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Inside-out Regulation of Ectodomain Cleavage of Cluster-of-Differentiation-44 (CD44) and of Neuregulin-1 Requires Substrate Dimerization*

  • Monika Hartmann
    Footnotes
    Affiliations
    From the Leibniz Institute for Age Research, Fritz Lipmann Institute, 07745 Jena, Germany and
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  • Liseth M. Parra
    Affiliations
    From the Leibniz Institute for Age Research, Fritz Lipmann Institute, 07745 Jena, Germany and

    the Harvard Institutes of Medicine, Renal Division, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusett 02115
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  • Anne Ruschel
    Affiliations
    From the Leibniz Institute for Age Research, Fritz Lipmann Institute, 07745 Jena, Germany and
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  • Christina Lindner
    Affiliations
    From the Leibniz Institute for Age Research, Fritz Lipmann Institute, 07745 Jena, Germany and
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  • Helen Morrison
    Affiliations
    From the Leibniz Institute for Age Research, Fritz Lipmann Institute, 07745 Jena, Germany and
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  • Andreas Herrlich
    Footnotes
    Affiliations
    the Harvard Institutes of Medicine, Renal Division, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusett 02115
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  • Peter Herrlich
    Correspondence
    Supported by Grant DFGHE551 from the German Research Foundation. To whom correspondence should be addressed: Leibniz Institute for Age Research, Beutenbergstr. 11, 07745 Jena, Germany. Tel.: 49-3641-656257;
    Footnotes
    Affiliations
    From the Leibniz Institute for Age Research, Fritz Lipmann Institute, 07745 Jena, Germany and
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grant R00DK077731 from NIDDK (to A. H.). This work was also supported by our institutions. The authors declare that they have no conflicts of interest with the contents of this article.
    ♦ This article was selected as a Paper of the Week.
    1 Supported by a fellowship from the Ernst-Jung Foundation.
    2 Both authors are co-senior authors.
Open AccessPublished:April 29, 2015DOI:https://doi.org/10.1074/jbc.M114.610204
      Ectodomain shedding of transmembrane precursor proteins generates numerous life-essential molecules, such as epidermal growth factor receptor ligands. This cleavage not only releases the regulatory growth factor, but it is also the required first step for the subsequent processing by γ-secretase and the release of gene regulatory intracellular fragments. Signaling within the cell modifies the cytoplasmic tails of substrates, a step important in starting the specific and regulated cleavage of a large number of studied substrates. Ectodomain cleavage occurs, however, on the outside of the plasma membrane and is carried out by membrane-bound metalloproteases. How the intracellular domain modification communicates with the ectodomain of the substrate to allow for cleavage to occur is unknown. Here, we show that homodimerization of a cluster-of-differentiation-44 or of pro-neuregulin-1 monomers represents an essential pre-condition for their regulated ectodomain cleavage. Both substrates are associated with their respective metalloproteases under both basal or cleavage-stimulated conditions. These interactions only turn productive by specific intracellular signal-induced intracellular domain modifications of the substrates, which in turn regulate metalloprotease access to the substrates' ectodomain and cleavage. We propose that substrate intracellular domain modification induces a relative rotation or other positional change of the dimerization partners that allow metalloprotease cleavage in the extracellular space. Our findings fill an important gap in understanding substrate-specific inside-out signal transfer along cleaved transmembrane proteins and suggest that substrate dimerization (homo- or possibly heterodimerization) might represent a general principle in ectodomain shedding.
      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.

      Introduction

      Proteolytic processing of transmembrane proteins yields a number of regulatory molecules with significance for the organism (e.g. growth factors, cytokines, decoy receptors, or nuclear transcription factors). Ectodomain cleavage by membrane-bound metalloproteases represents the initiating step required for the subsequent processing by γ-secretase and the release of the intracellular fragment. Regulation of ectodomain cleavage is highly important for homeostasis of the organism, most apparent in the release of active growth factors from their transmembrane precursors, which needs to be tightly regulated in respect to both its overall abundance and time course (
      • van der Vorst E.P.
      • Keijbeck A.A.
      • de Winther M.P.
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      A disintegrin and metalloproteases: molecular scissors in angiogenesis, inflammation and atherosclerosis.
      ,
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      • Bischoff R.
      Active metalloproteases of the a disintegrin and metalloprotease (ADAM) family: biological function and structure.
      ). Cleavage is controlled on several levels as follows: expression of both the enzymes and the precursor substrates; their transport to the cell surface and their glycosylation; and by activation and inactivation of the metalloprotease (
      • Hartmann M.
      • Herrlich A.
      • Herrlich P.
      Who decides when to cleave an ectodomain?.
      ). It has been shown that cleavage is also regulated by specific intracellular domain (ICD)
      The abbreviations used are: ICD
      intracellular domain
      MEF
      mouse embryonic fibroblast
      ADAM
      a disintegrin and metalloprotease
      CD44
      cluster-of-differentiation-44
      AngII
      angiotensin II
      CHAPSO
      3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid
      PFA
      paraformaldehyde
      DAPT
      difluorophenylacetylalanylphenylglycine t-butyl ester
      BiFC
      bimolecular fluorescence complementation
      Fw
      forward
      Rev
      reverse
      FKBP
      FK506-binding protein.
      substrate modifications or protein interactions (
      • Dang M.
      • Armbruster N.
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      • Hartmann M.
      • Bell G.W.
      • Root D.E.
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      Regulated ADAM17-dependent EGF family ligand release by substrate-selecting signaling pathways.
      ,
      • Dang M.
      • Dubbin K.
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      Epidermal growth factor (EGF) ligand release by substrate-specific a disintegrin and metalloproteases (ADAMs) involves different protein kinase C (PKC) isoenzymes depending on the stimulus.
      ,
      • Hartmann M.
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      • Schubert S.
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      Tumor suppressor Nf2 blocks cellular migration by inhibiting ectodomain cleavage of Cd44.
      ) that induce protease accessibility of the substrate's ectodomain, at least in the substrates examined so far (L-selectin (
      • Killock D.J.
      • Ivetić A.
      The cytoplasmic domains of TNFα-converting enzyme (TACE/ADAM17) and L-selectin are regulated differently by p38 MAPK and PKC to promote ectodomain shedding.
      ,
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      In vitro and in vivo characterization of molecular interactions between calmodulin, Ezrin/Radixin/Moesin, and L-selectin.
      ); NRG1 (
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      • Bell G.W.
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      • Lodish H.F.
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      Regulated ADAM17-dependent EGF family ligand release by substrate-selecting signaling pathways.
      ); TGFα precursor and CD44 (
      • Hartmann M.
      • Parra L.M.
      • Ruschel A.
      • Schubert S.
      • Li Y.
      • Morrison H.
