Somatostatin, also known as somatotropin-release inhibitory factor, is a cyclopeptide that exerts potent inhibitory actions on hormone secretion and neuronal excitability. Its physiologic functions ...are mediated by five G protein-coupled receptors (GPCRs) called somatostatin receptor (SST)1-5. These five receptors share common structural features and signaling mechanisms but differ in their cellular and subcellular localization and mode of regulation. SST
and SST
receptors have evolved as primary targets for pharmacological treatment of pituitary adenomas and neuroendocrine tumors. In addition, SST
is a prototypical GPCR for the development of peptide-based radiopharmaceuticals for diagnostic and therapeutic interventions. This review article summarizes findings published in the last 25 years on the physiology, pharmacology, and clinical applications related to SSTs. We also discuss potential future developments and propose a new nomenclature.
Mutations in SHANK3, coding for a large scaffold protein of excitatory synapses in the CNS, are associated with neurodevelopmental disorders including autism spectrum disorders and intellectual ...disability (ID). Several cases have been identified in which the mutation leads to truncation of the protein, eliminating C‐terminal sequences required for post‐synaptic targeting of the protein. We identify here a patient with a truncating mutation in SHANK3, affected by severe global developmental delay and intellectual disability. By analyzing the subcellular distribution of this truncated form of Shank3, we identified a nuclear localization signal (NLS) in the N‐terminal part of the protein which is responsible for targeting Shank3 fragments to the nucleus. To determine the relevance of Shank3 for nuclear signaling, we analyze how it affects signaling by β‐catenin, a component of the Wnt pathway. We show that full length as well as truncated variants of Shank3 interact with β‐catenin via the PDZ domain of Shank3, and the armadillo repeats of β‐catenin. As a result of this interaction, truncated forms of Shank3 and β‐catenin strictly co‐localize in small intra‐nuclear bodies both in 293T cells and in rat hippocampal neurons. On a functional level, the sequestration of both proteins in these nuclear bodies is associated with a strongly repressed transcriptional activation by β‐catenin owing to interaction with the truncated Shank3 fragment found in patients. Our data suggest that truncating mutations in SHANK3 may not only lead to a reduction in Shank3 protein available at postsynaptic sites but also negatively affect the Wnt signaling pathway.
Full‐length Shank3 interacts with β‐catenin at postsynaptic sites. Mutations in the SHANK3 gene found in patients with autism and intellectual disability lead to expression of a truncated Shank3 protein. The lack of Shank3 synaptic targeting elements leads to recruitment of Shank3 and β‐catenin to nuclear bodies, where activation of the Wnt signaling pathway by β‐catenin is blocked by the truncated Shank3 fragment.
Mutations in the X‐linked gene coding for the calcium‐/calmodulin‐dependent serine protein kinase (CASK) are associated with severe neurological disorders ranging from intellectual disability (in ...males) to mental retardation and microcephaly with pontine and cerebellar hypoplasia. CASK is involved in transcription control, in the regulation of trafficking of the post‐synaptic NMDA and α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptors, and acts as a presynaptic scaffolding protein. For CASK missense mutations, it is mostly unclear which of CASK’s molecular interactions and cellular functions are altered and contribute to patient phenotypes. We identified five CASK missense mutations in male patients affected by neurodevelopmental disorders. These and five previously reported mutations were systematically analysed with respect to interaction with CASK interaction partners by co‐expression and co‐immunoprecipitation. We show that one mutation in the L27 domain interferes with binding to synapse‐associated protein of 97 kDa. Two mutations in the guanylate kinase (GK) domain affect binding of CASK to the nuclear factors CASK‐interacting nucleosome assembly protein (CINAP) and T‐box, brain, 1 (Tbr1). A total of five mutations in GK as well as PSD‐95/discs large/ZO‐1 (PDZ) domains affect binding of CASK to the pre‐synaptic cell adhesion molecule Neurexin. Upon expression in neurons, we observe that binding to Neurexin is not required for pre‐synaptic localization of CASK. We show by bimolecular fluorescence complementation assay that Neurexin induces oligomerization of CASK, and that mutations in GK and PDZ domains interfere with the Neurexin‐induced oligomerization of CASK. Our data are supported by molecular modelling, where we observe that the cooperative activity of PDZ, SH3 and GK domains is required for Neurexin binding and oligomerization of CASK.
