Phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] 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 2-subunit in vitro and in native protein extracts. Endocytic cargo protein binding to 2 leads to a potent stimulation of PIPK activity. These data thus identify a positive feedback loop consisting of endocytic cargo proteins, AP-2 , and PIPK type I which may provide a specific pool of PI(4,5)P 2 dedicated to clathrin͞AP-2-dependent receptor internalization.phosphoinositides ͉ sorting motifs ͉ clathrin ͉ endocytosis P hosphoinositides have pleiotropic functions in cell physiology including the regulation of signal transduction, membrane traffic (1), and the organization of the actin cytoskeleton (2). Phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P 2 ], a phosphoinositide concentrated at the plasma membrane, is required for the generation of diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP 3 ), remodelling of the actin cytoskeleton (2, 3), clathrin-dependent (4) and -independent pathways of cell entry (5), the exo-endocytic cycling of synaptic and neurosecretory vesicles (6-8), and serves as a substrate for the synthesis of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P 3 ]. Biochemical, genetic, and cell biological data suggest that PI(4,5)P 2 plays an essential role in clathrin-dependent endocytosis of plasma membrane proteins including nutrient (4) and growth factor receptors, postsynaptic ion channels, as well as synaptic vesicle proteins (9-11). Endocytic adaptor proteins like the heterotetrameric AP-2 complex (via its ␣ and 2 subunits) (12, 13), AP180͞CALM, epsin (14), Dab2, and HIP1͞1R as well as the large GTPase dynamin (15) all bind directly to PI(4,5)P 2 . In addition to serving as a membrane attachment site for endocytic proteins, PI(4,5)P 2 also plays an active role by allosterically eliciting a conformational change within AP-2 that is required for its interaction with sorting signals of transmembrane proteins (16) and to stably associate with the plasmalemma (17). Impairment of PI(4,5)P 2 hydrolysis by deletion of the phosphoinositide-phosphatase synaptojanin (18-20) results in the accumulation of clathrin-coated pits and vesicles, suggesting that the membrane concentration of PI(4,5)P 2 controls the stability of endocytic clathrin coats.Three isoforms of type I phosphatidylinositol 4-phosphate 5-kinase (PIPK) have been identified in mammals (␣, ␤, ␥), which generate PI(4,5)P 2 by phosporylating PI(4)P at the D-5 position of the inositol ring (21,22). Although isoform-specific functions of type I PIPKs have been shown to contribute to distinct cellular processes such as formation of focal adhesions (23,24), at present it is largely unclear how the s...

The regulation of the number of ␥2-subunit-containing GABAA receptors (GABAARs) present at synapses is critical for correct synaptic inhibition and animal behavior. This regulation occurs, in part, by the controlled removal of receptors from the membrane in clathrin-coated vesicles, but it remains unclear how clathrin recruitment to surface ␥2-subunit-containing GABAARs is regulated. Here, we identify a ␥2-subunit-specific Yxx-type-binding motif for the clathrin adaptor protein, AP2, which is located within a site for ␥2-subunit tyrosine phosphorylation. Blocking GABAAR-AP2 interactions via this motif increases synaptic responses within minutes. Crystallographic and biochemical studies reveal that phosphorylation of the Yxx motif inhibits AP2 binding, leading to increased surface receptor number. In addition, the crystal structure provides an explanation for the high affinity of this motif for AP2 and suggests that ␥2-subunit-containing heteromeric GABAARs may be internalized as dimers or multimers. These data define a mechanism for tyrosine kinase regulation of GABAAR surface levels and synaptic inhibition.endocytosis ͉ phosphorylation ͉ structure ͉ synaptic transmission ͉ tyrosine kinase T he GABA A receptor (GABA A R), a ligand-gated ion channel, mediates the majority of fast inhibitory synaptic transmission in the mammalian CNS. Identifying the molecular mechanisms important for regulating these receptors is essential for our understanding of how synaptic inhibition and neuronal excitability are controlled. GABA A Rs are pentameric heterooligomers assembled from seven subunit classes (␣1-6, ␤1-3, ␥1-3, ␦, , , and ). It is generally assumed that the majority of GABA A Rs in the brain are assembled from at least 2 ␣-, 2 ␤-, and 1 ␥2-subunits (1). The GABA A R ␥2-subunit confers important pharmacological, functional, and membrane-trafficking properties to GABA A Rs, including benzodiazepine sensitivity, the selective targeting of GABA A Rs to inhibitory postsynaptic domains, and correct animal behavior (2, 3). The phosphorylation of tyrosine (Y) residues within the ␥2-subunit intracellular domain (ICD) at Y 365 and Y 367 increases GABA A R function. However, the mechanisms that underlie this regulation remain unclear (4, 5). Furthermore, it has recently been demonstrated that altered membrane trafficking of ␥2-subunit-containing GABA A Rs may underlie certain pathological conditions, such as the generation of pharmacoresistance and self-sustaining seizures in status epilepticus and the increased excitotoxicity in ischemia (6-8). Currently, little is known regarding the molecular mechanisms and protein interactions that underlie ␥2-subunit-dependent regulation of receptor membrane trafficking under normal or pathological conditions.A potential mechanism to regulate synaptic inhibition is to alter the number of surface and synaptic GABA A Rs. This surface receptor number can be determined, in part, by receptor endocytosis and the interaction with the clathrin adaptor protein (AP2) complex (9, 10). The AP2 complex...

