Abstract 1577: Inside-out regulation of ectodomain protease accessibility in the release of cytokines

Abstract Growth factors of the EGF family and numerous other growth factors and cytokines are produced as transmembrane precursor proteins and released by proteolysis. Also growth factor receptors, adhesion molecules, and possibly all membrane proteins carrying ectodomains are subject to proteolytic cleavage. Because these reactions produce many regulatory molecules, such cleavages must be precisely regulated. Only a small number of enzymes, metalloproteases, serve to perform defined cleavages of ectodomains. Most efforts to understand regulation of ectodomain cleavage have been directed towards elucidating protease function and activity, predominantly of proteases of the ADAM (A-Disintegrin-And-Metalloprotease) family. Given that a very limited number of ADAMs need to handle hundreds of substrates, regulation of enzyme activity does not generate the specificity and precise timing of life-essential processes as growth factor releases. We have hypothesized that the specificity can only be determined on the level of the substrates. In a large screen we have identified intracellular regulatory components that affect the extracellular cleavage of EGF family members (Dang et al., 2013). These data revealed substrate-specific pathways. Substrates of the same enzyme, ADAM17, the precursors of TGF-α, heparin-binding EGF, and amphiregulin, are regulated by PKC-α and the PKC-regulated protein phosphatase 1 inhibitor 14D, while neuregulin release requires PKC-δ. By examining the ectodomain cleavage of NRG1 (an ADAM17 substrate) and CD44 (an ADAM10 substrate), we have explored in detail the mechanism of regulation. By comparing two metalloprotease-cleaved substrates, we show that 1) regulation occurs on the level of the substrates 2) regulated cleavage is executed after the substrate has reached the plasma membrane 3) modification of the intracellular domains alters protease accessibility of the substrate's ectodomains 4) inside-out signal transfer from the C-terminus to the ectodomain of the substrate requires substrate dimerization. Our data are related to several aspects of cancer development and progression. It has been postulated that inhibiting metalloproteases could inhibit growth and metastasis of cancer cells. Expectedly, such inhibitors cannot generate enough specificity and numerous side effects are to be expected. Intracellular specific pathways as addressed by our data offer more specific targets of interference. Another interesting aspect: the adhesion molecule and co-receptor CD44 including its alternative splice forms play tumor-promoting roles. Interestingly, CD44 ectodomain cleavage is inhibited by the tumor suppressor protein merlin (neurofibromatosis type 2), suggesting that CD44 ectodomain cleavage might have a role in cancer progression. Citation Format: Monika Hartmann, Liseth Parra, Sandra Schubert, Yong Li, Helen Morrison, Christoph Kaether, Andreas Herrlich, Peter Herrlich. Inside-out regulation of ectodomain protease accessibility in the release of cytokines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1577. doi:10.1158/1538-7445.AM2014-1577