Pathfinding axons change responses to guidance cues at intermediate targets. During midline crossing, spinal cord commissural axons acquire responsiveness to class 3 Semaphorins and Slits, which regulate their floor plate exit and restrict their post-crossing trajectory into a longitudinal pathway. We found that Sonic Hedgehog (Shh) could activate the repulsive response of pre-crossing axons to Semaphorins. Blocking Shh function with a monoclonal antibody to Shh, 5E1, in 'open-book' explants or by expressing a dominant-negative form of Patched-1, Ptch1(Delta loop2), or a Smoothened (Smo) shRNA construct in commissural neurons resulted in severe guidance defects, including stalling and knotting inside the floor plate, recrossing, randomized anterior-posterior projection and overshooting after crossing, reminiscent of Neuropilin-2 mutant embryos. Enhancing protein kinase A activity in pre-crossing axons diminished Shh-induced Semaphorin repulsion and caused profound midline stalling and overshooting/wandering of post-crossing axons. Therefore, a morphogen, Shh, can act as a switch of axon guidance responses.

Background: Intracellular domain (ICD) modifications regulate extracellular ectodomain cleavage by metalloproteases. How this inside-out signal is relayed is unknown. Results: Cleavage requires substrate homodimerization; ICD modifications likely induce a relative positional change of the dimerization partners, allowing cleavage. Conclusion: Substrate dimerization might be a general requirement for cleavage. Significance: Our results fill an important gap in understanding growth factor release by ectodomain cleavage.

Ectodomain cleavage (shedding) of transmembrane proteins by metalloproteases (MMP) generates numerous essential signaling molecules, but its regulation is not totally understood. CD44, a cleaved transmembrane glycoprotein, exerts both antiproliferative or tumor-promoting functions, but whether proteolysis is required for this is not certain. CD44-mediated contact inhibition and cellular proliferation are regulated by counteracting CD44 C-terminal interacting proteins, the tumor suppressor protein merlin (NF2) and ERM proteins (ezrin, radixin, moesin). We show here that activation or overexpression of constitutively active merlin or downregulation of ERMs inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced [as well as serum, hepatocyte growth factor (HGF), or plateletderived growth factor (PDGF)] CD44 cleavage by the metalloprotease ADAM10, whereas overexpressed ERM proteins promoted cleavage. Merlin-and ERM-modulated Ras or Rac activity was not required for this function. However, latrunculin (an actin-disrupting toxin) or an ezrin mutant which is unable to link CD44 to actin, inhibited CD44 cleavage, identifying a cytoskeletal C-terminal link as essential for induced CD44 cleavage. Cellular migration, an important tumor property, depended on CD44 and its cleavage and was inhibited by merlin. These data reveal a novel function of merlin and suggest that CD44 cleavage products play a tumor-promoting role. Neuregulin, an EGF ligand released by ADAM17 from its proform NRG1, is predominantly involved in regulating cellular differentiation. In contrast to CD44, release of neuregulin from its pro-form was not regulated by merlin or ERM proteins. Disruption of the actin cytoskeleton however, also inhibited NRG1 cleavage. This current study presents one of the first examples of substrate-selective cleavage regulation.

bEctodomain cleavage by A-disintegrin and -metalloproteases (ADAMs) releases many important biologically active substrates and is therefore tightly controlled. Part of the regulation occurs on the level of the enzymes and affects their cell surface abundance and catalytic activity. ADAM-dependent proteolysis occurs outside the plasma membrane but is mostly controlled by intracellular signals. However, the intracellular domains (ICDs) of ADAM10 and -17 can be removed without consequences for induced cleavage, and so far it is unclear how intracellular signals address cleavage. We therefore explored whether substrates themselves could be chosen for proteolysis via ICD modification. We report here that CD44 (ADAM10 substrate), a receptor tyrosine kinase (RTK) coreceptor required for cellular migration, and pro-NRG1 (ADAM17 substrate), which releases the epidermal growth factor (EGF) ligand neuregulin required for axonal outgrowth and myelination, are indeed posttranslationally modified at their ICDs. Tetradecanoyl phorbol acetate (TPA)-induced CD44 cleavage requires dephosphorylation of ICD serine 291, while induced neuregulin release depends on the phosphorylation of several NRG1-ICD serines, in part mediated by protein kinase C␦ (PKC␦). Downregulation of PKC␦ inhibits neuregulin release and reduces ex vivo neurite outgrowth and myelination of trigeminal ganglion explants. Our results suggest that specific selection among numerous substrates of a given ADAM is determined by ICD modification of the substrate. Many transmembrane proteins on the cell surface are subject to proteolytic cleavage of their ectodomains, predominantly by metalloproteases (ectodomain shedding) (1-3). Ectodomain shedding regulates numerous important molecules involved in signal transfer between the extracellular space and the cell's interior and thus influences many cellular functions (1, 3). This includes, for example, the biological availability of epidermal growth factor (EGF) receptor ligands such as neuregulin (NRG1) (4, 5) and the modulation of complex cellular phenotypes required for contact inhibition of cells involving the hyaluronic acid receptor CD44 (4). NRG1 regulates neurite outgrowth and myelination but also has important functions in the development of other organs, for instance, the heart (6-9). When bound to hyaluronan, CD44 triggers a proliferation-inhibitory pathway (10-12). On the other hand, cancer stem cells carry CD44 (13-15), and, in this context, CD44 promotes tumor growth and metastasis (16-21), likely via alternative splice forms of CD44 that act as growth factor-enriching coreceptors for receptor tyrosine kinases (RTKs) (22,23).Inappropriate proteolysis of a number of shed substrates is associated with diseases when cleavage is either upregulated or reduced (24, 25). Equally, total knockout of substrates leads to significant phenotypes (26,27). This indicates that ectodomain cleavage requires tight regulation. How ectodomain cleavage is regulated and made substrate specific is largely unknown to date.The metallop...

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Release of cytokines, growth factors and other life-essential molecules from precursors by a-disintegrin-and-metalloproteases (ADAMs) is regulated with high substrate-specificity. We hypothesized that this is achieved by cleavage-regulatory intracellular-domain (ICD)-modifications of the precursors. We show here that cleavage-stimuli-induced specific ICD-modifications cause structural substrate changes that enhance ectodomain sensitivity of neuregulin-1 (NRG1; epidermal-growth-factor) or CD44 (receptor-tyrosine-kinase (RTK) co-receptor) to chymotrypsin/trypsin or soluble ADAM. This inside-out signal transfer required substrate homodimerization and was prevented by cleavage-inhibitory ICD-mutations. In chimeras, regulation could be conferred to a foreign ectodomain, suggesting a common higher-order structure. We predict that substrate-specific protease-accessibility-regulation controls release of numerous ADAM substrates.

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

<p>Supplemental Figures S1-S7: Supplemental Figure S1: The effect of γ-secretase inhibition on ectodomain cleavage Supplemental Figure S2. Regulated cleavage targets substrates after surface expression Supplemental Figure S3: Interaction between CD44 and Merlin. Supplemental Figure S4: The tumor suppressor merlin (Nf2) inhibits CD44 cleavage. Supplemental Figure S5. Exemplified analysis of the constructs used in the migration assay of Figure 7A and Supplemental Figure 6 Supplemental Figure S6: Examplified photographs of one of the scratch assays with MEFs as shown in Figure 7A. Supplemental Figure S7: Loading diagrams of Figures 3B and 4C</p>