Spatial regulation of Galphai protein signaling in clathrin-coated membrane microdomains containing GAIP.
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Regulators of G-protein signaling (RGS) proteins are GTPase-activating proteins (GAPs) that bind to Galpha subunits and attenuate G protein signaling, but where these events occur in the cell is not yet established. Here we investigated, by immunofluorescence labeling and deconvolution analysis, the site at which endogenous Galpha-interacting protein (GAIP) (RGS19) binds to Galphai3-YFP and its fate after activation of delta-opioid receptor (DOR). In the absence of agonist, GAIP is spatially segregated from Galphai3 and DOR in clathrin-coated domains (CCPs) of the cell membrane (PM), whereas Galphai3-YPF and DOR are located in non-clathrin-coated microdomains of the PM. Upon addition of agonist, Galphai3 partially colocalizes with GAIP in CCPs at the PM. When endocytosis is blocked by expression of a dynamin mutant [dyn(K44A)], there is a striking overlap in the distribution of DOR and Galphai3-YFP with GAIP in CCPs. Moreover, Galphai3-YFP and GAIP form a coprecipitable complex. Our results support a model whereby, after agonist addition, DOR and Galphai3 move together into CCPs where Galphai3 and GAIP meet and turn off G protein signaling. Subsequently, Galphai3 returns to non-clathrin-coated microdomains of the PM, GAIP remains stably associated with CCPs, and DOR is internalized via clathrin-coated vesicles. This constitutes a novel mechanism for regulation of Galpha signaling through spatial segregation of a GAP in clathrin-coated pits. (+info)
Cooperative interactions of simian immunodeficiency virus Nef, AP-2, and CD3-zeta mediate the selective induction of T-cell receptor-CD3 endocytosis.
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The Nef proteins of human immunodeficiency virus and simian immunodeficiency virus (SIV) bind the AP-1 and AP-2 clathrin adaptors to downmodulate the expression of CD4 and CD28 by recruiting them to sites of AP-2 clathrin-dependent endocytosis. Additionally, SIV Nef directly binds the CD3-zeta subunit of the CD3 complex and downmodulates the T-cell receptor (TCR)-CD3 complex. We report here that SIV mac239 Nef induces the endocytosis of TCR-CD3 in Jurkat T cells. SIV Nef also induces the endocytosis of a chimeric CD8-CD3-zeta protein containing only the CD3-zeta cytoplasmic domain (8-zeta), in the absence of other CD3 subunits. Thus, the interaction of SIV Nef with CD3-zeta likely mediates the induction of TCR-CD3 endocytosis. In cells expressing SIV Nef and 8-zeta, both proteins colocalize with AP-2, indicating that Nef induces 8-zeta internalization via this pathway. Surprisingly, deletion of constitutively strong AP-2 binding determinants (CAIDs) in SIV Nef had little effect on its ability to induce TCR-CD3, or 8-zeta endocytosis, even though these determinants are required for the induction of CD4 and CD28 endocytosis via this pathway. Fluorescent microscopic analyses revealed that while neither the mutant SIV Nef protein nor 8-zeta colocalized with AP-2 when expressed independently, both proteins colocalized with AP-2 when coexpressed. In vitro binding studies using recombinant SIV Nef proteins lacking CAIDs and recombinant CD3-zeta cytoplasmic domain demonstrated that SIV Nef and CD3-zeta cooperate to bind AP-2 via a novel interaction. The fact that Nef uses distinct AP-2 interaction surfaces to recruit specific membrane receptors demonstrates how Nef independently selects distinct types of target receptors and recruits them to AP-2 for endocytosis. (+info)
ARF6 stimulates clathrin/AP-2 recruitment to synaptic membranes by activating phosphatidylinositol phosphate kinase type Igamma.
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Clathrin-mediated endocytosis of synaptic vesicle membranes involves the recruitment of clathrin and AP-2 adaptor complexes to the presynaptic plasma membrane. Phosphoinositides have been implicated in nucleating coat assembly by directly binding to several endocytotic proteins including AP-2 and AP180. Here, we show that the stimulatory effect of ATP and GTPgammaS on clathrin coat recruitment is mediated at least in part by increased levels of PIP2. We also provide evidence for a role of ADP-ribosylation factor 6 (ARF6) via direct stimulation of a synaptically enriched phosphatidylinositol 4-phosphate 5-kinase type Igamma (PIPKIgamma), in this effect. These data suggest a model according to which activation of PIPKIgamma by ARF6-GTP facilitates clathrin-coated pit assembly at the synapse. (+info)
Tyrosine phosphorylation of the beta2 subunit of clathrin adaptor complex AP-2 reveals the role of a di-leucine motif in the epidermal growth factor receptor trafficking.
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Tyrosine phosphorylation of the beta2 subunit of clathrin adaptor complex AP-2 was detected in three types of cells treated with epidermal growth factor (EGF). The tyrosine phosphorylation was observed during recruitment of EGF receptors into coated pits at 4 degrees C and reached maximum at 37 degrees C at post-recruitment stages of endocytosis. An inhibitor of EGF receptor kinase completely abolished this phosphorylation in all cell types, whereas the inhibitor of Src family kinases partially inhibited beta2 phosphorylation in A-431 cells but not in HeLa cells. By using beta2 subunit tagged with yellow fluorescent protein that is effectively assembled into AP-2 complex, the major phosphorylation site of beta2 was mapped to Tyr-6. Analysis of cells expressing dominant-interfering mutant mu2 subunit of AP-2 suggested that beta2 phosphorylation is partially mediated by the receptor interaction with the mu2 subunit. Mutation of leucine residues 1010 and 1011 motif in the EGF receptor resulted in the severe inhibition of beta2 tyrosine phosphorylation. From these data, we propose that interactions of the EGF receptor with AP-2 mediated by the receptor 974YRAL and di-leucine motifs may contribute to beta2 tyrosine phosphorylation. Surprisingly, mutation of the Leu-1010/Leu-1011 motif resulted in impaired degradation of EGF receptors, suggesting the role of this motif in lysosomal targeting of the receptor. (+info)
Phosphatidylinositol phosphate 5-kinase Ibeta recruits AP-2 to the plasma membrane and regulates rates of constitutive endocytosis.
