Abstract
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.
Highlights
Intracellular domain (ICD) modifications regulate extracellular ectodomain cleavage by metalloproteases
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
Reagents—DNA oligonucleotides were purchased from Metabion (Munich, Germany); phorbol 12-myristate 13-acetate (TPA) and difluorophenylacetylalanylphenylglycine t-butyl ester (DAPT) were from Enzo Life Sciences GmbH (Lörrach, Germany); angiotensin II peptide and the AP20187 dimerizer were from ARIAD Pharmaceuticals, Inc. (Cambridge, MA); batimastat (BB94) was from Calbiochem, and GM-6001 was from Enzo (Farmingdale, NY)
Summary
Intracellular domain (ICD) modifications regulate extracellular ectodomain cleavage by metalloproteases. 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 [23, 24] 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 [25]? 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). We further show that dimerization or oligomerization of substrate monomers is a pre-condition for induced cleavage
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