The class II phosphoinositide 3-kinase C2alpha is activated by clathrin and regulates clathrin-mediated membrane trafficking

Phosphoinositides play key regulatory roles in vesicular transport pathways in eukaryotic cells. Clathrin-mediated membrane trafficking has been shown to require phosphoinositides, but little is known about the enzyme(s) responsible for their formation. Here we report that clathrin functions as an adaptor for the class II PI 3-kinase C2alpha (PI3K-C2alpha), binding to its N-terminal region and stimulating its catalytic activity, especially toward phosphorylated inositide substrates. Further, we show that endogenous PI3K-C2alpha is localized in coated pits and that exogenous expression affects clathrin-mediated endocytosis and sorting in the trans-Golgi network. These findings provide a mechanistic basis for localized inositide generation at sites of clathrin-coated bud formation, which, with recruitment of inositide binding proteins and subsequent synaptojanin-mediated phosphoinositide hydrolysis, may regulate coated vesicle formation and uncoating.     

Phosphoinositide 3-kinase C2alpha links clathrin to microtubule-dependent movement

Phosphoinositide 3-kinase C2alpha (PI3K-C2alpha) is a type II PI-3-kinase that has been implicated in several important membrane transport and signaling processes. We previously found that overexpression of PI3K-C2alpha inhibits clathrin-mediated membrane trafficking and induces proliferation of novel clathrin-coated structures within the cytoplasm. Using fluorescently tagged fusions of PI3K-C2alpha and clathrin, we explored the behavior of these structures in intact cells. Both proteins are present in the structures, and using rapid image acquisition and fluorescence photoactivation probes, we find that they exhibit localized, rapid mobility (5-20 microm/s). The movement is micro-tubule-based as revealed by use of inhibitors, and PI3K-C2alpha accumulates on microtubules rapidly and reversibly following cytoplasmic acidification, which also blocks movement. Dynactin mediates the movement of these clathrin-PI3K-C2alpha structures, since disruption of dynactin function by overexpression of its p50 subunit also inhibits movement. Finally, immunoprecipitation experiments reveal an interaction between endogenous PI3K-C2alpha and dynactin subunits. Together, these results reveal a molecular linkage between PI3K-C2alpha and the microtubule motor machinery, with implications for membrane trafficking in intact cells.     

The class II phosphoinositide 3-kinase PI3K-C2alpha is concentrated in the trans-Golgi network and present in clathrin-coated vesicles

In recent years, a large family of phosphoinositide 3-kinase (PI3K) isozymes has been characterized and cloned. Several of these PI3K enzymes have overlapping tissue distributions and it remains unclear if and how their 3-phosphoinositide products elicit differential, intracellular effects. One possibility is that the PI3K enzymes display a restricted distribution within the cell to produce their 3-phospholipid products in specific, subcellular compartments. In the present study we characterize the subcellular distribution of the novel class II PI3K isozyme PI3K-C2alpha in several mammalian cell types. Differential centrifugation of COS-1 and U937 cells together with Western blot analysis demonstrated that PI3K-C2alpha is constitutively associated with phospholipid membranes. Centrifugation of rat brain homogenates and Western blotting revealed that in contrast to the class IA PI3K enzymes, PI3K-C2alpha could be co-purified with a population of clathrin-coated vesicles (CCVs). Furthermore, a PI3K activity refractory to wortmannin treatment was detected in CCV preparations consistent with the presence of the PI3K-C2alpha isozyme. These biochemical observations were supported by immunofluorescence analysis that revealed PI3K-C2alpha to have a punctate distribution and an enrichment of immunoreactivity within a perinuclear site consistent with its presence in the endoplasmic reticulum or Golgi apparatus. Dual label immunofluorescence demonstrated that in this region, the distribution of PI3K-C2alpha closely paralleled that of gamma-adaptin, a component of the AP-1 adaptor that is present in the trans-Golgi and the trans-Golgi network (TGN) resident protein TGN-46. Neither the phospholipid association nor the subcellular localization of PI3K-C2alpha was dependent upon either its COOH-terminal PX or C2 domains. Mutants lacking these domains demonstrated a similar distribution to the wild type enzyme when expressed as recombinant proteins. Treatment of cells with brefeldin A disrupted the perinuclear staining pattern of both PI3K-C2alpha and the AP-1 complex demonstrating that the localization of both molecules at the TGN is dependent upon ADP-ribosylation factor GTPase activity.     

