, receptor-beta-arrestin complexes lacking the beta-arrestin/AP-2 interactions are not effectively compartmentalized in punctated areas of the plasma membrane. These results reveal that the binding of a receptor-beta-arrestin complex to AP-2, not to clathrin, is necessary for the initial targeting of beta(2)-adrenergic receptor to clathrin-coated pits.

Adaptor Protein Complex alpha Subunits↗

PubMed2000

Epsin binds to clathrin by associating directly with the clathrin-terminal domain. Evidence for cooperative binding through two discrete sites.

Epsin is a recently identified protein that appears to play an important role in clathrin-mediated endocytosis. The central region of epsin 1, the so-called DPW domain, binds to the heterotetrameric AP-2 adaptor complex by associating directly with the globular appendage of the alpha subunit. We have found that this central portion of epsin 1 also associates with clathrin. The interaction with clathrin is direct and not mediated by epsin-bound AP-2. Alanine scanning mutagenesis shows that clathrin binding depends on the sequence (257)LMDLADV located within the epsin 1 DPW domain. This sequence, related to the known clathrin-binding sequences in the adaptor beta subunits, amphiphysin, and beta-arrestin, facilitates the association of epsin 1 with the terminal domain of the clathrin heavy chain. Unexpectedly, inhibiting the binding of AP-2 to the GST-epsin DPW fusion protein by progressively deleting DPW triplets but leaving the LMDLADV sequence intact, diminishes the association of clathrin in parallel with AP-2. Because the beta subunit of the AP-2 complex also contains a clathrin-binding site, optimal association with soluble clathrin appears to depend on the presence of at least two distinct clathrin-binding sites, and we show that a second clathrin-binding sequence (480)LVDLD, located within the carboxyl-terminal segment of epsin 1, also interacts with clathrin directly. The LMDLADV and LVDLD sequences act cooperatively in clathrin recruitment assays, suggesting that they bind to different sites on the clathrin-terminal domain. The evolutionary conservation of similar clathrin-binding sequences in several metazoan epsin-like molecules suggests that the ability to establish multiple protein-protein contacts within a developing clathrin-coated bud is an important aspect of epsin function.

Adaptor Protein Complex alpha Subunits↗

PubMed2005

Phospho-dependent binding of the clathrin AP2 adaptor complex to GABAA receptors regulates the efficacy of inhibitory synaptic transmission.

The efficacy of synaptic inhibition depends on the number of gamma-aminobutyric acid type A receptors (GABA(A)Rs) expressed on the cell surface of neurons. The clathrin adaptor protein 2 (AP2) complex is a critical regulator of GABA(A)R endocytosis and, hence, surface receptor number. Here, we identify a previously uncharacterized atypical AP2 binding motif conserved within the intracellular domains of all GABA(A)R beta subunit isoforms. This AP2 binding motif (KTHLRRRSSQLK in the beta3 subunit) incorporates the major sites of serine phosphorylation within receptor beta subunits, and phosphorylation within this site inhibits AP2 binding. Furthermore, by using surface plasmon resonance, we establish that a peptide (pepbeta3) corresponding to the AP2 binding motif in the GABA(A)R beta3 subunit binds to AP2 with high affinity only when dephosphorylated. Moreover, the pepbeta3 peptide, but not its phosphorylated equivalent (pepbeta3-phos), enhanced the amplitude of miniature inhibitory synaptic current and whole cell GABA(A)R current. These effects of pepbeta3 on GABA(A)R current were occluded by inhibitors of dynamin-dependent endocytosis supporting an action of pepbeta3 on GABA(A)R endocytosis. Therefore phospho-dependent regulation of AP2 binding to GABA(A)Rs provides a mechanism to specify receptor cell surface number and the efficacy of inhibitory synaptic transmission.

Adaptor Protein Complex 2↗