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Regulation of Receptor-Mediated Endocytosis by a GTPase and a Kinase

S.L. Schmid, S.D. Conner, H. Damke, M. Ishido, T. Houghton, A. Jones, M. Leonard, T. Schröter, S. Sholly, F. Soulet, B.D. Song

Receptor-mediated endocytosis is essential for the efficient uptake of nutrients and other macromolecules into cells and for the regulation of signaling by cell-surface receptors. The process occurs at clathrin-coated pits, which concentrate receptor-ligand complexes, deform the membrane, invaginate, and eventually pinch off, forming clathrin-coated vesicles (CCVs). Clathrin is a self-assembling molecule that forms a curved polygonal lattice to drive membrane invagination.

Adaptor protein-2 (AP2) complexes are composed of 4 subunits: a, b, m2, and s2. The complexes are targeted to the plasma membrane through protein and lipid interactions involving the a subunit. Interactions between the b subunit and clathrin trigger clathrin assembly, forming coated pits. The m2 subunit interacts directly with tyrosine-based sorting motifs on the cytoplasmic tails of surface receptors to concentrate receptors into the assembling coated pit. AP2 complexes also interact with several accessory proteins, perhaps coordinating the activities of the proteins in receptor-mediated endocytosis.

Taking a biochemical approach, we developed and use cell-free assays that faithfully reconstitute discrete events in receptor-mediated endocytosis to discover new components of the endocytic machinery and to probe the hierarchy of interactions leading to coat assembly, cargo selection, vesicle budding, membrane fission, and CCV uncoating. Our results this past year revealed new insights into how CCV formation is regulated.

Because of the central role of AP2 in controlling receptor-mediated endocytosis, we were interested in discovering how the assembly and function of this protein are regulated. Using a phage display approach, we identified a multidomain serine/threonine kinase, adaptor-associated kinase-1 (AAK1), that binds to the N-terminal appendage domain of the subunit of AP2. AAK1 colocalizes with clathrin and AP2 in nonneuronal cells and is concentrated at the synapse of primary neurons in culture. AAK1 copurifies with CCVs isolated from either bovine brain or rat liver and with AP2 complexes extracted from CCVs.

In vitro phosphorylation studies indicated that purified, baculovirus-expressed AAK1 specifically phosphorylates the m2 subunit of AP2. In collaboration with D. Ricotta and S. Höning, University of Göttingen, Göttingen, Germany, we identified threonine 156, which is critical for m2 function in vivo, as the phosphorylation site. Phosphorylation of m2 in vitro enhanced the affinity of AP2 complexes for tyrosine-based sorting motifs 25-fold. Addition of AAK1 to a perforated cell assay that reconstitutes receptor-mediated endocytosis inhibited AP2-dependent early events in CCV formation. AAK1 is homologous to members of the ark/prk family of kinases that regulate both cortical actin dynamics and endocytosis in yeast. These data suggest that AAK1 plays an important role in regulating AP2 function and receptor-mediated endocytosis in vivo.

The GTPase dynamin is a major regulator of endocytic formation of CCVs. Dynamin self-assembles in the presence of nonhydrolyzable GTP analogs into helical stacks of rings that localize to the necks of endocytic intermediates. We showed that dynamin self-assembly stimulates its GTPase activity and identified a domain, the GTPase effector domain (GED), that functions as an assembly-dependent GTPase-activating protein.

We identified mutations in GED that abrogate the ability of dynamin to self-assemble and consequently its assembly-stimulated GTPase activity. Inhibition of receptor-mediated endocytosis caused by overexpression of these new GED mutants was indistinguishable from inhibition due to dominant-negative GTPase-defective mutants of dynamin suggesting that dynamin self-assembly is required for receptor-mediated endocytosis.

We are also developing a new assay for CCVs that involves the use of highly purified plasma membrane sheets from rat liver (Fig. 1). In this assay, isolated membrane sheets are incubated in the presence of cytosol, ATP, and GTP, and vesicles released are separated from the large membrane sheets by differential centrifugation. Inclusion into the vesicle fraction requires an active sorting event, because we detected 3 different receptors, receptors for low-density lipoprotein-related protein, transferrin, and asialoglycoprotein, which are concentrated in coated pits, in the pellet. In contrast, the abundant rat liver plasma membrane protein SR-BP1, which is excluded from coated pits, was not detected. The appearance of receptors for low-density lipoprotein-related protein in the vesicular fraction required ATP, GTP, and cytosol and was dependent on dynamin and clathrin.

The assay is powerful, because both cytosol and membranes can be frozen and stored at -70°C and retain function. Membranes can be stripped of endogenous clathrin and AP2 and vesicle formation reconstituted by addition of cytosol. The development of this assay is a prerequisite for defining the minimal cytosolic components required and for determining the hierarchy of events that lead to the formation of CCVs.

PUBLICATIONS

Conner, S.D., Schmid. S.L. Identification of an adaptor-associated kinase, AAK1, as a regulator of clathrin-mediated endocytosis. J. Cell Biol. 156:921, 2002.

Ricotta, D., Conner, S.D., Schmid, S.L., von Figura, K., Höning, S. Phosphorylation of the AP2 m subunit by AAK1 mediates high affinity binding to membrane protein sorting signals. J. Cell Biol. 156:791, 2002.

Schmid, S.L. Conventional and unconventional aspects of dynamin GTPases. In: Handbook of Cellular Signaling: G Proteins. Bradshaw, R., Dennis, E. (Eds.). Academic Press, San Diego, in press.

Schmid, S.L., Sorkin, A.D. Days and knights discussing membrane dynamics in endocytosis: meeting report from the Euresco/EMBL Membrane Dynamics in Endocytosis, 6-11 October in Tomar, Portugal. Traffic 3:77, 2002.