Endocytic cargo such as the transferrin receptor is incorporated into clathrin-coated pits by associating, via tyrosine-based motifs, with the AP2 complex. Cargo–AP2 interactions occur via the μ2 subunit of AP2, which needs to be phosphorylated for endocytosis to occur. The most likely role for μ2 phosphorylation is in cargo recruitment because μ2 phosphorylation enhances its binding to internalization motifs. Here, we investigate the control of μ2 phosphorylation. We identify clathrin as a specific activator of the μ2 kinase and, in permeabilized cells, we show that ligand sequestration, driven by exogenous clathrin, results in elevated levels of μ2 phosphorylation. Furthermore, we show that AP2 containing phospho-μ2 is mainly associated with assembled clathrin in vivo, and that the level of phospho-μ2 is strongly reduced in a chicken B cell line depleted of clathrin heavy chain. Our results imply a central role for clathrin in the regulation of cargo selection via the modulation of phospho-μ2 levels.

Reversible phosphorylation has long been an attractive mechanism to control cycles of coat assembly and disassembly during clathrin-mediated endocytosis. Many of the coat proteins are phosphorylated in vivo and in vitro. Our work has focused on the role of phosphorylation of the $#x03BC;2 subunit of AP-2 (adaptor protein 2), which appears to be necessary for efficient cargo recruitment. Studies to probe the regulation of $#x03BC;2 phosphorylation demonstrated that clathrin is a specific activator of the $#x03BC;2 kinase, and, in permeabilized cells, cargo sequestration, driven by exogenously added clathrin, results in elevated levels of $#x03BC;2 phosphorylation. Furthermore, phosphorylated $#x03BC;2 is mainly associated with assembled clathrin in vivo and its steady-state level is strongly reduced in cells depleted of clathrin heavy chain. Our results imply a central role for clathrin in the regulation of cargo selection via modulation of phospho-$#x03BC;2 levels. This is therefore a novel regulatory role for clathrin that is independent of its structural role and that provides elegant spatial control of AP-2 and cargo interactions, ensuring that AP-2 is only activated at the correct cellular location and in the correct functional context. Ongoing studies are exploring further the roles of reversible phosphorylation in the coated vesicle cycle.

Clathrin-coated pits and vesicles represent the major ports of entry into most eukaryotic cells. As well as performing housekeeping functions (e.g., allowing cells to take up essential nutrients), the endocytic pathway participates in a number of tissue-specific events such as synaptic-vesicle recycling, control of morphogen gradients during development, downregulation of receptor tyrosine kinases, and immune surveillance (Conner and Schmid, 2003). To understand the role played by clathrin-mediated uptake, it is therefore essential to have robust endocytosis assays in intact cells (Basic Protocol 1). The clathrin-coated vesicle cycle requires a complicated interplay of proteins and lipids that is regulated in space and time. Reconstitution assays in permeabilized cells (Basic Protocol 2 and Alternate Protocols 1 and 2) provide a powerful approach to understanding how this complex process is regulated. Support Protocols 1 to 9 describe the preparation of cells and reagents for the assays. NOTE: All solutions and equipment coming into contact with live cells must be sterile, and aseptic technique should be used accordingly. NOTE: All cell culture incubations should be carried out in a 37 • C, 5% CO 2 humidified incubator unless otherwise indicated. BASIC PROTOCOL 1 MEASUREMENT OF RECEPTOR-MEDIATED ENDOCYTOSIS IN INTACT CELLS A number of methods have been described to measure the internalization of ligands such as transferrin (Hopkins and Trowbridge, 1983). In general, they rely on the ability to distinguish cell surface from intracellular pools of ligand. Routinely, the authors use transferrin that has been biotinylated via a cleavable disulfide linkage (B-SS-Tfn) as a reporter molecule to distinguish between intracellular and cell surface ligand by accessibility either to avidin or to the small membrane-impermeant thiol-reducing reagent, sodium 2-mercaptoethanesulfonate (MesNa). Transferrin is a ligand of choice to study the core machinery because the transferrin receptor is abundant on most cells, the route followed by transferrin when bound to its receptor is well characterized (Hopkins and Trowbridge, 1983), and transferrin is inexpensive and easy to modify by biotinylation or iodination. It is possible to use other ligands, however (see Background Information). Essential prerequisites are a reasonable number of receptor molecules on the cell surface and effective methods to label the receptor with either ligand or antibody or by cell surface labeling. This protocol describes a method whereby avidin and the small membrane-impermeant reducing agent MesNa quench B-SS-Tfn at the cell surface so that the internalized B-SS-Tfn may be measured. Following addition of avidin or MesNa, these reagents are themselves functionally inactivated. Biocytin (a conjugate of biotin and lysine) is added to bind to remaining binding sites on avidin, which is tetrameric. Excess MesNa is removed by reaction with iodoacetamide (IAA). In each case, the cells are solubilized and all of the transferrin is captured on an ELISA plate coate...