Simultaneous Binding of PtdIns(4,5)P ₂ and Clathrin by AP180 in the Nucleation of Clathrin Lattices on Membranes

Abstract

hours after transfection. COS-7 cells were incubated with EGF (0.1 g/ml) [biotinylated, complexed to Texas Red-streptoavidin (Molecular Probes, Eugene, OR)] in binding buffer [20 mM Hepes-NaOH ( pH 7.5), 130 mM NaCl, and 0.1% bovine serum albumin] at 4°C for 60 min. Internalization of EGF was allowed by incubation in Dulbecco's modified Eagle's medium at 37°C for 10 min, then excess EGF was removed with 0.2 M AcOH ( pH 2.5) and 0.5 M NaCl at 4°C for 5 min. Cells were fixed in 3.7% formaldehyde, permeabilized with 0.2% Triton X-100, and immunostained with a polyclonal antibody to myc (Santa Cruz Biotechnology, Santa Cruz, CA) and fluorescein isothiocyanate-conjugated antibody to rabbit (Organon Teknika, Boxtel, Netherlands). Internalization of EGF was observed by confocal microscopy (Bio-Rad). 37. We thank Y. Watanabe (Ehime University, Japan) for providing us with various synthetic phosphoinositides.

The phospholipid code: a key component of dying cell recognition, tumor progression and host–microbe interactions

“…For example, adaptor protein 180, a key component of clathrin-mediated endocytosis, can interact with both PI(4,5)P 2 and clathrin simultaneously to facilitate the formation of clathrin lattice at the plasma membrane. 125 Likewise, the Ca 2+ -sensing protein, synaptotagmin, mediates neurotransmitter release via interacting with PI(4,5)P 2 and t-SNARE at the pre-synaptic axon terminal of neurons. 126 Similarly, assembly of factors required for early endosome fusion involves the recruitment of early endosome antigen 1 protein to phosphatidylinositol 3-phosphate (PI(3)P) located at the cytosolic leaflet of endosome via its FYVE domain.…”

RNAi-mediated Hip1R Silencing Results in Stable Association between the Endocytic Machinery and the Actin Assembly Machinery

“…Furthermore, Hip1R contains an AP180-homology (ANTH) domain, which is also present in several other proteins involved in clathrin-mediated endocytosis. ANTH domains are known to interact with phosphatidylinositol-4, 5-biphosphate at the plasma membrane (Ford et al, 2001). Hip1R belongs to a conserved family of proteins that includes yeast Sla2p/End4p and mammalian Hip1.…”

Effects of synaptotagmin reveal two distinct mechanisms of agonist‐stimulated internalization of the M4 muscarinic acetylcholine receptor

“…Of course, the fact that the PH domain reduces receptor internalization indicates that the normal mode of internalization also requires the availability of PIP 2 . Since it is known that several proteins crucial to the process of clathrinmediated endocytosis, such as AP-2, AP-180 and dynamin, are recruited to the plasma membrane at least in part by binding to PIP 2 (Ford et al, 2001;Takei & Haucke, 2001), this is perhaps unsurprising.…”

“…Hence, the endocytosis of a G protein-coupled receptor in response to agonist stimulation involves the co-ordinated operation of a number of proteins. Furthermore, the phospholipid phosphatidylinositol-4,5-bisphosphate (PIP 2 ) interacts with both AP-2 and dynamin (as well as other components of the endocytotic machinery), and is also likely to play a key role, perhaps by initiation of coated pit formation at the plasma membrane (Ford et al, 2001;Takei & Haucke, 2001).…”