Mapping of Eps15 Domains Involved in Its Targeting to Clathrin-coated Pits

130

178

HM1.24/Bst2/CD317 is a protein highly expressed in multiple myeloma cells and has unique topology with two membrane anchor domains, an NH 2 -terminal transmembrane domain and a glycosylphosphatidylinositol attached to the COOH terminus. We show here that human HM1.24 is localized not only on the cell surface but also in the trans-Golgi network and/or recycling endosomes, where it resides in detergent-resistant microdomains, lipid rafts. In contrast to other glycosylphosphatidylinositol-anchored proteins, HM1.24 was internalized from lipid rafts on the cell surface by clathrin-mediated endocytosis. Interestingly, a non-canonical tyrosine-based motif, which contains two tyrosine residues, Tyr-6 and Tyr-8, present in the NH 2 -terminal cytoplasmic tail, was essential for endocytosis through interaction with an ␣-adaptin, but not 2-subunit, of the AP-2 complex. Indeed, an appendage domain of ␣-adaptin was identified as a protein interacting with the cytoplasmic tail of HM1.24. Furthermore, overexpression of the appendage domain of ␣-adaptin in cells depleted of ␣-adaptin could rescue the clathrin-mediated endocytosis of HM1.24 but not of the transferrin receptor. Taken together, our findings suggest that clathrin-dependent endocytosis of human HM1.24 from the cell surface lipid rafts is mediated by direct interaction with ␣-adaptin.

Section: ␣-Adaptin-dependent Hm124 Endocytosismentioning

confidence: 78%

HM1.24 Is Internalized from Lipid Rafts by Clathrin-mediated Endocytosis through Interaction with α-Adaptin

Masuyama

Kuronita

Tanaka

et al. 2009

130

178

“…To determine whether ␤-arrestin and TRHR traffic together as a complex after receptor stimulation in our model, their relative cellular distributions were studied by immunofluorescence microscopy. The images shown focus on the plasma membrane of cells that are adherent to the coverslip as described previously (32). Under basal conditions, ␤-arrestin2 displayed a characteristic diffuse cytosolic distribution (39) (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…For immunofluorescence studies, HeLa cells were seeded on coverslips in six-well plates, transfected with 2 g of VSV-TRHR and 0.2 g ␤-arrestin2-GFP the following day, and used for immunofluorescence 1 day post-transfection. Cells were fixed and processed for fluorescence microscopy as described previously (32). Endocytosis of Alexa594-conjugated Tf was performed on HeLa cells grown on coverslips 1 day after transfection.…”

Section: Methodsmentioning

confidence: 99%

Recruitment of Activated G Protein-coupled Receptors to Pre-existing Clathrin-coated Pits in Living Cells

Scott¹,

Benmerah²,

Muntaner³

et al. 2002

Self Cite

101

The process of clathrin-mediated endocytosis tightly regulates signaling of the superfamily of seven-transmembrane G protein-coupled receptors (GPCRs). A fundamental question in the cell biology of membrane receptor endocytosis is whether activated receptors can initiate the formation of clathrin-coated pits (CPs) or whether they are simply mobilized to pre-existing CPs. Here, using various approaches, including a dynamic assay to monitor the distribution of CPs and GPCR-␤-arrestin complexes in live HeLa cells, we demonstrate for the first time that activated GPCRs do not initiate the de novo formation of CPs but instead are targeted to pre-existing CPs.Many cell surface receptors and membrane proteins are internalized through specialized structures of the plasma membrane, called clathrin-coated pits (CPs), 1 which gradually invaginate and ultimately detach from the plasma membrane forming clathrin-coated vesicles. The endocytic machinery comprises two major structural proteins, clathrin and the adapter protein complex AP2, which plays a central role in CP assembly. A number of more recently identified cytosolic or integral-membrane proteins regulate CP formation and the fission of clathrin-coated vesicles. In the conventional view of constitutive endocytosis, readily accessible tyrosine-or di-leucine-based endocytic motifs, present in the cytoplasmic tails of membrane proteins that are to be internalized, interact with the AP2 adapter complex. In the case of ligand-induced endocytosis, these endocytic signals are cryptic and thought to be unmasked upon receptor activation by its cognate ligand (1-3).G protein-coupled receptors (GPCRs), one of the largest families of membrane receptors (4), represent a different model of ligand-induced endocytosis through CPs due to their use of specific adapter proteins called ␤-arrestins that form a bridge between activated GPCRs and the clathrin coat (5). Endocytosis of GPCRs plays an important role in the regulation of their signaling cycle (6). Agonist-stimulated GPCRs initiate cell responses by modulating effector molecules via the activation of heterotrimeric G proteins. These receptors are then rapidly phosphorylated by specific kinases. This phosphorylation promotes the binding of cytosolic nonvisual arrestins (␤-arrestin1 and ␤-arrestin2) to GPCRs (7) resulting in their desensitization (8, 9), a transient state during which receptors become refractory to any further stimulation. To recover a full signaling function, GPCRs have to be internalized (10), dephosphorylated in the endosomal compartment (11), and then recycled back to the plasma membrane (12, 13). Previous studies have indicated that nonvisual arrestins play the role of adapter molecules during this process (14). The overexpression of arrestins can rescue an endocytosis-defective mutant of the ␤ 2 -adrenergic receptor, a member of the GPCR family and the overexpression of dominant negative forms of arrestins inhibits GPCR internalization (15). In addition, activated GPCR-arrestin complexes concentrate in punc...

“…Immunocytochemistry-Immunocytochemistry was performed as described previously (26,27). Primary antibodies included anti-transferrin receptor (TfR) (CD71) and anti-␥-adaptin 100.3 and anti-␤-adaptins 100.1 mouse monoclonal antibodies (Sigma), anti-CALM (C-18) and anti-Epsin (R-20) goat polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), anti-clathrin (derived from the CON.1 hybridoma (ATCC, Manassas, VA)) and anti-Eps15 (3T, a gift of Dr. Pier Di Fiore, European Institute of Oncology, Milan, Italy).…”

Section: Methodsmentioning

confidence: 99%

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

Rappoport

Taha

Lemeer

et al. 2003

Self Cite

Numerous biologically relevant substrates are selectively internalized via clathrin-mediated endocytosis. At the plasma membrane the AP-2 complex plays a major role in clathrin coat formation, interacting with both cargo and clathrin. Utilizing simultaneous dual-channel total internal reflection fluorescence microscopy we have analyzed components of the AP-2 complex (␣-and ␤2-adaptin) during clathrin-mediated endocytosis. Although in static images enhanced green fluorescent protein-tagged AP-2 markers significantly co-localized with clathrin and other components of clathrin-coated pits, AP-2 did not seem to be present in clathrin spots that appeared to undergo internalization or motility parallel to the plane of the plasma membrane. Two populations of clathrin at the plasma membrane seem to exist, the dynamic and the static, and AP-2 appears to be only found within the latter. These results suggest that colocalized clathrin/AP-2 puncta may represent loci for coated pit production and that previous models that assumed AP-2 was retained within clathrin coats during endocytosis may need to be re-evaluated.