Class II phosphoinositide 3-kinases are downstream targets of activated polypeptide growth factor receptors

The class II phosphoinositide 3-kinases (PI3K) PI3K-C2alpha and PI3K-C2beta are two recently identified members of the large PI3K family. Both enzymes are characterized by the presence of a C2 domain at the carboxy terminus and, in vitro, preferentially utilize phosphatidylinositol and phosphatidylinositol 4-monophosphate as lipid substrates. Little is understood about how the catalytic activity of either enzyme is regulated in vivo. In this study, we demonstrate that PI3K-C2alpha and PI3K-C2beta represent two downstream targets of the activated epidermal growth factor (EGF) receptor in human carcinoma-derived A431 cells. Stimulation of quiescent cultures with EGF resulted in the rapid recruitment of both enzymes to a phosphotyrosine signaling complex that contained the EGF receptor and Erb-B2. Ligand addition also induced the appearance of a second, more slowly migrating band of PI3K-C2alpha and PI3K-C2beta immunoreactivity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Since both PI3K enzymes can utilize Ca(2+) as an essential divalent cation in lipid kinase assays and since the catalytic activity of PI3K-C2alpha is refractory to the inhibitor wortmannin, these properties were used to confirm the recruitment of each PI3K isozyme to the activated EGF receptor complex. To examine this interaction in greater detail, PI3K-C2beta was chosen for further investigation. EGF and platelet-derived growth factor also stimulated the association of PI3K-C2beta with their respective receptors in other cells, including epithelial cells and fibroblasts. The use of EGF receptor mutants and phosphopeptides derived from the EGF receptor and Erb-B2 demonstrated that the interaction with recombinant PI3K-C2beta occurs through E(p)YL/I phosphotyrosine motifs. The N-terminal region of PI3K-C2beta was found to selectively interact with the EGF receptor in vitro, suggesting that it mediates the association of this PI3K with the receptor. However, the mechanism of this interaction remains unclear. We conclude that class II PI3K enzymes may contribute to the generation of 3' phosphoinositides following the activation of polypeptide growth factor receptors in vivo and thus mediate certain aspects of their biological activity.     

Adaptor and clathrin exchange at the plasma membrane and trans-Golgi network

We previously demonstrated, using fluorescence recovery after photobleaching, that clathrin in clathrin-coated pits at the plasma membrane exchanges with free clathrin in the cytosol, suggesting that clathrin-coated pits are dynamic structures. We now investigated whether clathrin at the trans-Golgi network as well as the clathrin adaptors AP2 and AP1 in clathrin-coated pits at the plasma membrane and trans-Golgi network, respectively, also exchange with free proteins in the cytosol. We found that when the budding of clathrin-coated vesicle is blocked without significantly affecting the structure of clathrin-coated pits, both clathrin and AP2 at the plasma membrane and clathrin and AP1 at the trans-Golgi network exchange rapidly with free proteins in the cytosol. In contrast, when budding of clathrin-coated vesicles was blocked at the plasma membrane or trans-Golgi network by hypertonic sucrose or K(+) depletion, conditions that markedly affect the structure of clathrin-coated pits, clathrin exchange was blocked but AP2 at the plasma membrane and both AP1 and the GGA1 adaptor at the trans-Golgi network continue to rapidly exchange. We conclude that clathrin-coated pits are dynamic structures with rapid exchange of both clathrin and adaptors and that adaptors are able to exchange independently of clathrin when clathrin exchange is blocked.     

Phosphatidylinositol 3-kinase C2alpha is essential for ATP-dependent priming of neurosecretory granule exocytosis

Neurotransmitter release and hormonal secretion are highly regulated processes culminating in the calcium-dependent fusion of secretory vesicles with the plasma membrane. Here, we have identified a role for phosphatidylinositol 3-kinase C2alpha (PI3K-C2alpha) and its main catalytic product, PtdIns3P, in regulated exocytosis. In neuroendocrine cells, PI3K-C2alpha is present on a subpopulation of mature secretory granules. Impairment of PI3K-C2alpha function specifically inhibits the ATP-dependent priming phase of exocytosis. Overexpression of wild-type PI3K-C2alpha enhanced secretion, whereas transfection of PC12 cells with a catalytically inactive PI3K-C2alpha mutant or a 2xFYVE domain sequestering PtdIns3P abolished secretion. Based on these results, we propose that production of PtdIns3P by PI3K-C2alpha is required for acquisition of fusion competence in neurosecretion.     