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Overexpression of phosphatidylinositol phosphate 5-kinase (PIP5KI) isoforms alpha, beta, or gamma in CV-1 cells increased phosphatidylinositol 4,5-bisphosphate (PIP2) levels by 35, 180, and 0%, respectively. Endocytosis of transferrin receptors, association of AP-2 proteins with membranes, and the number of clathrin-coated pits at the plasma membrane increased when PIP2 increased. When expression of PIP5KIbeta was inhibited with small interference RNA in HeLa cells, expression of PIP5KIalpha was also reduced slightly, but PIP5KIgamma expression was increased. PIP2 levels and internalization of transferrin receptors dropped 50% in these cells; thus, PIP5KIgamma could not compensate for loss of PIP5KIbeta. When expression of PIP5KIalpha was reduced, expression of both PIP5KIbeta and PIP5KIgamma increased and PIP2 levels did not change. A similar increase of PIP5KIalpha and PIP5KIbeta occurred when PIP5KIgamma was inhibited. These results indicate that constitutive endocytosis in CV-1 and HeLa cells requires (and may be regulated by) PIP2 produced primarily by PIP5KIbeta. (+info)
Xenopus autosomal recessive hypercholesterolemia protein couples lipoprotein receptors with the AP-2 complex in oocytes and embryos and is required for vitellogenesis.
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ARH is required for normal endocytosis of the low density lipoprotein (LDL) receptor in liver and mutations within this gene cause autosomal recessive hypercholesterolemia in humans. xARH is a localized maternal RNA in Xenopus with an unknown function in oogenesis and embryogenesis. Like ARH, xARH contains a highly conserved phosphotyrosine binding domain and a clathrin box. To address the function of xARH, we examined its expression pattern in development and used pull-down experiments to assess interactions between xARH, lipoprotein receptors and proteins in embryo extracts. xARH was detected concentrated at the cell periphery as well as in the perinuclear region of oocytes and embryos. In pull-down experiments, the xARH phosphotyrosine binding domain interacted with the LDL and vitellogenin receptors found in Xenopus oocytes and embryos. Mutations within the receptor internalization signal specifically abolished this interaction. The xARH C-terminal region pulled-down several proteins from embryo extracts including alpha- and beta-adaptins, subunits of the AP-2 endocytic complex. Mutations within either of the two Dvarphi(F/W) motifs found in xARH abolished binding to alpha- and beta-adaptins. Expression of a dominant negative mutant of xARH missing the clathrin box and one functional Dvarphi(F/W) motif severely inhibited endocytosis of vitellogenin in cultured oocytes. The data indicate that xARH acts as an adaptor protein linking LDL and vitellogenin receptors directly with the AP-2 complex. In oocytes, we propose that xARH mediates the uptake of lipoproteins from the blood for storage in endosomes and later use in the embryo. Our findings point to an evolutionarily conserved function for ARH in lipoprotein uptake. (+info)
Differential requirements for AP-2 in clathrin-mediated endocytosis.
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AP-2 complexes are key components in clathrin-mediated endocytosis (CME). They trigger clathrin assembly, interact directly with cargo molecules, and recruit a number of endocytic accessory factors. Adaptor-associated kinase (AAK1), an AP-2 binding partner, modulates AP-2 function by phosphorylating its mu2 subunit. Here, we examined the effects of adenoviral-mediated overexpression of WT AAK1, kinase-dead, and truncation mutants in HeLa cells, and show that AAK1 also regulates AP-2 function in vivo. WT AAK1 overexpression selectively blocks transferrin (Tfn) receptor and LRP endocytosis. Inhibition was kinase independent, but required the full-length AAK1 as truncation mutants were not inhibitory. Although changes in mu2 phosphorylation were not detected, AAK1 overexpression significantly decreased the phosphorylation of large adaptin subunits and the normally punctate AP-2 distribution was dispersed, suggesting that AAK1 overexpression inhibited Tfn endocytosis by functionally sequestering AP-2. Surprisingly, clathrin distribution and EGF uptake were unaffected by AAK1 overexpression. Thus, AP-2 may not be stoichiometrically required for coat assembly, and may have a more cargo-selective function in CME than previously thought. (+info)
Clathrin-mediated endocytosis in AP-2-depleted cells.
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We have used RNA interference to knock down the AP-2 mu2 subunit and clathrin heavy chain to undetectable levels in HeLaM cells. Clathrin-coated pits associated with the plasma membrane were still present in the AP-2-depleted cells, but they were 12-fold less abundant than in control cells. No clathrin-coated pits or vesicles could be detected in the clathrin-depleted cells, and post-Golgi membrane compartments were swollen. Receptor-mediated endocytosis of transferrin was severely inhibited in both clathrin- and AP-2-depleted cells. Endocytosis of EGF, and of an LDL receptor chimera, were also inhibited in the clathrin-depleted cells; however, both were internalized as efficiently in the AP-2-depleted cells as in control cells. These results indicate that AP-2 is not essential for clathrin-coated vesicle formation at the plasma membrane, but that it is one of several endocytic adaptors required for the uptake of certain cargo proteins including the transferrin receptor. Uptake of the EGF and LDL receptors may be facilitated by alternative adaptors. (+info)