title: Inhibition of the receptor-binding function of clathrin adaptor protein AP-2 by dominant-negative mutant mu2 subunit and its effects on endocytosis.
abstract: Although interactions between the mu2 subunit of the clathrin adaptor protein complex AP-2 and tyrosine-based internalization motifs have been implicated in the selective recruitment of cargo molecules into coated pits, the functional significance of this interaction for endocytosis of many types of membrane proteins remains unclear. To analyze the function of mu2-receptor interactions, we constructed an epitope-tagged mu2 that incorporates into AP-2 and is targeted to coated pits. Mutational analysis revealed that Asp176 and Trp421 of mu2 are involved in the interaction with internalization motifs of TGN38 and epidermal growth factor (EGF) receptor. Inducible overexpression of mutant mu2, in which these two residues were changed to alanines, resulted in metabolic replacement of endogenous mu2 in AP-2 complexes and complete abrogation of AP-2 interaction with the tyrosine-based internalization motifs. As a consequence, endocytosis of the transferrin receptor was severely impaired. In contrast, internalization of the EGF receptor was not affected. These results demonstrate the potential usefulness of the dominant-interfering approach for functional analysis of the adaptor protein family, and indicate that clathrin-mediated endocytosis may proceed in both a mu2-dependent and -independent manner.
authors.affiliation: Department of Medicine, University of California at San Diego, La Jolla, CA 92093, USA.
authors.forename: A
authors.initials: A
authors.lastname: Nesterov
authors.forename: R E
authors.initials: RE
authors.lastname: Carter
authors.forename: T
authors.initials: T
authors.lastname: Sorkina
authors.forename: G N
authors.initials: GN
authors.lastname: Gill
authors.forename: A
authors.initials: A
authors.lastname: Sorkin
mesh_terms: D033961:Adaptor Protein Complex 1; D033962:Adaptor Protein Complex 2; D033963:Adaptor Protein Complex 3; D033965:Adaptor Protein Complex alpha Subunits; D033983:Adaptor Protein Complex mu Subunits; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D002451:Cell Compartmentation; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism; D004705:Endocytosis; D066246:ErbB Receptors / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D011485:Protein Binding; D015202:Protein Engineering; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011990:Receptors, Transferrin / Q000378:metabolism
chemical_list: C499064:AP1M2 protein, human; C510070:AP3M2 protein, human; D033961:Adaptor Protein Complex 1; D033962:Adaptor Protein Complex 2; D033963:Adaptor Protein Complex 3; D033965:Adaptor Protein Complex alpha Subunits; D033983:Adaptor Protein Complex mu Subunits; D033942:Adaptor Proteins, Vesicular Transport; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; D011990:Receptors, Transferrin; C499065:adaptor protein complex 1, mu 2 subunit; C438886:adaptor protein complex 2, mu 2 subunit; D066246:ErbB Receptors