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First published online March 7, 2007
doi: 10.1242/10.1242/jcs.03403

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Different ADAMs have distinct influences on Kit ligand processing: phorbol-ester-stimulated ectodomain shedding of Kitl1 by ADAM17 is reduced by ADAM19

¹ Developmental Biology Program, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA
² Department of Orthopedic Surgery, Keio University, School of Medicine, Tokyo 160-8582, Japan
³ Department of Biochemical and Analytical Pharmacology, GlaxoSmithKline Inc., Research Triangle Park, NC 27709, USA
⁴ Arthritis and Tissue Degeneration Program, Hospital for Special Surgery, New York, NY 10021, USA
⁵ Departments of Medicine and of Physiology and Biophysics, Weill Medical College of Cornell University, New York, NY 10021, USA

View larger version (35K): [in this window] [in a new window]   Fig. 1. Effect of overexpression of ADAM19 on Kitl1 shedding. (A) Wild-type ADAM19 (lanes 1 and 2) or ADAM19 carrying an inactivating E>A mutation in its catalytic site (A19 EA) (lanes 3 and 4) were transfected into COS-7 cells together with AP-tagged Kitl1 (upper panel) or TGF (lower panel). Kitl1 shedding was stimulated with 25 ng/ml PMA as described in the Materials and Methods. Briefly, medium was collected before and after addition of PMA in the same well for 1 hour and analyzed on 8% SDS-PAGE. Comparison of cell lysates expressing Kitl1 or TGF and either ADAM19 or the E>A mutant showed similar expression levels of pro-TGF and two forms of Kitl1 (black and open arrowhead), whereas much less of the slowest migrating form of Kitl1 (indicated by an asterisk) was seen in cells expressing ADAM19 compared with cells expressing the E>A mutant. (B) Western-blot analysis of the expression of Kitl1 (upper panel) and ADAM19 or ADAM19 E>A (lower panel) in COS-7 cells. The slowest migrating form of Kitl1, which is not present in the sample overexpressing ADAM19, is indicated by an asterisk.

View larger version (42K): [in this window] [in a new window]   Fig. 2. ADAM17 is required for PMA- and PV-stimulated shedding of Kitl1 in mEFs, whereas ADAMs 8, 9, 10, 12 and 15 are dispensable. (A) Kitl1 shedding was analyzed in primary mEFs isolated from E13.5 wild-type controls (lanes 1, 2, 5, 6, 9 and 10) or from corresponding Adam8–/–, Adam9/12/15–/–, Adam17–/– embryos (lanes 3, 4, 7, 8, 11 and 12). Adam10–/– cells (lanes 3, 4, 7, 8, 11 and 12) and Adam10+/– controls (shown under wild type in lanes 1, 2, 5, 6, 9 and 10) were immortalized since Adam10–/– embryos die at E9.5. Whereas PMA- and PV-induced shedding was comparable between Adam8–/– and Adam9/12/15–/– mEFs and cells isolated from their respective wild-type controls, as well as between Adam10–/– and Adam10+/– cells, PMA-dependent increase in shedding of Kitl1 was abolished (lane 4) and PV-induced shedding was significantly reduced in Adam17–/– cells (lane 8) compared with cells from wild-type controls (lanes 2 and 6). In all Adam–/– and wild-type cells analyzed here, the BB94-sensitive component of constitutive Kitl1 shedding was unaffected (lanes 10 and 12 compared with lanes 9 and 11, respectively), suggesting that other metalloproteases besides the ADAMs tested here are responsible for constitutive Kitl1 shedding. (B) PMA-stimulated Kitl1 shedding in Adam17–/– cells (lane 2) could be rescued by coexpression of mouse ADAM17 (lane 4). A western blot of the cell lysates of Adam17–/– cells expressing Kitl1 (lane 5) or Kitl1 and ADAM17 (lane 6) is shown on the right. Overexpression of ADAM17 reduces the slowest migrating form of Kitl1 in Adam17–/– cells. All experiments were repeated four times with essentially identical results.

View larger version (44K): [in this window] [in a new window]   Fig. 3. Effect of overexpressed ADAM19 on levels of mature Kitl1. (A) COS-7 cells were transfected with AP-tagged Kitl1 (top panel), or with Kitl1 together with either the catalytically inactive ADAM19E>A mutant (second panel from the top), or wild-type ADAM19 (third panel from the top), or wild-type ADAM19 in the presence of the proteasome inhibitor MG132 (lower panel) labeled with [35S]-methionine for 30 minutes, and chased in OPTI-MEM medium. The cell lysates were collected at the indicated time points, immunoprecipitated with anti-Kitl antibody, and the immunoprecipitated material was separated by 8% SDS-PAGE and visualized by autoradiography. A black arrow indicates the mature form of Kitl1 (125 KDa), an asterisk is next to the intermediate form I (112.5 KDa) and a black arrowhead points to the intermediate form II (100 KDa). It should be noted that immunoprecipitated AP-tagged Kitl1 runs as a monomer on SDS-PAGE since it is reduced and boiled in SDS sample buffer. Therefore, it has a different mass than in the in-gel AP-assay shown in Fig. 1A, where AP-tagged Kitl1 runs as a dimer since the material is not boiled before SDS-PAGE. (B) Effect of ADAM19 on Kitl1 that can be biotinylated on the cell surface. Kitl1 was expressed in COS-7 cells together with either ADAM19 or ADAM19E>A, and cell surface proteins were labeled with a non-membrane-permeable biotinylation reagent as described in the Materials and Methods. The upper panel shows a western blot of Kitl1 in cell lysates, and the lower panel shows cell-surface biotinylated material that was immunoprecipitated with an antibody against the alkaline phosphatase moiety attached to Kitl1, transferred to nitrocellulose and probed with HRP-labeled streptavidin (avidin). The amount of Kitl1 that can be cell-surface biotinylated is reduced in the presence of ADAM19.

