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.