The AP-2 Complex Is Excluded from the Dynamic Population of Plasma Membrane-associated Clathrin

Rappoport, Joshua Z.; Taha, Bushra W.; Lemeer, Simone; Benmerah, Alexandre; Simon, Sanford M.

doi:10.1074/jbc.c300390200

Cited by 64 publications

(112 citation statements)

References 35 publications

(30 reference statements)

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“…These events have been interpreted as CCV budding, movement, and uncoating. Because the GFP-␣ is not incorporated into the moving spots, it has been hypothesized that AP-2 is somehow swept aside as the vesicle buds (Rappoport et al, 2003(Rappoport et al, , 2005. Our results suggest an alternative interpretation.…”

Section: Discussionmentioning

confidence: 60%

“…We initially inserted a GFP tag into our ␣ constructs as a way of localizing them, because GFP-tagged ␣ had been previously used for live cell imaging studies (Rappoport et al, 2003;Keyel et al, 2004;Rappoport et al, 2005). Some of the results of these studies have been puzzling, and our data may help to explain them.…”

Section: Discussionmentioning

confidence: 99%

“…Interestingly, however, the first tagged ␣ construct that we made behaved very differently. Others have inserted GFP tags at either the N terminus or the C terminus of ␣ for live cell imaging studies (Rappoport et al, 2003;Keyel et al, 2004;Rappoport et al, 2005), and initially we inserted a C-terminal GFP tag ( Figure 8A). Although we were unable to isolate a completely homogeneous population of cells, we generated cell lines in which ϳ60% of the cells were expressing tagged ␣, and Western blotting showed selective disappearance of endogenous ␣A and ␣C after siRNA knockdown ( Figure 8B).…”

Section: Gfp-tagged ␣ Subunitmentioning

confidence: 99%

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Functional Analysis of AP-2 α and μ2 Subunits

Motley

Berg

Taylor

et al. 2006

MBoC

100

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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 ␣ and 2 subunits and then used siRNA knockdowns to deplete the endogenous proteins. Mutating the PtdIns(4,5)P2 binding site of ␣, the phosphorylation site of 2, or the YXX⌽ binding site of 2 impairs AP-2 function, as assayed by transferrin uptake. In contrast, removing the C-terminal appendage domain of ␣, or mutating the PtdIns(4,5)P2 binding site of 2, has no apparent effect. However, adding a C-terminal GFP tag to ␣ 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 structurefunction analyses, and as a way of testing tagged constructs for function in vivo.

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Section: Discussionmentioning

confidence: 60%

Section: Discussionmentioning

confidence: 99%

Section: Gfp-tagged ␣ Subunitmentioning

confidence: 99%

See 1 more Smart Citation

Functional Analysis of AP-2 α and μ2 Subunits

Motley

Berg

Taylor

et al. 2006

MBoC

100

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“…The number of dots was expressed as density of dots on the cell surface (dots/m 2 of surface). For the quantitation of EGFR/clathrin colocalization and the number of CCPs, HeLa cells were transfected with dsRed-clathrin (Rappoport et al, 2003) using lipofectAMINE reagent (Invitrogen, Carlsbad, CA), incubated for 16 h in SFM, placed on ice, and washed with prechilled SFM. Cells were then incubated with anti-EGFR (13A9 antibody) in SFM, in the presence or in the absence of EGF (100 ng/ml), 60 min at 0°C on a rocker, washed, fixed, and stained with an anti-mouse fluorochrome-labeled antibody.…”

mentioning

confidence: 99%

Relationships between EGFR Signaling–competent and Endocytosis-competent Membrane Microdomains

Puri

Tosoni

Comai

et al. 2005

MBoC

143

124

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Membrane microdomains, the so-called lipid rafts, function as platforms to concentrate receptors and assemble the signal transduction machinery. Internalization, in most cases, is carried out by different specialized structures, the clathrincoated pits. Here, we show that several endocytic proteins are efficiently recruited to morphologically identified plasma membrane lipid rafts, upon activation of the epidermal growth factor (EGF) receptor (EGFR), a receptor tyrosine kinase. Analysis of detergent-resistant membrane fractions revealed that the EGF-dependent association of endocytic proteins with rafts is as efficient as that of signaling effector molecules, such as Grb2 or Shc. Finally, the EGFR, but not the nonsignaling transferrin receptor, could be localized in nascent coated pits that almost invariably contained raft membranes. Thus, specialized membrane microdomains have the ability to assemble both the molecular machineries necessary for intracellular propagation of EGFR effector signals and for receptor internalization. INTRODUCTIONOn engagement of receptor tyrosine kinases (RTKs) by their cognate ligands, their intrinsic kinase activity is stimulated with ensuing receptor autophosphorylation and recruitment/activation of the signal transduction machinery, in turn responsible for several effector functions. Concurrently, activated receptors trigger their own endocytosis, whose ultimate goal is to extinguish signaling through removal of receptors from the cell surface (Carpenter, 2000). In the case of the epidermal growth factor (EGF) receptor (EGFR) the localization of the two processes is well characterized (Jorissen et al., 2003). Signaling occurs within specialized membrane microdomains, lipid rafts (Simons and Toomre, 2000;Maxfield, 2002), whereas endocytosis occurs mostly through the clathrin-coated pits (CCPs; Conner and Schmid, 2003).Membrane rafts are cholesterol-and sphingolipid-rich membrane regions characterized by higher order and lower buoyant density than bulk plasma membrane (Simons and Toomre, 2000;Sprong et al., 2001;Kusumi et al., 2004). These structures are also characterized by their insolubility in some detergents at 4°C (DRM, detergent resistant membranes; Brown and Rose, 1992). Several transmembrane receptors have been reported to associate with membrane rafts (Cheng et al., 1999;Krauss and Altevogt, 1999;Mineo et al., 1999;Lamaze et al., 2001;Giurisato et al., 2003), including the EGFR . The association of receptors with lipid rafts is thought to be functional to the activation of signaling cascades (Cheng et al., 1999;Waugh et al., 1999;Drevot et al., 2002;Matveev and Smart, 2002;Pierce, 2002;Stoddart et al., 2002;del Pozo et al., 2004). Accordingly, specific signaling (Gingras et al., 1998;Iwabuchi et al., 1998;Michaely et al., 1999;Kindzelskii et al., 2004) and adaptor proteins (e.g., shc and grb2; Biedi et al., 2003;Ridyard and Robbins, 2003;Yang et al., 2004) have been found associated to rafts. However, lipid rafts are rather small, possibly containing only few molecules (P...

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“…However, the role for AP-2 during the post-internalization phase has been controversial. Rappoport et al initially showed that AP-2 is lost from the CCV during internalization (Rappoport, Taha, Lemeer, et al, 2003), although Keyel et al later proposed that AP-2 accompanies CCVs into the cytosol, suggesting its possible regulatory role the downstream sorting machinery (Keyel, et al, 2004). However, more detailed TIRF image analysis confirmed the former hypothesis, and showed that while AP-2 was co-localized to static CCPs in the membrane, it was absent from those CCPs observed to disappear into the cytosol (Rappoport, et al, 2005).…”

Section: Understanding Clathrin Mediated Endocytosis Via Tirf Microscopymentioning

confidence: 99%