By using the yeast two-hybrid system we identified a novel protein from the human brain interacting with the C terminus of somatostatin receptor subtype 2. This protein termed somatostatin receptor interacting protein is characterized by a novel domain structure, consisting of six N-terminal ankyrin repeats followed by SH3 and PDZ domains, several proline-rich regions, and a C-terminal sterile ␣ motif. It consists of 2185 amino acid residues encoded by a 9-kilobase pair mRNA; several splice variants have been detected in human and rat cDNA libraries. Sequence comparison suggests that the novel multidomain protein, together with cortactinbinding protein, forms a family of cytoskeletal anchoring proteins. Fractionation of rat brain membranes indicated that somatostatin receptor interacting protein is enriched in the postsynaptic density fraction. The interaction of somatostatin receptor subtype 2 with its interacting protein was verified by overlay assays and coimmunoprecipitation experiments from transfected human embryonic kidney cells. Somatostatin receptor subtype 2 and the interacting protein display a striking overlap of their expression patterns in the rat brain. Interestingly, in the hippocampus the mRNA for somatostatin receptor interacting protein was not confined to the cell bodies but was also observed in the molecular layer, suggesting a dendritic localization of this mRNA.Targeting of neurotransmitter receptors to postsynaptic or presynaptic sites is an area that has been widely studied in recent years; a large body of evidence has accumulated showing that many receptors are anchored at their specifc site of action by specialized anchoring proteins, which may link receptors to components of the synaptic structure or the cytoskeleton (1, 2). This is true for inhibitory as well as excitatory receptors of the family of ligand-gated ion channels. For the second large family of neurotransmitter receptors, the seven transmembrane domain G-protein-coupled receptors, only very recently have some proteins been identified that may be involved in anchoring or linkage to the cytoskeleton. These include the homer proteins, which are tightly associated with metabotropic glutamate receptors via a PDZ 1 domain in homer and the C terminus of the mGluRs (3). However, for the large majority of G-protein-coupled receptors, no intracellular associated proteins have been identified so far beyond those proteins which are necessary for signal transduction and functional regulation of the receptors, i.e. the G-proteins and proteins of the arrestin family (4).We have begun to address this issue for members of the somatostatin receptor family (SSTRs). SSTRs are widely expressed in neuronal tissue and modulate synaptic responses by interacting with inhibitory G-proteins in presynaptic as well as postsynaptic compartments of neurons (e.g. Refs. 5-8). Recently we have used the yeast two-hybrid system to screen for proteins intracellularly associated with SSTR2, one of the major SSTR subtypes in the mammalian brain. Here we show t...

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. Sitedirected 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.Insect development and behavior are largely controlled by hormones and neurotransmitters often identified using a diverse array of bioassays. Besides the biogenic amines and the steroid-like hormones, insect hormones have been frequently classified as neuropeptides, which are widely distributed throughout the invertebrate kingdom (1, 2). Despite the large number of neuropeptides, the number of known cognate receptors in insects is still rather limited, with only a few examples in Drosophila that have been cloned based on homology with mammalian G-protein-coupled receptors (GPCRs) 1 (i.e. neuropeptide Y-like and tachykinin-like receptors) (3, 4). With the completion of the Drosophila genome project, a more thorough analysis of neuropeptide/receptor relations in insects is now possible. Whereas this genome data base allows the identification of peptide hormones previously isolated from other insect species as part of larger precursors (5), Drosophila GPCR-like sequences have been predicted mostly based on structural analogy of the transmembrane regions to mammalian neuropeptide receptor groups (6).Structural evidence for the existence of ligands identical or similar to their mammalian neuropeptide counterparts are lacking when searching the Drosophila genome data base. This may indicate that in insects these receptors are activated by an entirely different set of ligands. This view is supported by data reported here on the identification of two novel GPCRs from Drosophila melanogaster, termed Drostar1 and -2, which are structurally related to the mam...

Background: Missense mutations in the SHANK3 gene have been detected in autism patients. Results: A mutation in the conserved SPN region of Shank3 improves ligand binding to the ankyrin repeats. Conclusion:The SPN domain regulates accessibility of the ankyrin repeats through an intramolecular interaction. Significance: Autism-associated mutations of Shank3 result in gain-of-function with respect to specific interaction partners.

The five different rat somatostatin receptor subtypes (SSTR1-SSTR5) were coexpressed with a subunit of G-protein gated inwardly rectifying potassium channel (GIRK1) in Xenopus oocytes. SSTR2-SSTR5, but not SSTR1 coupled efficiently to the activation of GIRK currents when stimulated by SST14 or SST28. A comparison of the dose-response curves and of the maximum currents obtained indicates that SSTR2 couples most efficiently to this effector, supporting the notion that SSTR2 is involved in activation of potassium conductances by SST in vivo.

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.

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.

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.