Abstract
Shank proteins are major scaffolds of the postsynaptic density of excitatory synapses. Mutations in SHANK genes are associated with autism and intellectual disability. The relevance of missense mutations for these pathologies is unclear. Several missense mutations in SHANK3 affect the N-terminal region, consisting of the Shank/ProSAP N-terminal (SPN) domain and a set of Ankyrin (Ank) repeats. Here we identify a novel SHANK3 missense mutation (p.L270M) in the Ankyrin repeats in patients with an ADHD-like phenotype. We functionally analysed this and a series of other mutations, using biochemical and biophysical techniques. We observe two major effects: (i) a loss of binding to δ-catenin (e.g. in the p.L270M variant), and (ii) interference with the intramolecular interaction between N-terminal SPN domain and the Ank repeats. This also interferes with binding to the α-subunit of the calcium-/calmodulin dependent kinase II (αCaMKII), and appears to be associated with a more severe neurodevelopmental pathology.
Competing Interest Statement
The authors have declared no competing interest.
Abbreviations
- Abi-1
- Abl interactor 1
- ADHD
- Attention deficit hyperactivity disorder
- Ank
- ankyrin repeats
- ANOVA
- analysis of variance
- ASD
- autism spectrum disorder
- CaMKII
- Ca2+/calmodulin-dependent protein kinase II
- cDNA
- complementary deoxyribonucleic acid
- chk
- chicken
- DAPI
- 4’,6-Diamidino-2-phenylindole
- DIV
- Days in vitro
- DSF
- differential scanning fluorimetry
- DTT
- Dithiothreitol
- EDTA
- Ethylenediaminetetraacetic acid
- EGFP
- enhanced green fluorescent protein
- FRET
- Förster energy resonance trasfer
- GFP
- green fluorescent protein
- GKAP
- guanylate kinase-associated protein
- GTP
- guanosine triphosphate
- HA
- Human influenza hemagglutinin
- HBS
- Hepes buffered salt solution
- HBSS
- Hank’s balanced salt solution
- HEK
- Human embryonic kidney
- HRP
- horseradish peroxidase
- ICC
- immunocytochemistry
- ID
- intellectual disability
- IgG
- immunoglobulin G
- IP
- immunoprecipitation
- IRSp53
- insulin receptor substrate 53
- MAP2
- microtubule-associated protein 2
- mRFP
- monomeric red fluorescent protein
- ns
- not significant
- PAGE
- polyacrylamide gel electrophoresis
- PBS
- phosphate-buffered saline
- PCR
- polymerase chain reaction
- PDZ
- PSD-95/DLG/ZO1
- PSD
- post-synaptic density
- PSD-95
- postsynaptic density protein 95
- SAM
- sterile alpha motif
- SAPAP
- SAP90/PSD-95-associated protein
- SD
- standard deviation
- SDS
- sodium dodecyl sulfate
- Shank
- SH3 and multiple ankyrin repeats
- SPN
- Shank3/ProSAP N-terminal
- SUMO
- Small Ubiquitin-like Modifier
- TBS-T
- tris-buffered saline-Tween 20
- Ubl
- ubiquitin like
- WES
- Whole exom sequencing
- WB
- Western blot
- WT
- wild type