Ca2+-regulated pool of phosphatidylinositol-3-phosphate produced by phosphatidylinositol 3-kinase C2alpha on neurosecretory vesicles

Phosphatidylinositol-3-phosphate [PtdIns(3)P] is a key player in early endosomal trafficking and is mainly produced by class III phosphatidylinositol 3-kinase (PI3K). In neurosecretory cells, class II PI3K-C2alpha and its lipid product PtdIns(3)P have recently been shown to play a critical role during neuroexocytosis, suggesting that two distinct pools of PtdIns(3)P might coexist in these cells. However, the precise characterization of this additional pool of PtdIns(3)P remains to be established. Using a selective PtdIns(3)P probe, we have identified a novel PtdIns(3)P-positive pool localized on secretory vesicles, sensitive to PI3K-C2alpha knockdown and relatively resistant to wortmannin treatment. In neurosecretory cells, stimulation of exocytosis promoted a transient albeit large increase in PtdIns(3)P production localized on secretory vesicles sensitive to PI3K-C2alpha knockdown and expression of PI3K-C2alpha catalytically inactive mutant. Using purified chromaffin granules, we found that PtdIns(3)P production is controlled by Ca(2+). We confirmed that PtdIns(3)P production from recombinantly expressed PI3K-C2alpha is indeed regulated by Ca(2+). We provide evidence that a dynamic pool of PtdIns(3)P synthesized by PI3K-C2alpha occurs on secretory vesicles in neurosecretory cells, demonstrating that the activity of a member of the PI3K family is regulated by Ca(2+) in vitro and in living neurosecretory cells.     

The N-terminus of phosphoinositide 3-kinase-C2beta regulates lipid kinase activity and binding to clathrin

The class II phosphoinositide 3-kinase (PI3K)-C2beta is recruited to polypeptide growth factor receptors following ligand stimulation. In contrast to the class I A p85/p110 heterodimers, this interaction is dependent upon proline residues present within the N-terminal sequence of the 3-phosphoinositide kinase. However, the mechanism by which PI3K-C2beta catalytic activity is regulated currently remains unknown. In many tumours, increased expression of ErbB receptors confers a poor prognosis. We demonstrate that increased expression of EGFR enhanced its association with PI3K-C2beta following stimulation with EGF. Deletion of the first proline rich region within the N-terminus precluded recruitment of PI3K-C2beta to activated EGFR. Although deletion of the first proline rich motif also rendered the enzyme catalytically inactive, further deletions (residues 1-148 and 1-261) that removed the second and third proline rich motifs increased kinase activity. These data confirm a regulatory role for the N-terminus of class II PI3K enzymes suggesting that catalytic activity is regulated by factors that associate with this region during recruitment to activated growth factor receptors. Using an N-terminal PI3K-C2beta-GST fusion protein, clathrin heavy chain was affinity purified from A431 cell lysates. Association of PI3K-C2beta with clathrin was confirmed by co-immunoprecipitation from cell lysates while intracellular co-localisation of PI3K-C2beta and clathrin was confirmed by confocal microscopy. Our findings demonstrate for the first time that the PI3K-C2beta isoform associates with clathrin and thus provides a link between receptor mediated intracellular signalling and clathrin coated vesicle transport.     

Suppression of the alpha-isoform of class II phosphoinositide 3-kinase gene expression leads to apoptotic cell death

Phosphoinositide 3-kinases (PI3Ks) have known to be key enzymes activating intracellular signaling molecules when a number of growth factors bind to their cell surface receptors. PI3Ks are divided into three classes (I, II, and III) and enzymes of each class have different tissue-specificities and physiological functions. Class II PI3Ks consist of three isoforms (alpha,beta,gamma). Although the alpha-isoform (PI3K-C2alpha) is considered ubiquitous and preferentially activated by insulin rather than the beta-isoform, the physiological significance of PI3K-C2alpha is poorly understood. The present study aimed to determine whether PI3K-C2alpha is associated with the suppression of apoptotic cell death. Different sense- and antisense oligonucleotides (ODNs) were synthesized based on the sequence of C2 domain of PI3K-C2alpha gene. Transfection of CHO-IR cells with two different antisense ODNs clearly reduced the protein content as well as mRNA levels of PI3K-C2alpha whereas neither the nonspecific mock- nor sense ODNs affected. The decrease of PI3K-C2alpha gene expression was paralleled by cellular changes indicating apoptotic cell death such as nuclear condensation, formation of apoptotic bodies, and DNA fragmentation. PI3K-C2alpha mRNA levels were also reduced when cells were incubated in growth factor-deficient medium. Supplementing growth factors (serum or insulin) into medium lead to an increase of PI3K-C2alpha mRNA levels. This finding strongly suggests that PI3K-C2alpha is a crucial survival factor.     