The pathogenic mechanism underlying several patient‐derived missense mutations of the MAGUK CASK remains unclear. MAGUKs have been shown to oligomerize when binding to their PDZ‐ligand through a conformational change in their PDZ, SH3 and GK domains. We demonstrate here that WT CASK also oligomerizes when bound to its PDZ‐ligand Neurexin, but that several of the 10 studied CASK mutant variants fail to bind to Neurexin and/or to oligomerize. This suggests that the loss of Neurexin‐induced oligomerization may be a key pathogenic mechanism underlying CASK missense mutations.
Shank proteins are major scaffolds of the postsynaptic density of excitatory synapses. Mutations in SHANK genes are associated with autism and intellectual disability. The effects of missense ...mutations on Shank3 function, and therefore the pathomechanisms are 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: (1) a loss of binding to δ-catenin (e.g. in the p.L270M variant), and (2) 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.
The calcium-/calmodulin dependent serine protein kinase (CASK) belongs to the membrane-associated guanylate kinases (MAGUK) family of proteins. It fulfils several different cellular functions, ...ranging from acting as a scaffold protein to transcription control, as well as regulation of receptor sorting. CASK functions depend on the interaction with a variety of partners, for example neurexin, liprin-alpha, Tbr1 and SAP97. So far, it is uncertain how these seemingly unrelated interactions and resulting functions of CASK are regulated. Here, we show that alternative splicing of CASK can guide the binding affinity of CASK isoforms to distinct interaction partners. We report seven different variants of CASK expressed in the fetal human brain. Four out of these variants are not present in the NCBI GenBank database as known human variants. Functional analyses showed that alternative splicing affected the affinities of CASK variants for several of the tested interaction partners. Thus, we observed a clear correlation of the presence of one splice insert with poor binding of CASK to SAP97, supported by molecular modelling. The alternative splicing and distinct properties of CASK variants in terms of protein-protein interaction should be taken into consideration for future studies.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Members of the SH3- and ankyrin-rich repeat (SHANK) protein family are considered as master scaffolds of the post-synaptic density of glutamatergic synapses. Several missense mutations within the ...canonical SHANK3 isoform have been proposed as causative for the development of autism spectrum disorders (ASDs). However, there is a surprising paucity of data linking missense mutation-induced changes in protein structure and dynamics to the occurrence of ASD-related synaptic phenotypes. In this proof-of-principle study, we focus on two ASD-associated point mutations, both located within the same domain of SHANK3 and demonstrate that both mutant proteins indeed show distinct changes in secondary and tertiary structure as well as higher conformational fluctuations. Local and distal structural disturbances result in altered synaptic targeting and changes of protein turnover at synaptic sites in rat primary hippocampal neurons.
In neuronal cells, many membrane receptors interact via their intracellular, C-terminal tails with PSD-95/
discs large
/ZO-1 (PDZ) domain proteins. Some PDZ proteins act as scaffold proteins. In ...addition, there are a few PDZ proteins such as Gopc which bind to receptors during intracellular transport. Gopc is localized at the trans-Golgi network (TGN) and binds to a variety of receptors, many of which are eventually targeted to postsynaptic sites. We have analyzed the role of Gopc by knockdown in primary cultured neurons and by generating a conditional Gopc knockout (KO) mouse line. In neurons, targeting of neuroligin 1 (Nlgn1) and metabotropic glutamate receptor 5 (mGlu5) to the plasma membrane was impaired upon depletion of Gopc, whereas NMDA receptors were not affected. In the hippocampus and cortex of Gopc KO animals, expression levels of Gopc-associated receptors were not altered, while their subcellular localization was disturbed. The targeting of mGlu5 to the postsynaptic density was reduced, coinciding with alterations in mGluR-dependent synaptic plasticity and deficiencies in a contextual fear conditioning paradigm. Our data imply Gopc in the correct subcellular sorting of its associated mGlu5 receptor in vivo.