View larger version (21K): [in this window] [in a new window]   Fig. 4. Kitl2 shedding by ADAM17 and ADAM19. (A) The schematic presentation of AP-tagged wild-type Kitl1 and Kitl2. Arrows indicate the positions of the major proteolytic cleavage site at A164 and A165 in Kitl1. One of these sites, A165, corresponds to the Kitl1 cleavage site used by ADAM17 and ADAM19 in vitro [(23,26) see also Table 1]. Exon 6, which contains the putative major cleavage site, is spliced out in Kitl2. AP, alkaline phosphatase tag; CD, cytoplasmic domain; ECD, extracellular domain; JM, juxtamembrane domain; MH, Myc-His tag; TM, transmembrane domain. (B) AP-tagged Kitl2 was either expressed alone or together with wild-type ADAM19 or the catalytically inactive ADAM19E>A mutant (A19 EA) (lanes 3 and 4) in COS-7 cells, and shedding was stimulated with 25 ng/ml PMA. ADAM19 did not affect Kitl2 shedding into the medium or the levels of mature Kitl2 in cell lysates (indicated by an asterisk) compared with ADAM19E>A or cells expressing only Kitl2. (C) Kitl2 shedding in Adam17–/– and wild-type control mEFs. Both constitutive and PV-stimulated shedding of Kitl2 was strongly reduced in Adam17–/– cells (lanes 3 and 4) compared with wild-type control mEFs (lanes 1 and 2). The defect in Kitl2 shedding in Adam17–/– cells could be rescued by overexpressed wild-type mouse ADAM17 (lanes 7 and 8), confirming that ADAM17 is the major sheddase for both constitutive and PMA-stimulated shedding of Kitl2.

View larger version (46K): [in this window] [in a new window]   Fig. 5. Evaluation of how mutations in the juxtamembrane domain affect Kitl shedding. (A) Mutants carrying deletions in the juxtamembrane domain of Kitl1 or Kitl2 were generated as described in the Materials and Methods in order to identify the cleavage site(s) for ADAM17. The designations of individual deletion mutants are listed on the left. All expression constructs used in this study contain an N-terminal AP-tag and a C-terminal Myc-His tag. Exon 6, which contains the putative major cleavage site, is spliced out in Kitl2. TM, transmembrane domain. Two different kinds of vertical lines indicate the boundary for exons, and the boundary between the extracellular and transmembrane domain, respectively. (B) COS-7 cells were transfected with AP-tagged wild-type Kitl1, Kitl2 or various mutant plasmids, labeled with [35S]-methionine for 30 minutes and chased for 6 hours. Cell lysates were collected, immunoprecipitated with anti-Kitl antibody and treated with Endo H (E, lane 2) or PNGase F (P, lane 3) at 37°C overnight, as described in the Materials and Methods. Untreated sample was loaded in lane 1. Asterisks, black arrowheads and white arrowheads indicate the mature form (125 KDa), intermediate form II (112.5 KDa) and intermediate form I (100 KDa) of Kitl, respectively. The mature forms of Kitl in cells transfected with Kitl2 mutants were resistant to treatment with Endo H to a similar degree as wild-type Kitl2. Since Endo H resistance is acquired during passage through the medial Golgi compartment, these results indicate that similar amounts of the mutant and wild-type proteins were released from the ER and migrated through the medial Golgi compartment. This strongly suggests that the mutants giving rise to Endo H-resistant mature forms are properly folded, and that the lack of shedding of KL2SSTL/KAAK is not caused by retention of this mutant in the ER. Since the KL1DM mutant did not acquire resistance to Endo H it was not used for further shedding analysis. (C) Effects of deletions on Kitl shedding. AP-tagged deletion variants were transfected in wild-type (lanes 1 and 2) or Adam17–/– mEFs (lanes 3 and 4). Shedding was performed as described above, using 100 mM PV as a stimulus. Supernatants (lanes 1-4) were concentrated by ConA-Sepharose and analyzed on 8% SDS-PAGE as described in the Materials and Methods. An AP-analysis of cell lysates shows similar expression of mutant forms of Kitl2 in wild-type and Adam17–/– cells in cases in which shedding is abolished. Lower levels of Kitl2 mutants were seen in wild-type cell lysates following PV or PMA stimulation compared with Adam17–/– cells, presumably because the mutants accumulate in the absence of ADAM17.