Insulin activates the alpha isoform of class II phosphoinositide 3-kinase.

The novel class II phosphoinositide (PI) 3-kinases are characterized by the presence of a C-terminal C2 domain, but little is known about their regulation. We find insulin causes a rapid 2-3-fold increase in the activity of PI 3-kinase C2alpha (PI3K-C2alpha) in CHO-IR cells, 3T3-L1 adipocytes, and fully differentiated L5L6 myotubes. No insulin-induced activation of PI3K-C2alpha was observed in cell types known to have low responsiveness to insulin including HEK 293 cells, 3T3-L1 preadipocytes, and undifferentiated L5L6 myoblasts. The mechanism of activation of PI3K-C2alpha by insulin differs from that of class Ia PI 3-kinases in that insulin stimulation did not cause PI3K-C2alpha to associate with IRS-1 or insulin receptor. PI3K-C2alpha existed as a doublet, and insulin stimulation caused a redistribution from the lower molecular weight band to the higher molecular weight band, suggesting phosphorylation-induced bandshift. Consistent with this, in vitro phosphatase treatment reduced the intensity of the upper band back to that seen in unstimulated cells. This suggests that insulin-induced phosphorylation could play a role in regulation of the activity of PI3K-C2alpha. The finding that insulin activates PI3K-C2alpha in cell types known to possess a wide range of responses to insulin suggests that PI3K-C2alpha is a novel component of insulin-stimulated signaling cascades.     

Structural and membrane binding analysis of the Phox homology domain of phosphoinositide 3-kinase-C2alpha

Phox homology (PX) domains, which have been identified in a variety of proteins involved in cell signaling and membrane trafficking, have been shown to interact with phosphoinositides (PIs) with different affinities and specificities. To elucidate the structural origin of diverse PI specificities of PX domains, we determined the crystal structure of the PX domain from phosphoinositide 3-kinase C2alpha (PI3K-C2alpha), which binds phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). To delineate the mechanism by which this PX domain interacts with membranes, we measured the membrane binding of the wild type domain and mutants by surface plasmon resonance and monolayer techniques. This PX domain contains a signature PI-binding site that is optimized for PtdIns(4,5)P(2) binding. The membrane binding of the PX domain is initiated by nonspecific electrostatic interactions followed by the membrane penetration of hydrophobic residues. Membrane penetration is specifically enhanced by PtdIns(4,5)P(2). Furthermore, the PX domain displayed significantly higher PtdIns(4,5)P(2) membrane affinity and specificity when compared with the PI3K-C2alpha C2 domain, demonstrating that high affinity PtdIns(4,5)P(2) binding was facilitated by the PX domain in full-length PI3K-C2alpha. Together, these studies provide new structural insight into the diverse PI specificities of PX domains and elucidate the mechanism by which the PI3K-C2alpha PX domain interacts with PtdIns(4,5)P(2)-containing membranes and thereby mediates the membrane recruitment of PI3K-C2alpha.     

Role of cyclin G-associated kinase in uncoating clathrin-coated vesicles from non-neuronal cells