      • Herrlich A.
      • Herrlich P.
      Tumor suppressor Nf2 blocks cellular migration by inhibiting ectodomain cleavage of Cd44.
      ).
      L. M. Parra, M. Hartmann, S. Schubach, Y. Li, P. Herrlich, and A. Herrlich, submitted for publication.
      Based on our own results and published work of others, it appears that substrate ICDs are the end points of different intracellular signaling pathways specific for each substrate. These signals are mostly generated by extracellular stimulation of G-protein-coupled receptors or receptor tyrosine kinases (
      • Gooz M.
      ADAM-17: the enzyme that does it all.
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      Regulation of the proteolytic disintegrin metalloproteinases, the “Sheddases”.
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      The role of protease activity in ErbB biology.
      • Mendelson K.
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      Stimulation of platelet-derived growth factor receptor β (PDGFRβ) activates ADAM17 and promotes metalloproteinase-dependent cross-talk between the PDGFRβ and epidermal growth factor receptor (EGFR) signaling pathways.
      ) and often involve intracellular calcium influx and the release of diacylglycerol, which activates protein kinase C (
      • Arribas J.
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      Transforming growth factor-α and β-amyloid precursor protein share a secretory mechanism.
      ,
      • Fan H.
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      Ectodomain shedding of TGF-α and other transmembrane proteins is induced by receptor tyrosine kinase activation and MAP kinase signaling cascades.
      • Prenzel N.
      • Zwick E.
      • Daub H.
      • Leserer M.
      • Abraham R.
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      • Ullrich A.
      EGF receptor transactivation by G-protein-coupled receptors requires metalloproteinase cleavage of proHB-EGF.
      ). Thus, intracellular signaling pathways regulate a process (known as substrate protease accessibility) that occurs outside of the plasma membrane. Because many substrates are single-pass transmembrane proteins, this begs the following question. How can a single-pass transmembrane protein transmit and execute a structural change of the ectodomain via intracellular signal-induced ICD modification?
      Signal transfer along single-pass transmembrane proteins is not well understood. In the case of receptor tyrosine kinases, signal transfer occurs from outside to inside mediated by receptor dimers or trimers that are formed in the endoplasmic reticulum prior to transport to the plasma membrane (
      • Gadella Jr., T.W.
      • Jovin T.M.
      Oligomerization of epidermal growth factor receptors on A431 cells studied by time-resolved fluorescence imaging microscopy. A stereochemical model for tyrosine kinase receptor activation.
      • Tao R.-H.
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      All EGF(ErbB) receptors have preformed homo- and heterodimeric structures in living cells.
      ,
      • Chung I.
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      Spatial control of EGF receptor activation by reversible dimerization on living cells.
      • Lemmon M.A.
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      Cell signaling by receptor tyrosine kinases.
      ). Extracellular ligand binding causes phosphorylation at tyrosine residues in the cytoplasmic tails of the receptors. Crystal structures of the ligand-bound and -unbound molecules show the start and end conformations of both the extracellular and intracellular domains (
      • Ferguson K.M.
      • Berger M.B.
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      EGF activates its receptor by removing interactions that autoinhibit ectodomain dimerization.
      ,
      • Jura N.
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      Mechanism for activation of the EGF receptor catalytic domain by the juxtamembrane segment.
      ). But how is this ligand-induced signal transfer through the membrane accomplished? An interesting early proposal assumed that the ligand triggers rotation of one receptor monomer relative to the other (
      • Gadella Jr., T.W.
      • Jovin T.M.
      Oligomerization of epidermal growth factor receptors on A431 cells studied by time-resolved fluorescence imaging microscopy. A stereochemical model for tyrosine kinase receptor activation.
      ,
      • Moriki T.
      • Maruyama H.
      • Maruyama I.N.
      Activation of preformed EGF receptor dimers by ligand-induced rotation of the transmembrane domain.
      ). Very recent NMR and molecular simulation data of the EGF receptor propose a ligand-induced change in the orientation of the transmembrane helices relative to each other, a process that affects the positioning of juxtamembrane sections of the dimer at the inner side of the plasma membrane, allowing phosphorylation and activation of the receptor (
      • Endres N.F.
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      Conformational coupling across the plasma membrane in activation of the EGF receptor.
      ,
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      Architecture and membrane interactions of the EGF receptor.
      ).
      Could such outside-in signaling be a model for signaling in the opposite direction, as needed here for the inside-out signal transfer along ADAM substrates, and similar to that previously described for integrin heterodimers (
      • Hynes R.O.
      Integrins: bidirectional, allosteric signaling machines.
      )? One prediction of this model would be that two single-pass transmembrane molecules (including the substrate) needed to change their position relative to each other, allowing cleavage.
      We have tested this prediction by exploring ectodomain cleavage of the ADAM10 substrate CD44 (an adhesion molecule and stem cell marker) and the ADAM17 substrate NRG1 (proform of the epidermal growth factor (EGF) receptor ligand neuregulin). When bound to hyaluronan, CD44 triggers a proliferation-inhibitory pathway (
      • Jin H.
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      Tumorigenic transformation by CPI-17 through inhibition of a merlin phosphatase.
      ,
      • Tian X.
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      High-molecular-mass hyaluronan mediates the cancer resistance of the naked mole rat.
      • Morrison H.
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      • Gutmann D.H.
      • Ponta H.
      • Herrlich P.
      The NF2 tumor suppressor gene product, merlin, mediates contact inhibition of growth through interactions with CD44.
      ). However, depending on the context (
      • Al-Hajj M.
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      Prospective identification of tumorigenic breast cancer cells.
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      Highly purified CD44+ prostate cancer cells from xenograft human tumors are enriched in tumorigenic and metastatic progenitor cells.
      ,
      • Pietras A.
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      • Wee B.
      • Halliday J.J.
      • Pitter K.L.
      • Werbeck J.L.
      • Amankulor N.M.
      • Huse J.T.
      • Holland E.C.
      Osteopontin-CD44 signaling in the glioma perivascular niche enhances cancer stem cell phenotypes and promotes aggressive tumor growth.
      • Zöller M.
      CD44: can a cancer-initiating cell profit from an abundantly expressed molecule?.
      ), it can also promote tumor growth and metastasis (
      • Todaro M.
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      • Catalano V.
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      CD44v6 is a marker of constitutive and reprogrammed cancer stem cells driving colon cancer metastasis.
      • Godar S.
      • Ince T.A.
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      • Feldser D.
      • Donaher J.L.
      • Bergh J.
      • Liu A.
      • Miu K.
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      • Reinhardt F.
      • McAllister S.S.
      • Jacks T.
      • Weinberg R.A.
      Growth-inhibitory and tumor-suppressive functions of p53 depend on its repression of CD44 expression.
