AP180 and AP-2 Interact Directly in a Complex That Cooperatively Assembles Clathrin

Abstract

Clathrin-coated vesicles are involved in protein and lipid trafficking between intracellular compartments in eukaryotic cells. AP-2 and AP180 are the resident coat proteins of clathrin-coated vesicles in nerve terminals, and interactions between these proteins could be important in vesicle dynamics. AP180 and AP-2 each assemble clathrin efficiently under acidic conditions, but neither protein will assemble clathrin efficiently at physiological pH. We find that there is a direct, clathrin-independent interaction between AP180 and AP-2 and that the AP180-AP-2 complex is more efficient at assembling clathrin under physiological conditions than is either protein alone. AP180 is phosphorylated in vivo, and in crude vesicle extracts its phosphorylation is enhanced by stimulation of casein kinase II, which is known to be present in coated vesicles. We find that recombinant AP180 is a substrate for casein kinase II in vitro and that its phosphorylation weakens both the binding of AP-2 by AP180 and the cooperative clathrin assembly activity of these proteins. We have localized the binding site for AP-2 to amino acids 623-680 of AP180. The AP180/AP-2 interaction can be disrupted by a recombinant AP180 fragment containing the AP-2 binding site, and this fragment also disrupts the cooperative clathrin assembly activity of the AP180-AP-2 complex. These results indicate that AP180 and AP-2 interact directly to form a complex that assembles clathrin more efficiently than either protein alone. Phosphorylation of AP180, by modulating the affinity of AP180 for AP-2, may contribute to the regulation of clathrin assembly in vivo.Clathrin-coated vesicles mediate protein and lipid transport between intracellular compartments in the regulated secretory and endocytic pathways of all eukaryotic cells (reviewed in Ref. 1). The outermost layer of a clathrin-coated vesicle consists of clathrin that has been polymerized into an icosahedral cage (2). Between the outer clathrin shell and the vesicle membrane lie the clathrin assembly proteins (3). Clathrin assembly proteins belong to one of two gene families, the tetrameric AP 1 family and the monomeric AP family. Three tetrameric APs have been described and designated AP-1, AP-2, and AP-3. AP-1 and AP-2 were first characterized in 1987 as major clathrin-coated vesicle coat proteins (4, 5). AP-3 has only recently been identified (6 -9) and also appears to be associated with clathrin-coated vesicles (10). Two members of the monomeric AP family have been described: AP180 and CALM. AP180 was independently discovered in a number of laboratories and was originally referred to as AP180 (11, 12), pp155 (13), NP185 (14), or F1-20 (15, 16). AP180, pp155, NP185, and F1-20 were found to be identical (17, 18) and given the name AP-3. However, in view of the decision to designate the newest member of the tetrameric AP family AP-3 (7, 8), we refer to the monomeric AP-3 as AP180. AP180 localizes to synapses (19 -21), while CALM is expressed ubiquitously (22). AP-1, AP-2, and AP180 localize to clathrin-coate...

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

“…Furthermore, we suggest that the reason for AP-mediated assembly of clathrin to be inefficient at physiological pH is to allow assembly to be regulated by other proteins, such as Eps15. This is consistent with previous studies indicating that the AP180-AP2 interaction also serves to facilitate cooperative clathrin assembly at physiological pH (32).…”

Interaction of Two Structurally Distinct Sequence Types with the Clathrin Terminal Domain β-Propeller

“…In fact, there is already some experimental evidence to support this idea. Complexing AP-2 with AP180 leads to enhanced clathrin assembly activity (20), and we have previously shown that AP-2 recruitment to the central DPW region (residues 249 -401) of epsin 1 augments clathrin binding mediated by the epsin sequence 257 LM-DLADV (15). Here, we further characterized this epsin se-quence and a related clathrin-binding sequence from amphiphysin, PWDLW (21).…”

Direct interaction of Cbl with pTyr 1045 of the EGF receptor (EGFR) is required to sort the EGFR to lysosomes for degradation

“…Each protein has a PI(4,5)P 2 binding domain and both bind to AP-2 and clathrin. Both AP-2 and CALM/AP180 are able to stimulate clathrin assembly by them selves, but the interaction between AP-2 and CALM/AP180 increases the ability of AP-2 to assemble clathrin (Hao et al, 1999). ß-arrestin is another sorting adaptor involved in clathrin-mediated endocytosis.…”