Auxilin is a brain-specific DnaJ homolog that is required for Hsc70 to dissociate clathrin from bovine brain clathrin-coated vesicles. However, Hsc70 is also involved in uncoating clathrin-coated vesicles formed at the plasma membrane of non-neuronal cells suggesting that an auxilin homolog may be required for uncoating in these cells. One candidate is cyclin G-associated kinase (GAK), a 150-kDa protein expressed ubiquitously in various tissues. GAK has a C-terminal domain with high sequence similarity to auxilin; like auxilin this C-terminal domain consists of three subdomains, an N-terminal tensin-like domain, a clathrin-binding domain, and a C-terminal J-domain. Western blot analysis shows that GAK is present in rat liver, bovine testes, and bovine brain clathrin-coated vesicles. More importantly, liver clathrin-coated vesicles, which contain GAK but not auxilin, are uncoated by Hsc70, suggesting that GAK acts as an auxilin homolog in non-neuronal cells. In support of this view, the clathrin-binding domain of GAK alone induces clathrin polymerization into baskets and the combined clathrin-binding domain and J-domain of GAK supports uncoating of AP180-clathrin baskets by Hsc70 at pH 7 and induces Hsc70 binding to clathrin baskets at pH 6. Immunolocalization studies suggest that GAK is a cytosolic protein that is concentrated in the perinuclear region; it appears to be highly associated with the trans-Golgi where the budding of clathrin-coated vesicles occurs. We propose that GAK is a required cofactor for the uncoating of clathrin-coated vesicles by Hsc70 in non-neuronal cells.     

The α-isoform of class II phosphoinositide 3-kinase is more effectively activated by insulin receptors than IGF receptors, and activation requires receptor NPEY motifs

Little is known about the physiological role and mechanism of activation of class II phosphoinositide 3-kinases (PI3Ks), although it has been shown that the PI3K-C2alpha isoform is activated by insulin. Using chimaeric receptor constructs which can be activated independently of endogenous receptors in transfected cells, we found that PI3K-C2alpha activity was stimulated to a greater extent by insulin receptors than IGF receptors in 3T3-L1 adipocytes. Activation of PI3K-C2alpha required an intact NPEY motif in the receptor juxtamembrane domain. We conclude that PI3K-C2alpha is a candidate for participation in insulin-specific intracellular signalling.     

Clint: a novel clathrin-binding ENTH-domain protein at the Golgi

We have characterized a novel clathrin-binding 68-kDa epsin N-terminal homology domain (ENTH-domain) protein that we name clathrin interacting protein localized in the trans-Golgi region (Clint). It localizes predominantly to the Golgi region of epithelial cells as well as to more peripheral vesicular structures. Clint colocalizes with AP-1 and clathrin only in the perinuclear area. Recombinantly expressed Clint interacts directly with the gamma-appendage domain of AP-1, with the clathrin N-terminal domain through the peptide motif (423)LFDLM, with the gamma-adaptin ear homology domain of Golgi-localizing, gamma-adaptin ear homology domain 2, with the appendage domain of beta2-adaptin and to a lesser extent with the appendage domain of alpha-adaptin. Moreover, the Clint ENTH-domain asssociates with phosphoinositide-containing liposomes. A significant amount of Clint copurifies with rat liver clathrin-coated vesicles. In rat kidney it is preferentially expressed in the apical region of epithelial cells that line the collecting duct. Clathrin and Clint also colocalize in the apical region of enterocytes along the villi of the small intestine. Apart from the ENTH-domain Clint has no similarities with the epsins AP180/CALM or Hip1/1R. A notable feature of Clint is a carboxyl-terminal methionine-rich domain (Met(427)-Met(605)), which contains >17% methionine. Our results suggest that Clint might participate in the formation of clathrin-coated vesicles at the level of the trans-Golgi network and remains associated with the vesicles longer than clathrin and adaptors.     

The role of phosphoinositide 3-kinase C2alpha in insulin signaling

The members of the class II phosphoinositide 3-kinase (PI3K) family can be activated by several stimuli, indicating that these enzymes can regulate many intracellular processes. Nevertheless, to date, there has been no definitive identification of their in vivo product, their mechanism(s) of activation, or their precise intracellular roles. By metabolic labeling, we here identify phosphatidylinositol 3-phosphate as the sole in vivo product of the insulin-dependent activation of PI3K-C2alpha, confirming the emerging role of such a phosphoinositide in signaling. We demonstrate that activation of PI3K-C2alpha involves its recruitment to the plasma membrane and that activation is mediated by the GTPase TC10. This is the first report showing a membrane targeting-mediated mechanism of activation for PI3K-C2alpha and that a small GTP-binding protein can activate a class II PI3K isoform. We also demonstrate that PI3K-C2alpha contributes to maximal insulin-induced translocation of the glucose transporter GLUT4 to the plasma membrane and subsequent glucose uptake, definitely assessing the role of this enzyme in insulin signaling.     

