Figure 1.
Schematic representation of the domain structure of ADAM10.
(A) The cytoplasmic tail at the C-terminus harbors two proline-rich regions (PRR) that might enable binding to SH3 or WW domain-containing proteins. The intracellular parts of human and murine ADAM10 are highly conserved and differ in only one amino acid. (B) Modular composition, maturation and processing of ADAM10. The N-terminal signal sequence of the protease is needed for intracellular maturation (1→2). To generate an enzymatically active protease, the pro-domain has to be removed by a protein convertase such as furin (2→3). The catalytic metalloproteinase domain is the largest domain of ADAM10 and might be activated by different signals including substrate-induced conformational changes at the plasma membrane (3→4). The membrane-proximal region is important for adhesion and substrate recognition and contains a disintegrin, a cysteine-rich and an EGF-like domain. ADAM10 itself is subjected to proteolysis by ADAM9 or ADAM15 and the γ–secretase complex (4→5). The fate and functions of the soluble ectodomain or the resulting C-terminal fragments are still unclear. Notably, intracellular regions at all stages might interact with individual SH3 domain-containing interaction partners.
Table 1.
GST-hADAM10(697-748)-precipitated SH3 domains: Function and localization of putative interaction partners according to the UniProt Protein Knowledgebase (UniprotKB).
Table 2.
GST/GST-hADAM10(697–748)- and GST-interacting SH3 domains.
Figure 2.
Verification of the interaction between ADAM10 and EEN.
(A) In order to verify the potential interaction of ADAM10 with Endophilin-A2/EEN, HEK 293T cells were either left untransfected or transfected with HA-tagged murine ADAM10 alone or in combination with human Endophilin-A2/EEN. 18 h later, the cells were lysed and immunoprecipitations (IPs) were performed with monoclonal antibodies directed against the HA-tag (clone 3F10) or EEN (clone 2F5), respectively. Protein input for IPs was 1.8 or 2 mg of protein, respectively. Of note: at the employed exposure time, endogenous EEN is hardly detectable in the whole cell lysates containing a total of 10 µg of protein. (B) Pull down analyses were performed from PHA blasts (day 16) using a GST fusion protein containing the SH3 domain of EEN coupled to GST (EEN SH3) and GST as a control. The subsequent Western blot was probed with anti-ADAM10 (clone 11G2).
Figure 3.
Interactions between ADAM10 and non-receptor protein tyrosine kinases and adaptor proteins of the Grb2 family.
(A) Lysates from Jurkat T cells (here JFL) were used for pull down analyses with SH2 or SH3 domain fusion proteins of non-receptor PTKs as indicated. GST served as a negative control. Protein input of the whole cell lysate was 15 µg. 10 µg of the respective fusion proteins were used for precipitation from 1 ml of cell lysate with 3.2 mg/ml of protein. MAb 11G2 was used to detect ADAM10 after Western blotting. (B) Immunoprecipitations were performed from Jurkat T cells (here JE6-1) using 2 µg of mAbs against Lck (clones 4/129 and 4/215) or ADAM10 (clone 11G2). Protein G beads served as a control for unspecific binding. Input of the cellular lysate was 15 µg; precipitates were performed from 1 ml of lysate (1 mg/ml protein). MAb 11G2 was used to detect ADAM10 by Western blotting. (C) Jurkat cells (JE6-1) were lysed and one ml of lysate containing 900 µg/ml protein was subjected to precipitation using 10 µg GST or the GST fusion protein containing the intracellular part of human ADAM10 (hADAM10(697–748)). In parallel, ADAM10 was precipitated using 2 µg/ml of mAb 11G2 with protein G beads serving as a control. Precipitated proteins were separated by SDS-PAGE and blotted on nitrocellulose. The blot was stained with a polyclonal anti-Grb2 antibody and re-probed with a polyclonal anti-ADAM10 antiserum (“animal 1”). (D) C- and N-terminal SH3 domains of Grb2, GRAP and GRAP2 fused to GST (10 µg each) were used for precipitations from Jurkat T cells (here JFL; 2.2 mg/ml protein input per precipitation) with GST alone serving as a control. 15 µg protein of the whole cell lysate were included as a reference. ADAM10 was detected with mAb 11G2.
Figure 4.
The intracellular domains of ADAM10 and ADAM17 interact with sorting nexins and PACSINs.
(A) HEK 293T cells were either left untransfected or were transfected with a control vector (p12linker) or with HA-tagged SNX9, SNX18 or SNX33. 18 h later, cells were lysed and precipitations were performed with 25 µg of GST, GST-hADAM10(697–748) or GST-hADAM17(694–824), respectively. Western blots were developed using mAb 3F10 directed against the HA-tag of the sorting nexins. Lysates (20 µg/lane) were stained as a control. (B) HEK 293T cells were left untransfected or transfected with control vector (pcDNA3.1) or with myc-tagged PACSIN1, PACSIN2 or PACSIN3. 18 h post transfection, cells were lysed and precipitations were performed with 10 µg of GST, GST-hADAM10(697–748) or GST-hADAM17(694–824). Precipitated proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Blots were developed with mAb clone 46–0603 directed against the myc-tag. Of the whole cell lysates, 5 µg of protein were separated to verify efficient transfection. (C) Recombinant GST fusion proteins (15 µg) containing individual SH3 domains of PACSIN1-3 were used to precipitate endogenous ADAM10 from lysates of Jurkat T cells (JFL, 1 ml each; 2.2 mg/ml). GST served as a negative control. 10 µg of whole cell lysate was used as a control to detect ADAM10 using mAb 11G2.