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Targeted truncation of the ADAM17 cytoplasmic domain in mice results in protein destabilization and a hypomorphic phenotype
- Source :
- The Journal of Biological Chemistry, The journal of biological chemistry 296, 100733-(2021). doi:10.1016/j.jbc.2021.100733
- Publication Year :
- 2020
-
Abstract
- A disintegrin and metalloprotease 17 (ADAM17) is a cell-surface metalloprotease that serves as the principle sheddase for tumor necrosis factor α (TNFα), interleukin-6 receptor (IL-6R), and several ligands of the epidermal growth factor receptor (EGFR), regulating these crucial signaling pathways. ADAM17 activation requires its transmembrane domain, but not its cytoplasmic domain, and little is known about the role of this domain in vivo. To investigate, we used CRISPR-Cas9 to mutate the endogenous Adam17 locus in mice to produce a mutant ADAM17 lacking its cytoplasmic domain (Adam17Δcyto). Homozygous Adam17Δcyto animals were born at a Mendelian ratio and survived into adulthood with slightly wavy hair and curled whiskers, consistent with defects in ADAM17/EGFR signaling. At birth, Adam17Δcyto mice resembled Adam17−/− mice in that they had open eyes and enlarged semilunar heart valves, but they did not have bone growth plate defects. The deletion of the cytoplasmic domain resulted in strongly decreased ADAM17 protein levels in all tissues and cells examined, providing a likely cause for the hypomorphic phenotype. In functional assays, Adam17Δcyto mouse embryonic fibroblasts and bone-marrow-derived macrophages had strongly reduced ADAM17 activity, consistent with the reduced protein levels. Nevertheless, ADAM17Δcyto could be stimulated by PMA, a well-characterized posttranslational activator of ADAM17, corroborating that the cytoplasmic domain of endogenous ADAM17 is not required for its rapid response to PMA. Taken together, these results provide the first evidence that the cytoplasmic domain of ADAM17 plays a pivotal role in vivo in regulating ADAM17 levels and function.
- Subjects :
- 0301 basic medicine
Male
Cytoplasm
KI, knock-in
knock-in mutation in mice
Biochemistry
Mice
iRhom1 and 2, inactive Rhomboid proteins 1 and 2
ADAM17 Protein
membrane protein
myeloid cell
MFI, mean fluorescence intensity
Receptor
Sequence Deletion
TACE (TNFα converting enzyme)
Bone growth
mEF, mouse embryonic fibroblast
Chemistry
Protein Stability
ddc
Cell biology
BMDM, bone-marrow-derived macrophage
TNFα, tumor necrosis factor α
Transmembrane domain
Phenotype
Protein destabilization
ddc:540
DV, donor vector
Female
Signal transduction
metabolism [Fibroblasts]
Research Article
IL-6R, interleukin-6 receptor
PMA, phorbol 12-myristate 13-acetate
genetics [ADAM17 Protein]
ADAM17 (a disintegrin and metalloprotease 17)
03 medical and health sciences
metabolism [ADAM17 Protein]
cell surface enzyme
Protein Domains
CHX, cycloheximide
Gene knockin
Animals
Amino Acid Sequence
Molecular Biology
metabolism [Cytoplasm]
030102 biochemistry & molecular biology
Base Sequence
Cell Biology
chemistry [ADAM17 Protein]
Sheddase
Fibroblasts
EGFR, epidermal growth factor receptor
Mice, Inbred C57BL
030104 developmental biology
EPCAM, epithelial cell adhesion molecule
mES, mouse embryonic stem
CRISPR-Cas Systems
ADAM17, a disintegrin and metalloprotease 17
Subjects
Details
- ISSN :
- 1083351X
- Volume :
- 296
- Database :
- OpenAIRE
- Journal :
- The Journal of biological chemistry
- Accession number :
- edsair.doi.dedup.....48ddba9cdef79f06d77f3f2a019dddab