title: SHANK3 gene mutations associated with autism facilitate ligand binding to the Shank3 ankyrin repeat region.
abstract: Shank/ProSAP proteins are major scaffold proteins of the postsynaptic density; mutations in the human SHANK3 gene are associated with intellectual disability or autism spectrum disorders. We have analyzed the functional relevance of several SHANK3 missense mutations affecting the N-terminal portion of the protein by expression of wild-type and mutant Shank3 in cultured neurons and by binding assays in heterologous cells. Postsynaptic targeting of recombinant Shank3 was unaltered. In electrophysiological experiments, both wild-type and L68P mutant forms of Shank3 were equally effective in restoring synaptic function after knockdown of endogenous Shank3. We observed that several mutations affected binding to interaction partners of the Shank3 ankyrin repeat region. One of these mutations, L68P, improved binding to both ligands. Leu-68 is located N-terminal to the ankyrin repeats, in a highly conserved region that we identify here as a novel domain termed the Shank/ProSAP N-terminal (SPN) domain. We show that the SPN domain interacts with the ankyrin repeats in an intramolecular manner, thereby restricting access of either Sharpin or α-fodrin. The L68P mutation disrupts this blockade, thus exposing the Shank3 ankyrin repeat region to its ligands. Our data identify a new type of regulation of Shank proteins and suggest that mutations in the SHANK3 gene do not necessarily induce a loss of function, but may represent a gain of function with respect to specific interaction partners.
authors.affiliation: From the Institut für Humangenetik, Universitätskrankenhaus Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany.
authors.forename: Marie Germaine
authors.initials: MG
authors.lastname: Mameza
authors.forename: Elena
authors.initials: E
authors.lastname: Dvoretskova
authors.forename: Margarete
authors.initials: M
authors.lastname: Bamann
authors.forename: Hans-Hinrich
authors.initials: HH
authors.lastname: Hönck
authors.forename: Türkan
authors.initials: T
authors.lastname: Güler
authors.forename: Tobias M
authors.initials: TM
authors.lastname: Boeckers
authors.forename: Michael
authors.initials: M
authors.lastname: Schoen
authors.forename: Chiara
authors.initials: C
authors.lastname: Verpelli
authors.forename: Carlo
authors.initials: C
authors.lastname: Sala
authors.forename: Igor
authors.initials: I
authors.lastname: Barsukov
authors.forename: Alexander
authors.initials: A
authors.lastname: Dityatev
authors.forename: Hans-Jürgen
authors.initials: HJ
authors.lastname: Kreienkamp
mesh_terms: D000818:Animals; D017089:Ankyrin Repeat / Q000235:genetics*; D001321:Autistic Disorder / Q000235:genetics* / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D004594:Electrophysiology; D057809:HEK293 Cells; D006624:Hippocampus / Q000166:cytology; D006801:Humans; D007930:Leucine / Q000737:chemistry; D008024:Ligands; D051379:Mice; D008810:Mice, Inbred C57BL; D008840:Microfilament Proteins / Q000378:metabolism; D020125:Mutation, Missense; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000502:physiology*; D009474:Neurons / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D009435:Synaptic Transmission; D020798:Two-Hybrid System Techniques; D014452:Ubiquitins / Q000378:metabolism; D013049:Spectrin
chemical_list: D002352:Carrier Proteins; D007930:Leucine; D008024:Ligands; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; D014452:Ubiquitins; D013049:Spectrin; C492357:SHANK3 protein, human; C575183:SHARPIN protein, human
keywords: Ankyrin Repeats; Autism; Genetic Diseases; Mutant; PDZ Domain; Postsynaptic Density; Scaffold Proteins; Synapses
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