      ,
      • Matzke A.
      • Herrlich P.
      • Ponta H.
      • Orian-Rousseau V.
      A five-amino-acid peptide blocks Met- and Ron-dependent cell migration.
      ,
      • Günthert U.
      • Hofmann M.
      • Rudy W.
      • Reber S.
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      • Haussmann I.
      • Matzku S.
      • Wenzel A.
      • Ponta H.
      • Herrlich P.
      A new variant of glycoprotein CD44 confers metastatic potential to rat carcinoma cells.
      ,
      • Yu Q.
      • Toole B.P.
      • Stamenkovic I.
      Induction of apoptosis of metastatic mammary carcinoma cells in vivo by disruption of tumor cell surface CD44 function.
      • Zeilstra J.
      • Joosten S.P.
      • van Andel H.
      • Tolg C.
      • Berns A.
      • Snoek M.
      • van de Wetering M.
      • Spaargaren M.
      • Clevers H.
      • Pals S.T.
      Stem cell CD44v isoforms promote intestinal cancer formation in Apc(min) mice downstream of Wnt signaling.
      ). NRG1 cleavage is essential for myelination in the nervous system but also for normal development and function of the mammary gland and the heart (
      • Mei L.
      • Xiong W.-C.
      Neuregulin 1 in neural development, synaptic plasticity and schizophrenia.
      ).
      We report here that ADAM enzymes are pre-associated with their respective substrates at the plasma membrane prior to stimulation of cleavage. We further show that dimerization or oligomerization of substrate monomers is a pre-condition for induced cleavage.

      Results

      To analyze inside-out signaling for ectodomain cleavage, we focused on two ADAM substrates, CD44 (ADAM10) and NRG1 (ADAM17). N- and C-terminally double-tagged CD44 and NRG1 were transfected into RPM-MC cells, MEFs, NIH3T3, or HEK293T cells. We established the following experimental conditions and controls. Cleavage was induced using TPA (a phorbol ester, diacylglycerol mimic, and PKC activator) or, in cells carrying the AT1 receptor, angiotensin II (AngII; a GPCR ligand that induces PKC activation) and detected by measuring both the released ectodomain (solCD44E; solNRG1E) and the membrane-bound residual fragments (CD44ΔE; NRG1ΔE). To prevent loss of the membrane-bound cleavage product, we inhibited further processing by γ-secretase using DAPT. In some cases, cleavage was prevented by the addition of an ADAM inhibitor (batimastat) and, in the case of CD44, by the introduction of an uncleavable ICD mutant, CD44-KR-Mt (the so-called lysine-arginine-rich KR domain, the binding site for ERM (ezrin, radixin, and moesin) proteins and merlin) (
      • Morrison H.
      • Sherman L.S.
      • Legg J.
      • Banine F.
      • Isacke C.
      • Haipek C.A.
      • Gutmann D.H.
      • Ponta H.
      • Herrlich P.
      The NF2 tumor suppressor gene product, merlin, mediates contact inhibition of growth through interactions with CD44.
      ). Induced cleavage of the mutant was indeed reduced significantly (Fig. 1A; the C-terminal cleavage product CD44ΔE is shown). Furthermore, we confirmed that CD44 was an ADAM10 substrate using MEFs with disruption of the Adam10 gene (Fig. 1A) or down-regulation by siRNA (Fig. 1B); there was only marginal cleavage by ADAM17. For NRG1 cleavage, we recently described the necessity of ADAM17 (
      • Dang M.
      • Armbruster N.
      • Miller M.A.
      • Cermeno E.
      • Hartmann M.
      • Bell G.W.
      • Root D.E.
      • Lauffenburger D.A.
      • Lodish H.F.
      • Herrlich A.
      Regulated ADAM17-dependent EGF family ligand release by substrate-selecting signaling pathways.
      ). In some experiments, we observed increased levels of CD44fl after TPA stimulation. However, we proved that the induction of ectodomain cleavage by TPA does not involve de novo protein synthesis. Increased CD44 ectodomain cleavage was observed already after 15–20 min, which is too short for up-regulation of protein synthesis of either ADAM10 or CD44. In fact, blocking translation by cycloheximide did not interfere with TPA-stimulated CD44 ectodomain cleavage (Fig. 1C).
      Figure thumbnail gr1
      FIGURE 1.TPA-induced and ADAM10-dependent processing of CD44 depends on the CD44ICD. A and B, ADAM10 is a major protease acting on CD44. A, wild type (Wt) MEFs or MEFs with the disruption of either the Adam10 or Adam17 or both genes (A10−/−/A17−/−) were transfected with Myc-tagged CD44 (C-terminal c-Myc) wild type, the noncleavable mutant CD44-KR-Mt, or with an empty vector (V). The cells were grown at low cell density, and CD44 cleavage was stimulated by treatment with 100 ng/ml TPA for 30 min. DAPT (5 μm) was added to the cells to prevent degradation of the CD44ΔE cleavage product by γ-secretase. Control cells were treated with DMSO alone (solvent for TPA and DAPT). Subsequently, CD44 full-length (CD44fl) and the membrane-bound C-terminal cleavage product CD44ΔE were detected by c-Myc antibody. CD44fl forms a double band, most likely because of differential glycosylation. Induced CD44 cleavage occurs only in the Adam17 null MEFs but not in the cells with disruption of the Adam10 gene. B, RPM-MC cells transfected with doubly tagged CD44 (N-terminal FLAG and C-terminal c-Myc) were grown at low cell density. A, TPA treatment. Expression of ADAM10 (A10) or ADAM17 (A17) was down-regulated by siRNA (to 3.8 and 1.6% as calculated from the blot by ImageJ). Nontargeting siRNA (C) was used as a control. The released ectodomain was precipitated from culture supernatant by TCA prior to SDS-PAGE. Cleaved ectodomain (solCD44E), CD44fl, and CD44ΔE were detected by FLAG and c-Myc antibodies, respectively. The efficiency of siRNA knockdowns was monitored by detection of ADAM10 and ADAM17 proteins as indicated (seen are the pro- (P) and mature (M) forms). Only ADAM10 knockdown significantly reduced basal and induced release of solCD44E and CD44ΔE. B′, histogram shows mean values of relative level of solCD44E ± S.D. from three independent experiments. ns, p = 0.031081; ****, p < 0.0001; ns, not significant. C, RPM-MC cells transfected with doubly tagged CD44 (N-terminal FLAG, C-terminal c-Myc) were grown at low cell density. For inhibition of translation the cells were pre-incubated with 50 μg/ml of cycloheximide (CHX). CD44 cleavage was stimulated by treatment with 100 ng/ml TPA for 4 h. WB, Western blot.