Background
Somatostatin (SOM) receptor subtype 2 (SSTR2) is the major receptor subtype mediating SOM effects throughout the neuraxis. We previously demonstrated that the non-selective agonist D-Trp
8
...-SOM induces intracellular sequestration of SSTR2, whereas this receptor is maintained at the cell surface after treatment with the SSTR2-selective agonist L-779,976 in cells co-expressing SSTR2 and SSTR5.
Methods and results
In this study, we knocked-out SSTR5 in AtT20 cells endogenously expressing both SSTR2 and SSTR5 and used immuno-labeling and confocal microscopy to investigate the effect of SSTR5 on regulation of SSTR2 trafficking. Our results indicate that unlike D-Trp
8
-SOM-induced intracellular sequestration, L-779,976 stimulation results in the maintenance of SSTR2 at the cell surface regardless of whether SSTR5 is present or not. We then examined the trafficking pathways of SSTR2 upon stimulation by either agonist. We found that both D-Trp
8
-SOM and L-779,976 induce SSTR2 internalization via transferrin-positive vesicles. However, SSTR2 internalized upon L-779,976 treatment undergoes rapid recycling to the plasma membrane, whereas receptors internalized by D-Trp
8
-SOM recycle slowly after washout of the agonist. Furthermore, D-Trp
8
-SOM stimulation induces degradation of a fraction of internalized SSTR2 whereas L-779,976-dependent, rapid SSTR2 recycling appears to protect internalized SSTR2 from degradation. In addition, Octreotide which has preferential SSTR2 affinity, induced differential effects on both SSTR2 trafficking and degradation.
Conclusion
Our results indicate that the biased agonistic property of L-779,976 protects against SSTR2 surface depletion by rapidly initiating SSTR2 recycling while SSTR5 does not regulate L-779-976-dependent SSTR2 trafficking.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Many G-protein-coupled receptors carry C-terminal ligand motifs for PSD-95/discs large/ZO-1 (PDZ) domains; via interaction with PDZ domain-containing scaffold proteins, this allows for integration of ...receptors into signaling complexes. However, the presence of PDZ domain proteins attached to intracellular membranes suggests that PDZ-type interactions may also contribute to subcellular sorting of receptors. The protein interacting specifically with Tc10 (PIST; also known as GOPC) is a trans-Golgi-associated protein that interacts through its single PDZ domain with a variety of cell surface receptors. Here we show that PIST controls trafficking of the interacting β1-adrenergic receptor both in the anterograde, biosynthetic pathway and during postendocytic recycling. Overexpression and knockdown experiments show that PIST leads to retention of the receptor in the trans-Golgi network (TGN), to the effect that overexpressed PIST reduces activation of the MAPK pathway by β1-adrenergic receptor (β1AR) agonists. Receptors can be released from retention in the TGN by coexpression of the plasma membrane-associated scaffold PSD-95, which allows for transport of receptors to the plasma membrane. Stimulation of β1 receptors and activation of the cAMP pathway lead to relocation of PIST from the TGN to an endosome-like compartment. Here PIST colocalizes with SNX1 and the internalized β1AR and protects endocytosed receptors from lysosomal degradation. In agreement, β1AR levels are decreased in hippocampi of PIST-deficient mice. Our data suggest that PIST contributes to the fine-tuning of β1AR sorting both during biosynthetic and postendocytic trafficking.
Background: PIST/GOPC is a Golgi-associated protein that interacts with several G-protein-coupled receptors via its single PDZ domain.
Results: PIST retains β1-adrenergic receptors in intracellular compartments and interferes with receptor degradation after endocytosis.
Conclusion: PIST stabilizes the receptor in an intracellular compartment.
Significance: PDZ proteins associated with intracellular membranes confer specific features to the subcellular targeting of interacting receptors.
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