Assembly of Clathrin Coats Disrupts the Association between Eps15 and AP-2 Adaptors

Abstract

Eps15 is a phosphorylation substrate of the epidermal growth factor receptor kinase. In vivo, it is largely found in complex with AP-2, the plasma membrane clathrin adaptor protein complex. Although AP-2 is uniformly distributed across the surface of clathrin-coated pits and vesicles, Eps15 is preferentially found in the rims of endocytic clathrin-coated pits (1). This observation suggests that Eps15 may disengage from AP-2 during coat formation. Here we use two new anti-Eps15 antibodies to show that, contrary to our own earlier suggestion, coated vesicles isolated from brain do not contain detectable amounts of Eps15. Furthermore, when AP-2 complexes that are saturated with Eps15 are used for in vitro assembly of clathrin-AP-2 coats, normal structures are formed that contain the expected amounts of clathrin and AP-2, but the amount of Eps15 present is dramatically lower than that of AP-2. We propose that during coated pit formation, addition of clathrin to the growing edge at the rim of the pit releases Eps15 from AP-2.During the past decade, several groups have extensively studied the protein components and the mechanism responsible for the formation of clathrin-coated pits. Clathrin and its tetrameric adaptors AP-1 and AP-2 are among the best characterized structural elements of the coat (for recent reviews, see Refs. 2-4). The APs recruit clathrin, and they facilitate coat formation through its association with clathrin. The interaction involves a short segment located in the hinge region of the ␤ chain of AP-2 (5) and the terminal domain of clathrin. 1 Recently, it was suggested that Eps15 (6), a ϳ100-kDa molecule that forms complexes with AP-2 (7-9), is involved in the clathrin-mediated internalization pathway. Eps15-AP-2 complexes are found both in the cytosol and at the plasma membrane (1, 9, 10). In solution, the interaction between Eps15 and AP-2 involves a short segment toward the C terminus of Eps15 and the C-terminal ear domain of the ␣ chain of AP-2 (7, 8).Most of the Eps15 in the cell is bound to AP-2, whereas ϳ80% of AP-2 is free (1). The membrane-bound Eps15/AP-2 complexes are mainly found within clathrin-coated pits (1, 10), usually at the rims and growing edges of coated pits (1).The formation of coated pits at the plasma membrane requires AP-2 complexes and clathrin to be recruited to the membrane and a poorly understood nucleation event that initiates clathrin coat assembly. After nucleation, new clathrin and AP-2 complexes presumably add to the free edges of the pit, so that an AP-2 complex that is added to the pit early on will move progressively deeper into the pit. How is it, then, that Eps15 is concentrated at the edges of pits, regardless of the state of completion of the pit? We previously examined whether Eps15 is present in coated vesicles (1) and found a band of the approximately correct molecular weight in gels of purified bovine brain vesicle preparations. However, this band was not conclusively identified as Eps15, and in the absence of purified Eps15 quantitation was impossible...

Eps15 Homology Domain-NPF Motif Interactions Regulate Clathrin Coat Assembly during Synaptic Vesicle Recycling

“…Thus, Eps15 does not polymerize clathrin (Fig. 4B), which confirms a previous report (26). However, Eps15 dramatically stimulated clathrin assembly mediated by GST-C45, evident as a decrease in the amount of clathrin present in the supernatants (Fig.…”

“…The latter explanation appears attractive, given that Eps15 has been found to dissociate from AP-2 once clathrin coats form (26). This is also consistent with immunoelectron microscopy localization of Eps15 to the rims of coated pits (15).…”

The Interaction of Epsin and Eps15 with the Clathrin Adaptor AP-2 Is Inhibited by Mitotic Phosphorylation and Enhanced by Stimulation-dependent Dephosphorylation in Nerve Terminals

“…Epsin/MP90 and Eps15 may be two such proteins. Both Eps15 and epsin were detected at clathrincoated pits but were found not to be co-enriched, together with clathrin, in clathrin-coated vesicles (7,18), suggesting that they are accessory and not key intrinsic components of the clathrin coat. The phosphorylation-dependent block of the interaction of epsin and Eps15 with AP-2 may dissociate them from the clathrin coat and play a role in the inhibition of the invagination process.…”

“…Perturbation of the interactions of both Eps15 and epsin with AP-2, as well as disruption of the function of both proteins by antibody injection, block clathrin-mediated endocytosis (7, 13-16). It was proposed that Eps15 and epsin play an important role in clathrin-mediated endocytosis, possibly by participating in dynamic rearrangements of the clathrin coat during bud invagination and fission (7,17,18).…”

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

“…Finally, to confirm that ␤-arrestin2 is recruited to pre-existing CPs using a dynamic in vivo assay, we utilized a triple transfection model in which ␤-arrestin2-GFP and VSV-TRHR were co-expressed in combination with RFP-Eps15. Eps15 is a constitutive component of CPs (25,26) but not clathrin-coated vesicles (47) and could therefore be used as a marker of "pre-existing" CPs, i.e. CPs present before TRHR stimulation.…”