      As a first option of a protein dimer complex that would allow intracellular signaling to regulate protease action outside the plasma membrane, we considered protease-substrate association. To explore whether substrate and protease were associated with each other, we used co-immunoprecipitation (co-IP) and BiFC, a technique that documents protein-protein interaction with great specificity (for details of this method see under “Experimental Procedures”). Indeed both pairs, ADAM10/CD44 and ADAM17/NRG1, were associated with each other with or without TPA stimulation and independent of whether the enzyme was inhibited and/or the substrate mutated to be noncleavable. ADAM10 complemented fluorescence with either CD44 WT or the uncleavable CD44 KR mutant (CD44 KR-Mt) was observed with or without TPA stimulation (Fig. 2A, cleavage inhibited by batimastat). Adiponectin receptor is known to dimerize and was used as a positive control. The adiponectin receptor does not interact with ADAM10. We therefore used the appropriate construct pair, ADAM10 and adiponectin receptor, as negative control in BiFC assays. Quantitation is shown by column diagram in Fig. 2A′. Similarly, NRG1 was associated with ADAM17 as shown by co-immunoprecipitation, irrespective of PKC activation by TPA or AngII stimulation (Fig. 2E; cleavage inhibited by batimastat). PKCδ, which regulates TPA-induced NRG1 cleavage (
      • Dang M.
      • Armbruster N.
      • Miller M.A.
      • Cermeno E.
      • Hartmann M.
      • Bell G.W.
      • Root D.E.
      • Lauffenburger D.A.
      • Lodish H.F.
      • Herrlich A.
      Regulated ADAM17-dependent EGF family ligand release by substrate-selecting signaling pathways.
      ), only co-precipitated with NRG1 after TPA or AngII stimulation (Fig. 2E, lower panel; cleavage inhibited by batimastat), suggesting that the observed association of NRG1 with ADAM17 is specific and does not depend on PKCδ. ADAM10 (mainly the mature form) was co-immunoprecipitated together with CD44 WT or CD44 KR-Mt (Fig. 2, B and C) and vice versa (Fig. 2D). Consistently with the BiFC data, TPA did not enhance the association of overexpressed or endogenous CD44 and ADAM10 (Fig. 2, B and D, respectively).
      Association of both partners prior to cleavage would, in principle, fit with our hypothesis that their relative positional change induced by ICD modification might allow proteolysis. ADAMs, like CD44 and NRG1, indeed possess cytoplasmic tails. However, the necessity of the ADAM17 and ADAM10 ICDs for regulated cleavage has been put into question (
      • Schwarz J.
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      • Rabe B.
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      • Chalaris A.
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      ), and the ADAM17 ICD can be removed without compromising regulated cleavage (
      • Reddy P.
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      ).
      Carl Blobel (Hospital for Special Surgery, New York), personal communication.
      We therefore tested the effect of ADAM10 ICD deletion mutants on CD44 cleavage. None of the deletion mutants tested inhibited induced release of solCD44E (Fig. 3). This argues that no structural determinants in the ADAM10 ICD regulate cleavage. It is hence unlikely that ICD modifications of CD44 in a substrate/ADAM10 heterodimer could be converted into a rotation or other positioning of the substrate relative to the protease. In addition, this observation further highlights the importance of intracellular signaling input into the substrate's ICD or, possibly, into the ICDs of other associated membrane proteins for cleavage regulation.
      Figure thumbnail gr3
      FIGURE 3.Modification of the ICD of ADAM10 is not required for cleavage regulation. CD44Wt or KR-Mt was co-transfected into ADAM10-deficient MEF cells with either ADAM10 WT (A10) or one of two ADAM10 ICD deletion mutants (Δ1 and Δ2, as indicated in the schematic). ADAM10 WT and both ADAM10 mutants rescued TPA-induced cleavage of CD44 WT as indicated by release of CD44ΔE (upper panel) or solCD44E (lower panel). CD44 KR-Mt remained uncleavable under either condition (upper panel). The experiments were repeated three times for each mutant. WB, Western blot; V, empty vector; C, control.
      As the next possible option, we concentrated on whether our studied substrates could act as homodimers or oligomers in the plasma membrane, allowing relative positioning of the monomers to each other across the membrane. BiFC and co-IP of differently tagged monomers indeed showed that both substrates exist, at least in part, as homodimers in the plasma membrane. In the BiFC assay (see schematic in Fig. 2), CD44 WT or NRG1 WT monomers complemented fluorescence with or without TPA (Fig. 4, A and B). By co-precipitation of differently tagged monomers, C-terminal Myc-tagged NRG1 was associated with C-terminal GFP-tagged NRG1 (Fig. 4C). Interestingly, although NRG1 monomers were not covalently linked to each other in the dimer, CD44 monomers formed cysteine disulfide bonds and migrated in nonreducing SDS-PAGE as dimers. These dimers existed prior to and after TPA induction, and they could be dissociated by dithiothreitol (reducing gel; Fig. 6A, see also Fig. 8B).
      Figure thumbnail gr6
      FIGURE 6.Disruption of CD44 dimers reduces ectodomain cleavage. A, excess of soluble ectodomain prevents homodimerization and ectodomain cleavage. Soluble CD44E was co-expressed (visible as a 70-kDa band, upper panel). Cells, treatments, and cleavage detection were as in B. A′, histogram shows mean values of relative level of solCD44E ± S.D. and CD44fl dimer ± S.D. from three independent experiments. **, p = 0.003930; ****, p < 0.0001. B, antibody complexes attached to CD44 prevent dimerization and ectodomain cleavage. Treating cells with 20 μg/ml primary mouse CD44 ectodomain-specific 5G8 antibody alone increased dimerization and cleavage. Adding 20 μg/ml secondary polyclonal goat anti-mouse antibody to the primary antibody before incubation inhibited dimerization and cleavage, suggesting sterical hindrance of cleavage by the bound primary-secondary antibody complex. Dimers were detected on nonreducing SDS-polyacrylamide gels. B′, histogram shows mean values of relative level of solCD44E ± S.D. and CD44fl dimer ± S.D. from three independent experiments; solCD44E “−” (no antibody) versus1st” (primary antibody), p = 0.002470; CD44fldimer “−“ versus1st”, p = 0.006427. WB, Western blot; V, empty vector.