Endosomes and endosomal vesicles (EVs) rapidly move along cytoskeletal filaments allowing them to exchange proteins and lipids between different endosomal compartments, lysosomes, the trans-Golgi network (TGN), and the plasma membrane. The precise mechanisms that connect membrane traffic between the TGN and perinuclear endosomal compartments with motor-protein driven transport have largely remained elusive. Here we show that Gadkin (also termed ␥-BAR), a peripheral membrane protein localized to the TGN and to TGN-derived EVs, directly associates with the clathrin adaptor AP-1 and with the motor protein kinesin KIF5, thereby potentially regulating EV dynamics. Gadkin overexpression induced the dispersion of transferrin (Tf)-and Rab4-positive EVs to the cell periphery, whereas KIF5B-depleted cells displayed a perinuclear concentration. Functional experiments suggest that the role of Gadkin as a regulator of endosomal membrane traffic critically depends on complex formation with both AP-1 and KIF5. Our data thus provide a direct molecular link between TGN-derived EVs and the microtubule-based cytoskeleton.motor-protein driven transport ͉ clathrin adaptor AP-1 ͉ endosomal vesicles ͉ recycling T he endosomal system comprises a mosaic of dynamically interconnected organelles that fulfills a variety of important cell physiological functions ranging from the uptake, recycling, and degradation of nutrients, signaling molecules and cell surface receptors to the regulation of cell migration, differentiation, and morphogenesis (1-3). The endocytic pathway also intersects with the biosynthetic delivery of lysosomal enzymes at several stations, most notably at the trans-Golgi network (TGN)/ endosomal boundary (1). Endosomes and TGN-or endosomederived vesicles exhibit characteristic distribution patterns with Rab4-positive sorting endosomal vesicles (EVs) and tubular recycling endosomes (REs) typically concentrated in the pericentrosomal area (4). Early endosomes, by contrast, appear dispersed throughout the cytoplasm (5). Function and dynamics of endosomes and EVs requires a so far ill-defined interplay between organellar sorting adaptors, the cytoskeleton (6) and molecular motors (7). Here we show that ␥-BAR, a recently described accessory factor of the clathrin adaptor complex AP-1 at the TGN/endosomal interface (8), modulates the dynamics of transferrin (Tf)-and Rab4-positive EVs by directly associating with AP-1 and kinesin KIF5. Because ␥-BAR does not harbor a curvature-sensing BIN/amphiphysin/Rvs167 (BAR) domain, we refer to this protein as Gadkin, for ␥1-adaptin and kinesin interactor. The AP-1/Gadkin/KIF5 complex identified here provides a hitherto unknown molecular link between TGN-derived EVs and the microtubule-based cytoskeleton. Our work also suggests a surprising complexity of endosomal membrane dynamics and its integration with cargo sorting. Results Gadkin Localizes to the TGN and to Perinuclear EVs and Regulates EVPositioning. Gadkin has originally been identified as an AP-1 binding protein localized to the T...

␣-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors undergo constitutive and ligand-induced internalization that requires dynamin and the clathrin adaptor complex AP-2. We report here that an atypical basic motif within the cytoplasmic tails of AMPA-type glutamate receptors directly associates with 2-adaptin by a mechanism similar to the recognition of the presynaptic vesicle protein synaptotagmin 1 by AP-2. A synaptotagmin 1-derived AP-2 binding peptide competes the interaction of the AMPA receptor subunit GluR2 with AP-2 and increases the number of surface active glutamate receptors in living neurons. Moreover, fusion of the GluR2-derived tail peptide with a synaptotagmin 1 truncation mutant restores clathrin/AP-2-dependent internalization of the chimeric reporter protein. These data suggest that common mechanisms regulate AP-2-dependent internalization of pre-and postsynaptic membrane proteins.endocytosis ͉ postsynaptic ͉ sorting signal ͉ synaptic plasticity F ast neurotransmission at excitatory synapses is mediated by heterotetrameric ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors composed of combinations of four subunits (GluR1-4). AMPA receptors interact with different factors including the transmembrane protein stargazin (1), PDZ proteins GRIP1/ABP, SAP97, and PICK1, and NSF (2). Accumulating evidence suggests that rapid changes in functional postsynaptic AMPA receptor numbers are important means of controlling synaptic efficacy (2-5). AMPA receptors undergo constitutive and regulated clathrin-and dynamin-dependent endocytosis via distinct AMPA-or NMDAinduced signaling cascades (reviewed in refs. 2-5). How exactly AMPA receptor cargo is targeted for clathrin-mediated internalization remains an open question. One possibility is that AMPA receptors are recognized by endocytic adaptor proteins such as the clathrin adaptor complex AP-2, a major endocytic protein interaction hub (6-8). NMDA-induced AMPA receptor internalization can be blocked by overexpression of a GluR2 cytoplasmic tail (CT) peptide (pep2r) or by mutating the putative AP-2 binding motif within the GluR2 CT. Infusion of hippocampal CA1 neurons with the putative AP-2-blocking peptide prevents induction of long-term depression (LTD), suggesting that the association of GluR2 with AP-2 may be an important determinant for NMDA-induced LTD (7). Whether AP-2 directly binds to GluR2 CTs and via which of its four subunits is unknown.Here we have identified the molecular determinants responsible for binding of AP-2 to the CTs of AMPA-type glutamate receptors. We demonstrate that the 2 subunit of AP-2 interacts directly and with nanomolar affinity with a basic motif found in CTs of GluR1-3 and the presynaptic vesicle protein synaptotagmin 1. Our data thus suggest that common mechanisms regulate AP-2-dependent internalization of pre-and postsynaptic membrane proteins.