Structural deficits in key domains of Shank2 lead to alterations in postsynaptic nanoclusters and to a neurodevelopmental disorder in humans.

Postsynaptic scaffold proteins such as Shank, PSD-95, Homer and SAPAP/GKAP family members establish the postsynaptic density of glutamatergic synapses through a dense network of molecular interactions. Mutations in SHANK genes are associated with neurodevelopmental disorders including autism and intellectual disability. However, no SHANK missense mutations have been described which interfere with the key functions of Shank proteins believed to be central for synapse formation, such as GKAP binding via the PDZ domain, or Zn2+-dependent multimerization of the SAM domain. We identify two individuals with a neurodevelopmental disorder carrying de novo missense mutations in SHANK2. The p.G643R variant distorts the binding pocket for GKAP in the Shank2 PDZ domain and prevents interaction with Thr(-2) in the canonical PDZ ligand motif of GKAP. The p.L1800W variant severely delays the kinetics of Zn2+-dependent polymerization of the Shank2-SAM domain. Structural analysis shows that Trp1800 dislodges one histidine crucial for Zn2+ binding. The resulting conformational changes block the stacking of helical polymers of SAM domains into sheets through side-by-side contacts, which is a hallmark of Shank proteins, thereby disrupting the highly cooperative assembly process induced by Zn2+. Both variants reduce the postsynaptic targeting of Shank2 in primary cultured neurons and alter glutamatergic synaptic transmission. Super-resolution microscopy shows that both mutants interfere with the formation of postsynaptic nanoclusters. Our data indicate that both the PDZ- and the SAM-mediated interactions of Shank2 contribute to the compaction of postsynaptic protein complexes into nanoclusters, and that deficiencies in this process interfere with normal brain development in humans.

SHANK3 gene mutations associated with autism facilitate ligand binding to the Shank3 ankyrin repeat region.

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.

Targeting of δ-catenin to postsynaptic sites through interaction with the Shank3 N-terminus.

BACKGROUND: Neurodevelopmental disorders such as autism spectrum disorder (ASD) may be caused by alterations in genes encoding proteins that are involved in synapse formation and function. This includes scaffold proteins such as Shank3, and synaptic adhesion proteins such as Neurexins or Neuroligins. An important question is whether the products of individual risk genes cooperate functionally (exemplified in the interaction of Neurexin with Neuroligin isoforms). This might suggest a common pathway in pathogenesis. For the SHANK3 gene, heterozygous loss of function, as well as missense mutations have been observed in ASD cases. Several missense mutations affect the N-terminal part of Shank3 which contains the highly conserved Shank/ProSAP N-terminal (SPN) and Ankyrin repeat (Ank) domains. The role of these domains and the relevance of these mutations for synaptic function of Shank3 are widely unknown. METHODS: We used purification from a synaptic protein fraction, as well as a variety of biochemical and cell biological approaches to identify proteins which associate with the Shank3 N-terminus at postsynaptic sites. RESULTS: We report here that δ-catenin, which is encoded by CTNND2, an autism candidate gene, directly interacts with the Ank domain of Shank3 at postsynaptic sites through its Armadillo-repeat domain. The interaction is not affected by well-known posttranslational modifications of δ-catenin, i.e. by phosphorylation or palmitoylation. However, an ASD-associated mutation in the SPN domain of Shank3, L68P, significantly increases the interaction of Shank3 with δ-catenin. By analysis of postsynaptic fractions from mice, we show that the lack of SPN-Ank containing, large isoforms of Shank3 results in the loss of postsynaptic δ-catenin. Further, expression of Shank3 variants containing the N-terminal domains in primary cultured neurons significantly increased the presence of coexpressed δ-catenin at postsynaptic sites. LIMITATIONS: Work in model organisms such as mice, and in primary cultured neurons may not reproduce faithfully the situation in human brain neurons. Work in primary cultured neurons was also hampered by lack of a specific antibody for endogenous δ-catenin. CONCLUSIONS: Our data show that the interaction between Shank3 N-terminus and δ-catenin is required for the postsynaptic targeting of δ-catenin. Failure of proper targeting of δ-catenin to postsynaptic sites may contribute to the pathogenesis of autism spectrum disorder.

Subcellular sorting of the G-protein coupled mouse somatostatin receptor 5 by a network of PDZ-domain containing proteins.

PSD-95/discs large/ZO-1 (PDZ) domain proteins integrate many G-protein coupled receptors (GPCRs) into membrane associated signalling complexes. Additional PDZ proteins are involved in intracellular receptor trafficking. We show that three PDZ proteins (SNX27, PIST and NHERF1/3) regulate the mouse somatostatin receptor subtype 5 (SSTR5). Whereas the PDZ ligand motif of SSTR5 is not necessary for plasma membrane targeting or internalization, it protects the SSTR5 from postendocytic degradation. Under conditions of lysosomal inhibition, recycling of the SSTR5 to the plasma membrane does not depend on the PDZ ligand. However, recycling of the wild type receptor carrying the PDZ binding motif depends on SNX27 which interacts and colocalizes with the receptor in endosomal compartments. PIST, implicated in lysosomal targeting of some membrane proteins, does not lead to degradation of the SSTR5. Instead, overexpressed PIST retains the SSTR5 at the Golgi. NHERF family members release SSTR5 from retention by PIST, allowing for plasma membrane insertion. Our data suggest that PDZ proteins act sequentially on the GPCR at different stages of its subcellular trafficking.

Somatostatin receptor 5 is palmitoylated by the interacting ZDHHC5 palmitoyltransferase.

Many G-protein coupled receptors are palmitoylated in their C-terminal, intracellular regions. So far no enzymes responsible for this modification have been described. We identified an interaction of the membrane proximal helix 8 of somatostatin receptor 5 (SSTR5) with the N-terminal region of the putative palmitoyltransferase ZDHHC5 using the Ras recruitment interaction screening system. ZDHHC5 and SSTR5 are colocalized at the plasma membrane and can be efficiently coimmunoprecipitated from transfected cells. Coexpression of ZDHHC5 in HEK293 cells increased palmitoylation of SSTR5 whereas knock-down of endogenous ZDHHC5 by siRNAs decreased it. Our data identify the first palmitoyltransferase for a G-protein coupled receptor.

Functional annotation of two orphan G-protein-coupled receptors, Drostar1 and -2, from Drosophila melanogaster and their ligands by reverse pharmacology.

By combining a Drosophila genome data base search and reverse transcriptase-PCR-based cDNA isolation, two G-protein-coupled receptors were cloned, which are the closest known invertebrate homologs of the mammalian opioid/somatostatin receptors. However, when functionally expressed in Xenopus oocytes by injection of Drosophila orphan receptor RNAs together with a coexpressed potassium channel, neither receptor was activated by known mammalian agonists. By applying a reverse pharmacological approach, the physiological ligands were isolated from peptide extracts from adult flies and larvae. Edman sequencing and mass spectrometry of the purified ligands revealed two decapentapeptides, which differ only by an N-terminal pyroglutamate/glutamine. The peptides align to a hormone precursor sequence of the Drosophila genome data base and are almost identical to allatostatin C from Manduca sexta. Both receptors were activated by the synthetic peptides irrespective of the N-terminal modification. Site-directed mutagenesis of a residue in transmembrane region 3 and the loop between transmembrane regions 6 and 7 affect ligand binding, as previously described for somatostatin receptors. The two receptor genes each containing three exons and transcribed in opposite directions are separated by 80 kb with no other genes predicted between. Localization of receptor transcripts identifies a role of the new transmitter system in visual information processing as well as endocrine regulation.