      Figure thumbnail gr8
      FIGURE 8.Co-regulation of dimer formation and ectodomain cleavage. A, noncleavable CD44 mutants do not dimerize. RPM-MC cells were transfected with plasmids encoding doubly tagged wild type CD44 WT or KR-Mt or CD44 with mutations of serine 291 mimicking dephosphorylation or phosphorylation (S291A and S291D, respectively). Treatments were as in B. Dimers were detected on nonreducing SDS-polyacrylamide gels. The noncleavable mutants KR-Mt and S291D did not dimerize. Spontaneously cleaved CD44 S291A showed increased basal and induced dimerization. B and C, constitutively cleaved CD44 lacking its ICD predominantly occurs as a dimer. RPM-MC cells were transected with plasmid encoding CD44 with deletion of the entire cytoplasmic domain and treated as in B. An additional mutation of cysteine 286 to alanine was introduced into CD44ΔICD, and cleavage and dimerization of this mutant were also analyzed. Mutation of cysteine 286 reduced dimer formation but did not affect cleavage. Dimers were detected on nonreducing SDS-polyacrylamide gels. D, tumor suppressor merlin reduces dimerization of CD44. Plasmids encoding CD44 WT were co-transfected with plasmids encoding constitutively active merlin (S518A) or inactive merlin (S518D). RPM-MC cells were grown at low density, which maintains endogenous merlin inactive. Treatments and cleavage detection were as in B. Cleavage inhibitory constitutively active merlin (S518A) inhibited dimerization. The experiments in A–D were repeated three times. WB, Western blot; V, vector.
      Consequently, we asked whether the induced cleavage reaction depended on homodimerization of the substrates and whether only dimers were subject to ectodomain cleavage. To this end, we first mutated cysteines putatively responsible for the stabilization of CD44 dimers to alanines. Dimerization was prevented by double mutation of Cys-286 (C286A), which lies in the transmembrane domain, and of Cys-295 (C295A), which lies in the above described cleavage regulatory KR motif (ERM/merlin interaction motif) (see box Fig. 5); importantly, the mutations also reduced ectodomain cleavage of CD44 (Fig. 5, compare lanes 2 and 5 with 3 and 6). Although less efficient, mutation of only one cysteine also reduced dimer formation and cleavage (Fig. 5, lanes 9 and 12).
      Figure thumbnail gr5
      FIGURE 5.CD44 dimerization promotes ectodomain cleavage. A, CD44 dimerization is stabilized by disulfide bonds. RPM-MC cells were transfected with either CD44 WT, CD44 cysteine mutants (mutations as indicated in the box), or the constitutively cleaved mutant S291A. The constructs were tagged as in . By the use of a nonreducing gel, cysteine bridge-stabilized dimers are visualized. Cleavage was determined by detection of released ectodomains in the culture supernatant (solCD44E) and of the membrane-bound C-terminal cleavage products, which remained dimerized due to the two cysteines still present in the product (CD44ΔE dimer). Cysteine mutations decreased, whereas CD44 S291A increased basal and TPA induced dimerization and cleavage as compared with CD44 WT. The experiment was repeated three times. B, CD44 dimers are preferentially cleaved by ADAM10. Dimers, but not monomers, disappear upon induced cleavage by ADAM10. Cells, treatments, and cleavage detection were as in B. Where indicated, expression of ADAM10 (A10) or ADAM17 (A17) was down-regulated by siRNA. Nontargeting siRNA C was used as a control. ADAM protein knockdown (not shown) was monitored as in B. Reduced levels of dimers and concomitant increase of cleavage product are detected in cells expressing ADAM10 (control C and A17 lanes). Cleavage is nevertheless increased by treatment with TPA. The experiment was repeated three times. WB, Western blot; V, empty vector; C, control.
      Monomer-monomer interaction leading to dimerization is likely mediated by sequences in the ectodomain of CD44. We thus hypothesized that we should be able to prevent dimerization by adding an excess of soluble ectodomain, and if the dimers were the targets of cleavage, preventing dimerization should inhibit cleavage. This was in fact the case. Overexpression of the soluble CD44 ectodomain (co-transfection of construct with truncation proximal to the transmembrane domain, detectable as a major band around 70 kDa) prevented CD44fl dimerization and TPA-induced solCD44E release (Fig. 6A). We note that dimers did not by themselves trigger cleavage but that TPA stimulation was also required (FIGURE 5., FIGURE 6.A). Another hint for dimer-dependent cleavage was obtained by the use of antibodies. By treating cells with a bivalent CD44 antibody, we could detect a moderate enhancement of basal or TPA-stimulated dimer formation (nonreducing conditions) as well as the release of solCD44E (Fig. 6B, upper and middle panels). Dimer formation and cleavage were prevented by pre-incubation of the CD44 antibody with an isotype-specific secondary antibody, presumably because the larger antibody complex sterically hindered dimerization (Fig. 6B). Further evidence for preferential cleavage of dimers is provided by our observation that down-regulation of ADAM10 (but not of ADAM17) by siRNA leads to the accumulation of CD44 dimers; cleavage, however, required TPA (Fig. 5B).
      To corroborate the observation that dimer formation is not sufficient to induce cleavage, we forced dimerization of CD44 by C-terminal fusion of FKBP dimerization domains and addition of the FKBP ligand AP20187 (
      • Clackson T.
      • Yang W.
      • Rozamus L.W.
      • Hatada M.
      • Amara J.F.
      • Rollins C.T.
      • Stevenson L.F.
      • Magari S.R.
      • Wood S.A.
      • Courage N.L.
      • Lu X.
      • Cerasoli Jr., F.
      • Gilman M.
      • Holt D.A.
      Redesigning an FKBP-ligand interface to generate chemical dimerizers with novel specificity.
      ). AP20187 indeed led to increased CD44-FKBP dimerization. However, TPA was still needed to induce release of CD44-FKBP C-terminal cleavage products (CD44-FKBPΔE) (Fig. 7A). The same result was obtained when stringency of dimerization was enhanced by the addition of two FKBP domains (Fig. 7B). However, addition of AP20187 strongly enhanced TPA-stimulated cleavage (compare 3rd and 4th lanes in Fig. 7, A and B). Taken together, these data are suggestive of a role of dimerization/oligomerization as a pre-condition of induced ectodomain cleavage of CD44 and NRG1 and for a regulatory role of the ICD in dimer formation and dependent cleavage.
      Figure thumbnail gr7
      FIGURE 7.Enforced dimerization enhances TPA-induced ectodomain cleavage. A and B, RPM-MC cells were transfected with HA-tagged CD44 WT containing a single (A) or a double (B) copy of the FK506 dimerization domain of FKBP added to its ICD. Dimerization was induced by the artificial FKBP ligand AP20187 (100 nm) 16 h prior to TPA. Control cells were treated with ethanol or DMSO alone (ethanol was solvent for AP20187). FKPB increased dimerization (CD44-FKBP dimer, nonreducing gel, upper panel), but TPA stimulation was still required for cleavage (CD44-FKBPΔE, second panel from bottom). A′/B′, histogram shows mean values of relative level of CD44-FKBPΔE or CD44–2×FKBPΔE ± S.D. from three independent experiments; ****, p < .0001; ***, p = 0.000113. WB, Western blot.
