TY - JOUR

T1 - Cell-surface metalloprotease ADAM12 is internalized by a clathrin- and Grb2-dependent mechanism

AU - Hansen, Dorte Stautz

AU - Leyme, Anthony

AU - Grandal, Michael Vibo

AU - Albrechtsen, Reidar

AU - van Deurs, Bo

AU - Wewer, Ulla

AU - Kveiborg, Marie

N1 - © 2012 John Wiley & Sons A/S.

PY - 2012

Y1 - 2012

N2 - 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.

AB - 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.

KW - ADAM Proteins

KW - Breast Neoplasms

KW - Carcinoma

KW - Clathrin

KW - Endocytosis

KW - Endosomes

KW - Female

KW - GRB2 Adaptor Protein

KW - HEK293 Cells

KW - Humans

KW - Membrane Proteins

KW - Proline-Rich Protein Domains

KW - Protein Interaction Domains and Motifs

U2 - 10.1111/j.1600-0854.2012.01405.x

DO - 10.1111/j.1600-0854.2012.01405.x

M3 - Journal article

C2 - 22882974

VL - 13

SP - 1532

EP - 1546

JO - Traffic

JF - Traffic

SN - 1398-9219

IS - 11

ER -