title: Cell-surface metalloprotease ADAM12 is internalized by a clathrin- and Grb2-dependent mechanism.
abstract: ADAM12 (A Disintegrin And Metalloprotease 12), a member of the ADAMs family of transmembrane proteins, is involved in ectodomain shedding, cell-adhesion and signaling, with important implications in cancer. Therefore, mechanisms that regulate the levels and activity of ADAM12 at the cell-surface are possibly crucial in these contexts. We here investigated internalization and subsequent recycling or degradation of ADAM12 as a potentially important regulatory mechanism. Our results show that ADAM12 is constitutively internalized primarily via the clathrin-dependent pathway and is subsequently detected in both early and recycling endosomes. The protease activity of ADAM12 does not influence this internalization mechanism. Analysis of essential elements for internalization established that proline-rich regions in the cytoplasmic domain of ADAM12, previously shown to interact with Src-homology 3 domains, were necessary for proper internalization. These sites in the ADAM12 cytoplasmic domain interacted with the adaptor protein growth factor receptor-bound protein 2 (Grb2) and knockdown of Grb2 markedly reduced ADAM12 internalization. These studies establish that internalization is indeed a mechanism that regulates ADAM cell surface levels and show that ADAM12 internalization involves the clathrin-dependent pathway and Grb2.
authors.affiliation: Department of Biomedical Sciences & Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark.
authors.forename: Dorte
authors.initials: D
authors.lastname: Stautz
authors.forename: Anthony
authors.initials: A
authors.lastname: Leyme
authors.forename: Michael Vibo
authors.initials: MV
authors.lastname: Grandal
authors.forename: Reidar
authors.initials: R
authors.lastname: Albrechtsen
authors.forename: Bo
authors.initials: B
authors.lastname: van Deurs
authors.forename: Ulla
authors.initials: U
authors.lastname: Wewer
authors.forename: Marie
authors.initials: M
authors.lastname: Kveiborg
mesh_terms: D051722:ADAM Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D000072199:ADAM12 Protein; D001943:Breast Neoplasms / Q000737:chemistry / Q000201:enzymology; D002277:Carcinoma / Q000737:chemistry; D002966:Clathrin / Q000378:metabolism*; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D005260:Female; D051380:GRB2 Adaptor Protein / Q000032:analysis / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D055232:Proline-Rich Protein Domains; D054730:Protein Interaction Domains and Motifs
chemical_list: D002966:Clathrin; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D008565:Membrane Proteins; D051722:ADAM Proteins; D000072199:ADAM12 Protein; C000606480:ADAM12 protein, human