      These results predicted that cleavage regulatory ICD modifications would affect the ability of CD44 to dimerize. Indeed, the uncleavable CD44 mutants KR-Mt and S291D did not form dimers at all, whereas the constitutively cleaved S291A and the ICD deletion mutant CD44ΔICD formed dimers (Figs. 5A and 8A) or even oligomers (Fig. 8, B and C) irrespective of TPA stimulation. The presence of the reducing agent DTT in cell lysates could partially reduce dimers and oligomers (Fig. 8B, lower panel). Interestingly, CD44ΔICD dimerization did partially depend on stabilization by the putative Cys-286 disulfide bridge (Fig. 8B, lower panel, 3rd lane; for comparison see C286A in CD44 WT Fig. 5A), but dimerization was not completely reduced by mutation of Cys-286 because part of it is likely mediated by interactions between the ectodomains. This interaction of ectodomains was also the basis of the experiment in Fig. 6A. CD44ΔICD, in contrast to the full-length CD44 with S291A mutation (see CD44S291A in Fig. 5A), was constitutively cleaved irrespective of the degree of dimerization (Fig. 8B, upper panel shows the N-terminal ectodomain cleavage product, solCD44E; compare with lower dimerization panel). As predicted by our results so far, the ICD deletion protein apparently does not need a signal transfer through the membrane, and the ectodomain is apparently open for protease access. This is not the case when the “repressive” ICD is present. The signal transfer is required, and accordingly, both dimerization and cleavage are regulated. This is further highlighted in Fig. 8D. Binding of a tumor suppressor merlin (neurofibromatosis 2, Nf2) to the CD44 ICD blocks its ectodomain cleavage (
      • Hartmann M.
      • Parra L.M.
      • Ruschel A.
      • Schubert S.
      • Li Y.
      • Morrison H.
      • Herrlich A.
      • Herrlich P.
      Tumor suppressor Nf2 blocks cellular migration by inhibiting ectodomain cleavage of Cd44.
      ). A constitutively active mutant of merlin (S518A mutant) interfered with CD44 dimerization, consistent with our prediction of dimerization as a prerequisite for cleavage (Fig. 8D).

      Discussion

      In summary, our results suggest that CD44 and NRG1 cleavage, and possibly the cleavage of other ADAM substrates, is regulated by an “inside-out” signaling mechanism that requires prior substrate homodimerization and specific modification of the substrate's ICD. Dimerization is a precondition that permits signal transfer from the modified ICD to the ectodomain. According to our BiFC data, substantial amounts of CD44 exist as homodimers on the cell surface (Fig. 4, A′/B′). Dimerization of CD44 required stabilization by putative cysteine disulfide bridges in the transmembrane domain (Cys-286) and the juxtamembrane ICD (Cys-295) (Fig. 5A). However, we cannot rule out that the cysteine mutations affect ICD modifications or interactions with partner proteins and that these putative interferences are in fact responsible for the reduced cleavage. Furthermore, we showed that dimers are also mediated by ectodomain interactions. This was corroborated by the fact that excess soluble CD44E or the combined treatment with primary (anti-CD44) and secondary (isotype-specific) antibody inhibited dimerization and the induced cleavage of CD44 (Fig. 6). Dimerization was however not sufficient for cleavage, even when forced by FKBP domains (Fig. 7), as cleavage generally required the presence of a cleavage stimulus (FIGURE 5., FIGURE 6., FIGURE 7.) or at least the presence of a cleavage promoting modification (e.g. CD44 S291A in Fig. 8A). Cleavage inhibitory modifications consistently prevented dimerization (KR-Mt, S291D, and merlin S518A overexpression; Fig. 8, A and D). An early report on antibody-induced shedding of CD44 (
      • Bazil V.
      • Horejsí V.
      Shedding of the CD44 adhesion molecule from leukocytes induced by anti-CD44 monoclonal antibody simulating the effect of a natural receptor ligand.
      ) probably already indicated dimer-dependent cleavage.
      In a previous report, phorbol ester stimulated CD44 dimer formation (
      • Liu D.
      • Sy M.S.
      Phorbol myristate acetate stimulates the dimerization of CD44 involving a cysteine in the transmembrane domain.
      ), an effect that is barely visible in our experiments. However, consistent with our results on the importance of cysteines Cys-286 and Cys-295, CD44 dimers were also found to be stabilized by cysteine links (
      • Liu D.
      • Sy M.S.
      Phorbol myristate acetate stimulates the dimerization of CD44 involving a cysteine in the transmembrane domain.
      ). Nevertheless, our results suggest that the ectodomains also have a significant role in dimer formation, as shown by the high level of dimerization of the CD44 ICD deletion (no C295) (Fig. 8B) and also by the effective inhibition of CD44 WT cleavage by addition of soluble CD44 ectodomain (Fig. 6A).
      BiFC assays (Fig. 4B) indicate that NRG1 is also associated as dimers on the cell surface. In the case of NRG1, dimerization appears to involve the intracellular domain. Cross-linking studies of the NRG1α2c ICD expressed alone showed that it spontaneously dimerizes and that force dimerization of a previously cleavage-resistant NRG1α2c ICD deletion mutant rescued its cleavage (an Fc region was added instead of its ICD; Fc dimerizes by disulfide bonds) (
      • Liu X.
      • Hwang H.
      • Cao L.
      • Wen D.
      • Liu N.
      • Graham R.M.
      • Zhou M.
      Release of the neuregulin functional polypeptide requires its cytoplasmic tail.
      ). Based on our observations, we postulate that other ADAM10 or ADAM17 cleaved transmembrane proteins might also require dimerization for their cleavage. In fact, many membrane proteins are known to form homo- or heterodimers, e.g. ICAM1, angiotensin I-converting enzyme, and Alzheimer precursor protein (
      • Melis M.
      • Pace E.
      • Siena L.
      • Spatafora M.
      • Tipa A.
      • Profita M.
      • Bonanno A.
      • Vignola A.M.
      • Bonsignore G.
      • Mody C.H.
      • Gjomarkaj M.
      Biologically active intercellular adhesion molecule-1 is shed as dimers by a regulated mechanism in the inflamed pleural space.
      • Kaden D.
      • Munter L.-M.
      • Joshi M.
      • Treiber C.
      • Weise C.
      • Bethge T.
      • Voigt P.
      • Schaefer M.
      • Beyermann M.
      • Reif B.
      • Multhaup G.
      Homophilic interactions of the amyloid precursor protein (APP) ectodomain are regulated by the loop region and affect β-secretase cleavage of APP.
      ,
      • Eggert S.
      • Midthune B.
      • Cottrell B.
      • Koo E.H.
      Induced dimerization of the amyloid precursor protein leads to decreased amyloid-β protein production.
