Cells expressing alkaline phosphatase-tagged proEGF (AP-EGF) as well as catalytically inactive (CI) ADAM12, ADAM12 WT, or ADAM12 were tested for launch of AP-EGF in to the tradition moderate. a di-leucine theme, which is regarded as LY 344864 a potential mobile trafficking signal. Today’s research was motivated both from the potential relevance of the recorded mutation to tumor, as well for identifying the role from the di-leucine theme in ADAM12 trafficking. Manifestation of ADAM12 in mammalian cells proven quantitatively similar manifestation amounts and LY 344864 zymogen maturation as wild-type (WT) ADAM12, aswell as comparable mobile localizations. A cell surface area biotinylation assay proven that cell surface levels of ADAM12 WT and ADAM12 were similar and that internalization of the mutant occurred at the same rate and extent as for ADAM12 WT. Moreover, functional analysis revealed no differences in cell proliferation or ectodomain shedding of epidermal growth factor (EGF), a known ADAM12 substrate between WT and mutant ADAM12. These data suggest that the ADAM12 mutation is unlikely to be a driver (cancer causing)-mutation in breast LY 344864 cancer. Introduction ADAM12 is a member of the ADAMs (A Disintegrin And Metalloproteases) family of transmembrane zinc-dependent proteases with a characteristic domain structure (Fig. 1). ADAMs are involved in regulating integrin-mediated cell adhesion, cell signaling and the proteolytic release, known as ectodomain shedding of cell surface-associated substrates [1]C[4]. Several members of the ADAM family are highly expressed in a variety of human carcinomas, likely contributing to tumor development and/or progression through the release of epidermal growth factor receptor EGFR ligands or effects on cell-cell or cell-matrix adhesion [1], [3]. We, and others previously showed that ADAM12 expression was markedly upregulated in Rabbit Polyclonal to CD3 zeta (phospho-Tyr142) different cancers [5]C[9], and that the level of ADAM12 in urine from breast and bladder cancer patients correlated with disease status and stage [10], [11]. ADAM12 promotes tumor progression in transgenic mouse models of breast and prostate cancer [6], [12], [13] and several ADAMs are considered as promising targets for cancer therapy [14]C[16]. Despite accumulating evidence for involvement of ADAMs in cancer, only a few cancer-related ADAM mutations have been reported (see ref. [17] for complete list). Human ADAM12 exists in two naturally occurring splice variants; ADAM12-L resembling the prototypical transmembrane ADAM protein shown in figure 1 and ADAM12-S, a soluble splice variant, lacking the transmembrane domain and the cytoplasmic tail. In an analysis of genomic changes in breast cancer, three somatic heterozygous mutations were found in ADAM12, in the metalloprotease domain, in the disintegrin domain, and in the cytoplasmic domain (Fig. 1) [18]. Bioinformatic analysis predicted that only and were likely to be cancer-causing, since no changes were tolerated at these two positions [19]. Analysis of the and proteins strongly suggested misfolding of these mutants, since neither was secreted, both were retained LY 344864 in the endoplasmic reticulum (ER), and neither underwent zymogen maturation, a process mediated by LY 344864 furin that converts nascent 120-kDa ADAM12 to the mature 90-kDa form and occurs downstream of the ER [19]. The mutation is situated in the second (from the N-terminus) of two di-leucine motifs in ADAM12. Although the mutation was predicted to be inconsequential, it is of potential interest as it affects a di-leucine motif that is an important sorting signal in internalization and/or trafficking of several proteins (Fig. 1). Di-leucine motifs play critical roles in the sorting of many type I, type II, and multi-spanning transmembrane proteins, by associating with adaptor proteins, such as AP-complexes or Golgi-localized, -ear containing, ADP-ribosylation factor-binding proteins (GGAs) [20], [21]. Open in a separate window Figure 1 Schematic illustration of ADAM12 indicating the published breast cancer-associated mutations and the di-leucine motif in the cytoplasmic tail.Schematic illustration of the domain organization of ADAM12. S: Signal peptide; Pro: Prodomain; Met: Metalloprotease domain; Dis: Disintegrin domain; Cys: Cysteine-rich domain; EGF: EGF-like domain; TM: Transmembrane domain; Cyt: Cytoplasmic tail. Positions of the three breast cancer-associated mutations are indicated and the amino acid sequence containing the di-leucine motif is shown. We therefore sought to investigate if the mutation in the cytoplasmic domain of ADAM12 (since only ADAM12-L is affected, the nomenclature ADAM12 is used for the sake of simplicity) altered protein processing, trafficking and internalization with potential functional implications for cell proliferation and protein ectodomain shedding. Rigorous analysis suggested that the ADAM12 mutant was comparable to WT ADAM12 in every aspects analyzed, indicating that the di-leucine motif was not involved in the intracellular trafficking of ADAM12 and thus may not be contributing significantly to the breast cancer phenotype. Results and Discussion The ADAM12 wild-type and mutant are similarly expressed and processed To evaluate.