Interaction of furin in immature secretory granules from neuroendocrine cells with the AP-1 adaptor complex is modulated by casein kinase II phosphorylation.

The composition of secretory granules in neuroendocrine and endocrine cells is determined by two sorting events; the first in the trans-Golgi complex (TGN), the second in the immature secretory granule (ISG). Sorting from the ISG, which may be mediated by the AP-1 type adaptor complex and clathrin-coated vesicles, occurs during ISG maturation. Here we show that furin, a ubiquitously expressed, TGN/endosomal membrane endoprotease, is present in the regulated pathway of neuroendocrine cells where it is found in ISGs. By contrast, TGN38, a membrane protein that is also routed through the TGN/endosomal system does not enter ISGs. Furin, however, is excluded from mature secretory granules, suggesting that the endoprotease is retrieved from the clathrin-coated ISGs. Consistent with this, we show that the furin cytoplasmic domain interacts with AP-1, a component of the TGN/ISG-localized clathrin sorting machinery. Interaction between AP-1 and furin is dependent on phosphorylation of the enzyme's cytoplasmic domain by casein kinase II. Finally, in support of a requirement for the phosphorylation-dependent association of furin with AP-1, expression of furin mutants that mimic either the phosphorylated or unphosphorylated forms of the endoprotease in AtT-20 cells demonstrates that the integrity of the CKII sites is necessary for removal of furin from the regulated pathway.     

The Class II Phosphatidylinositol 3 kinase C2β Is Required for the Activation of the K+ Channel KCa3.1 and CD4 T-Cells

The Ca²⁺-activated K⁺ channel KCa3.1 is required for Ca²⁺ influx and the subsequent activation of T-cells. We previously showed that nucleoside diphosphate kinase beta (NDPK-B), a mammalian histidine kinase, directly phosphorylates and activates KCa3.1 and is required for the activation of human CD4 T lymphocytes. We now show that the class II phosphatidylinositol 3 kinase C2β (PI3K-C2β) is activated by the T-cell receptor (TCR) and functions upstream of NDPK-B to activate KCa3.1 channel activity. Decreased expression of PI3K-C2β by siRNA in human CD4 T-cells resulted in inhibition of KCa3.1 channel activity. The inhibition was due to decreased phosphatidylinositol 3-phosphate [PI(3)P] because dialyzing PI3K-C2β siRNA-treated T-cells with PI(3)P rescued KCa3.1 channel activity. Moreover, overexpression of PI3K-C2β in KCa3.1-transfected Jurkat T-cells led to increased TCR-stimulated activation of KCa3.1 and Ca²⁺ influx, whereas silencing of PI3K-C2β inhibited both responses. Using total internal reflection fluorescence microscopy and planar lipid bilayers, we found that PI3K-C2β colocalized with Zap70 and the TCR in peripheral microclusters in the immunological synapse. This is the first demonstration that a class II PI3K plays a critical role in T-cell activation.     

Phosphoinositide 3-kinase regulates beta2-adrenergic receptor endocytosis by AP-2 recruitment to the receptor/beta-arrestin complex

Internalization of beta-adrenergic receptors (betaARs) occurs by the sequential binding of beta-arrestin, the clathrin adaptor AP-2, and clathrin. D-3 phosphoinositides, generated by the action of phosphoinositide 3-kinase (PI3K) may regulate the endocytic process; however, the precise molecular mechanism is unknown. Here we demonstrate that betaARKinase1 directly interacts with the PIK domain of PI3K to form a cytosolic complex. Overexpression of the PIK domain displaces endogenous PI3K from betaARK1 and prevents betaARK1-mediated translocation of PI3K to activated beta2ARs. Furthermore, disruption of the betaARK1/PI3K interaction inhibits agonist-stimulated AP-2 adaptor protein recruitment to the beta2AR and receptor endocytosis without affecting the internalization of other clathrin dependent processes such as internalization of the transferrin receptor. In contrast, AP-2 recruitment is enhanced in the presence of D-3 phospholipids, and receptor internalization is blocked in presence of the specific phosphatidylinositol-3,4,5-trisphosphate lipid phosphatase PTEN. These findings provide a molecular mechanism for the agonist-dependent recruitment of PI3K to betaARs, and support a role for the localized generation of D-3 phosphoinositides in regulating the recruitment of the receptor/cargo to clathrin-coated pits.