      • Gordon K.
      • Balyasnikova I.V.
      • Nesterovitch A.B.
      • Schwartz D.E.
      • Sturrock E.D.
      • Danilov S.M.
      Fine epitope mapping of monoclonal antibodies 9B9 and 3G8 to the N domain of angiotensin-converting enzyme (CD143) defines a region involved in regulating angiotensin-converting enzyme dimerization and shedding.
      ), that would allow regulation similar to the one described here.
      Although we have not strictly ruled out that the antibodies or excess soluble ectodomain used in our studies also interfered with the ADAM-substrate interaction, the fact that ADAMs were found associated with their substrates even under conditions of absent proteolysis (e.g. due to mutation of the substrate) (Fig. 2) strengthens the argument that the substrates need to be made cleavage-competent to be in a productive interaction with the ADAMs. This is accomplished by the above-mentioned inside-out signaling process that makes the substrate accessible for cleavage.5 Our data suggest an additional mechanism of cleavage regulation on the level of the substrate. We do not conclude from our data that regulation of cleavage does not also occur on the protease level. Protease activity regulation has been well documented. Numerous reports have shown that the expression level, surface localization, and catalytic activity of ADAMs are regulated (
      • Le Gall S.M.
      • Maretzky T.
      • Issuree P.D.
      • Niu X.-D.
      • Reiss K.
      • Saftig P.
      • Khokha R.
      • Lundell D.
      • Blobel C.P.
      ADAM17 is regulated by a rapid and reversible mechanism that controls access to its catalytic site.
      ,
      • Xu P.
      • Liu J.
      • Sakaki-Yumoto M.
      • Derynck R.
      TACE activation by MAPK-mediated regulation of cell surface dimerization and TIMP3 Association.
      • Willems S.H.
      • Tape C.J.
      • Stanley P.L.
      • Taylor N.A.
      • Mills I.G.
      • Neal D.E.
      • McCafferty J.
      • Murphy G.
      Thiol isomerases negatively regulate the cellular shedding activity of ADAM17.
      ).
      Would cells carry enough ADAM molecules to permit silent associations with numerous substrates? Probably yes. The cleavage reactions trigger the release of highly active regulatory molecules. Therefore, it would suffice if only a minority of substrate molecules were in fact subjected to proteolysis. Indeed, only a small fraction of CD44 on the cell surface (some 15%) is subjected to cleavage (
      • Hartmann M.
      • Parra L.M.
      • Ruschel A.
      • Schubert S.
      • Li Y.
      • Morrison H.
      • Herrlich A.
      • Herrlich P.
      Tumor suppressor Nf2 blocks cellular migration by inhibiting ectodomain cleavage of Cd44.
      ). Correspondingly, according to the BiFC data of Fig. 2A, only a similar percentage of CD44fl (about 10%) is associated with ADAM10 in the absence of cleavage. It is not clear from the BiFC assay whether CD44 is associated with ADAM10 as dimer. Our other experiments support that dimers are preferentially cleaved by ADAM10 (Fig. 5B). NRG1 cleavage however, is particularly effective suggesting that for some substrates the association with ADAMs is close to 100%. Ectodomain release may be required rapidly. Thus, close proximity of enzyme and substrates would permit fast responses that are needed for factors involved in signaling. Induced cleavage has been detected in under 5 min in many studies (
      • Dang M.
      • Armbruster N.
      • Miller M.A.
      • Cermeno E.
      • Hartmann M.
      • Bell G.W.
      • Root D.E.
      • Lauffenburger D.A.
      • Lodish H.F.
      • Herrlich A.
      Regulated ADAM17-dependent EGF family ligand release by substrate-selecting signaling pathways.
      ,
      • Herrlich A.
      • Klinman E.
      • Fu J.
      • Sadegh C.
      • Lodish H.
      Ectodomain cleavage of the EGF ligands HB-EGF, neuregulin1-β, and TGF-α is specifically triggered by different stimuli and involves different PKC isoenzymes.
      ).
      In the examples reported here, the interaction of ADAMs and substrates is made productive by activation of intracellular signaling pathways that induce specific substrate ICD modifications on CD44 and NRG15 (
      • Dang M.
      • Armbruster N.
      • Miller M.A.
      • Cermeno E.
      • Hartmann M.
      • Bell G.W.
      • Root D.E.
      • Lauffenburger D.A.
      • Lodish H.F.
      • Herrlich A.
      Regulated ADAM17-dependent EGF family ligand release by substrate-selecting signaling pathways.
      ,
      • Hartmann M.
      • Parra L.M.
      • Ruschel A.
      • Schubert S.
      • Li Y.
      • Morrison H.
      • Herrlich A.
      • Herrlich P.
      Tumor suppressor Nf2 blocks cellular migration by inhibiting ectodomain cleavage of Cd44.
      ). We showed that these ICD modifications regulate accessibility of the substrate's ectodomain to the ADAM protease.5 Thus, our finding of substrate homodimerization as a prerequisite of cleavage of CD44 and NRG1 (FIGURE 4., FIGURE 5.) offers an explanation of how the intracellular ICD modification could affect the accessibility of the ectodomain, namely by ICD modification-induced relative positional change of the substrate dimerization partners to each other, e.g. by rotation or movement within the plane of the membrane, allowing access of the extracellular ADAM catalytic domain to the substrate's ectodomain. This conclusion is further supported by the fact that the observed effects of our CD44 ICD mutants on dimerization are consistent with their observed effects on cleavage; cleavage inhibitory ICD mutations inhibit dimerization, and cleavage activating ICD mutations enhance dimerization (FIGURE 5., FIGURE 8.).
      Besides the examples reported here, ectodomain accessibility regulation has also been described for another ADAM substrate pair. Notch and ADAM10 are also associated in a nonproductive manner prior to ligand binding to Notch (
      • Gordon W.R.
      • Vardar-Ulu D.
      • Histen G.
      • Sanchez-Irizarry C.
      • Aster J.C.
      • Blacklow S.C.
      Structural basis for autoinhibition of Notch.
      ,
      • Bozkulak E.C.
      • Weinmaster G.
      Selective use of ADAM10 and ADAM17 in activation of Notch1 signaling.
      • Musse A.A.
      • Meloty-Kapella L.
      • Weinmaster G.
      Notch ligand endocytosis: mechanistic basis of signaling activity.
      ). Interestingly, ligand binding followed by its endocytosis exerts a “pulling” force on the Notch ectodomain, inducing structural changes that facilitate ADAM10 cleavage (
      • Gordon W.R.
      • Vardar-Ulu D.
      • Histen G.
      • Sanchez-Irizarry C.
      • Aster J.C.
      • Blacklow S.C.
      Structural basis for autoinhibition of Notch.
