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.
The adhesion molecule and co-receptor of receptor tyrosine kinases, CD44, is expressed in all cells of the immune system, but also in numerous non-immune cells. CD44 plays roles in the cellular response to different pathogens. The molecular actions of CD44 during these processes are by and large still unknown. The CD44 molecule undergoes a sequential proteolytic cleavage which leads to the release of a soluble intracellular domain (CD44-ICD). Previous reports had shown that the CD44-ICD is taken up into the nucleus where it enhances transcription of specific target genes. By RNA profiling we identified a CD44-dependent transcriptional increase of interferon-responsive genes, among them IFI16. IFI16 is important in the innate immune response. It senses and binds pathogenic DNA and, together with cGAS, activates the cGAS-cGAMP-STING pathway and induces the expression of genes relevant for the response, e.g. IFN-β. Our results show that the enhancement of IFI16 expression depended on CD44 cleavage. A CD44-negative tumor cell line, embryonic fibroblasts and bone marrow-derived macrophages from cd44-/- mice were reduced in their response to IFN-γ, to viral DNA fragments and to Listeria monocytogenes infection. We could rescue the deficiency of CD44 negative RPM-MC cells and cd44-/- MEFs by expressing only the soluble CD44-ICD in the absence of any other CD44 domain. Expression of the CD44-ICD carrying a mutation that prevented the uptake into the nucleus, could not rescue the absence of CD44. This molecular aspect of regulation by CD44 may explain part of the immune phenotypes of mice with cd44 gene disruption.
<p>Supplemental Material and Methods. Cell lines, Transfections, Precipitation of Proteins by TCA-DOC , Co-immunoprecipitation (Co-IP) from Cell Lysates, Preparation of Cell Lysates</p>
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