      ,
      • Meloty-Kapella L.
      • Shergill B.
      • Kuon J.
      • Botvinick E.
      • Weinmaster G.
      Notch ligand endocytosis generates mechanical pulling force dependent on dynamin, epsins, and actin.
      ) and providing evidence for the existence of other modes of ectodomain cleavage accessibility regulation than those requiring substrate homodimerization reported here. Along these lines, certain ADAM17 substrates (and ADAM17 itself) are known to interact/heterodimerize with “third” proteins that could relay such a function, e.g. annexins (
      • Tsukamoto H.
      • Tanida S.
      • Ozeki K.
      • Ebi M.
      • Mizoshita T.
      • Shimura T.
      • Mori Y.
      • Kataoka H.
      • Kamiya T.
      • Fukuda S.
      • Higashiyama S.
      • Joh T.
      Annexin A2 regulates a disintegrin and metalloproteinase 17-mediated ectodomain shedding of pro-tumor necrosis factor-α in monocytes and colon epithelial cells.
      ,
      • Koumangoye R.B.
      • Nangami G.N.
      • Thompson P.D.
      • Agboto V.K.
      • Ochieng J.
      • Sakwe A.M.
      Reduced annexin A6 expression promotes the degradation of activated epidermal growth factor receptor and sensitizes invasive breast cancer cells to EGFR-targeted tyrosine kinase inhibitors.
      • Nakayama H.
      • Fukuda S.
      • Inoue H.
      • Nishida-Fukuda H.
      • Shirakata Y.
      • Hashimoto K.
      • Higashiyama S.
      Cell surface annexins regulate ADAM-mediated ectodomain shedding of proamphiregulin.
      ). In addition, other transmembrane proteins that interact with ADAM17 could potentially participate in such cleavage regulation, e.g. iRhoms (
      • Maretzky T.
      • McIlwain D.R.
      • Issuree P.D.
      • Li X.
      • Malapeira J.
      • Amin S.
      • Lang P.A.
      • Mak T.W.
      • Blobel C.P.
      iRhom2 controls the substrate selectivity of stimulated ADAM17-dependent ectodomain shedding.
      ).
      An interesting comparable case of inside-out signaling concerns the ectodomain ligand binding affinity of integrins (
      • Hynes R.O.
      Integrins: bidirectional, allosteric signaling machines.
      ,
      • Harburger D.S.
      • Calderwood D.A.
      Integrin signalling at a glance.
      ). Integrins are heterodimers of α and β chains. Similar to our proposed model for CD44 and NRG1 cleavage, interaction partners of the β chain ICD presumably trigger a conformational change of the ectodomains, requiring a change in the relative positioning of the heterodimerization partners. Interestingly, activation of integrin requires linkage to the actin cytoskeleton via talin (
      • Ginsberg M.H.
      • Partridge A.
      • Shattil S.J.
      Integrin regulation.
      ,
      • Wegener K.L.
      • Partridge A.W.
      • Han J.
      • Pickford A.R.
      • Liddington R.C.
      • Ginsberg M.H.
      • Campbell I.D.
      Structural basis of integrin activation by talin.
      • Anthis N.J.
      • Wegener K.L.
      • Ye F.
      • Kim C.
      • Goult B.T.
      • Lowe E.D.
      • Vakonakis I.
      • Bate N.
      • Critchley D.R.
      • Ginsberg M.H.
      • Campbell I.D.
      The structure of an integrin/talin complex reveals the basis of inside-out signal transduction.
      ). Similar to CD44 and ERM/merlin, binding and release of talin is regulated by phosphorylation/dephosphorylation of the integrin β chain. ERM proteins are also known to interact with actin, and we observed that this actin link function is required for cleavage of CD44, and we speculate that this may anchor and stabilize a CD44 dimer (
      • Hartmann M.
      • Parra L.M.
      • Ruschel A.
      • Schubert S.
      • Li Y.
      • Morrison H.
      • Herrlich A.
      • Herrlich P.
      Tumor suppressor Nf2 blocks cellular migration by inhibiting ectodomain cleavage of Cd44.
      ). However, we did not detect a clear effect of an ezrin actin link mutant on dimerization of CD44 (data not shown). Thus, the exact role of actin interaction for substrate cleavage has not yet been revealed.
      A schematic representation of our data is summarized in Fig. 9. Our results and the example of integrin heterodimer activation by an inside-out signaling mechanism in principle support the notion that dimerization of substrates may represent a more generally used regulatory mechanism of ectodomain cleavage; this mechanism allows intracellular signaling to induce inside-out signaling via modification of the substrate's ICD.
      Figure thumbnail gr9
      FIGURE 9.Schematic representation of cleavage regulation through dimerization. CD44 monomers and dimers co-exist on the cell surface. CD44 dimers are stabilized by putative cysteine bridges in the ICD and by putative ectodomain interactions. Our data suggest that ectodomain cleavage regulation depends on ICD modification and interaction with either ERM proteins or merlin on CD44 dimers. At high cell density merlin is dephosphorylated and active and bound to the phosphorylated CD44 ICD. Under low cell density or after TPA stimulation (PKC activator), Ser-291 is dephosphorylated, and phosphorylated/activated ERM proteins displace merlin. This releases a restrictive ICD conformation in the dimer and leads to a positional structural change of the dimerization partners that enable ectodomain accessibility to the ADAM protease. CD44 without its ICD is missing the restrictive ICD conformation and is spontaneously cleaved. A link of ERM proteins to the actin cytoskeleton is possibly important in this regulation. How can CD44S291 be dephosphorylated in response to TPA-dependent activation of PKC? PP1/2 serine phosphatase can indeed be activated by PKC and is regulated by endogenous PKC-activated inhibitors (
      • Bollen M.
      • Peti W.
      • Ragusa M.J.
      • Beullens M.
      The extended PP1 toolkit: designed to create specificity.
      ,
      • Eto M.
      Regulation of cellular protein phosphatase-1 (PP1) by phosphorylation of the CPI-17 family, C-kinase-activated PP1 inhibitors.
      ,
      • Liu Q.R.
      • Zhang P.W.
      • Lin Z.
      • Li Q.F.
      • Woods A.S.
      • Troncoso J.
      • Uhl G.R.
      GBPI, a novel gastrointestinal- and brain-specific PP1-inhibitory protein, is activated by PKC and inactivated by PKA.
      ).

      Acknowledgments

      The administration staff of the Fritz Lipmann Institute was very helpful. We thank Birgit Pavelka, out technician and lab manager. Nico Andreas helped with cell sorting. Jan C. Simon (University of Leipzig, Germany), Ion C. Cirstea (Fritz Lipmann Institute), and Paul Saftig (University of Kiel, Germany) provided plasmids and cell lines.

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