Background: PIST/GOPC is a Golgi-associated protein that interacts with several G-protein-coupled receptors via its single PDZ domain. Results: PIST retains ␤1-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.

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.

Progressive myoclonus epilepsies (PMEs) are inherited disorders characterized by myoclonus, generalized tonic-clonic seizures, and ataxia. One of the genes that is associated with PME is the ER-to-Golgi Qb-SNARE GOSR2, which forms a SNARE complex with syntaxin-5, Bet1 and Sec22b. Most PME patients are homo­zygous for a p.Gly144Trp mutation and develop similar clinical presentations. Recently, a patient who was compound heterozygous for p.Gly144Trp and a previously unseen p.Lys164del mutation was identified. Because this patient presented with a milder disease phenotype, we hypothesized that the p.Lys164del mutation may be less severe compared to p.Gly144Trp. To characterize the effect of the p.Gly144Trp and p.Lys164del mutations, both of which are present in the SNARE motif of GOSR2, we examined the corresponding mutations in the yeast ortholog Bos1. Yeasts expressing the orthologous mutants in Bos1 showed impaired growth, suggesting a partial loss of function, which was more severe for the Bos1 p.Gly176Trp mutation. Using anisotropy and gel filtration, we report that Bos1 p.Gly176Trp and p.Arg196del are capable of complex formation, but with partly reduced activity. Molecular dynamics (MD) simulations showed that the hydrophobic core, which triggers SNARE complex formation, is compromised due to the glycine-to-tryptophan substitution in both GOSR2 and Bos1. In contrast, the deletion of residue p.Lys164 (or p.Arg196del in Bos1) interferes with the formation of hydrogen bonds between GOSR2 and syntaxin-5. Despite these perturbations, all SNARE complexes stayed intact during longer simulations. Thus, our data suggest that the milder course of disease in compound heterozygous PME is due to less severe impairment of the SNARE function.

BET1 is required, together with its SNARE complex partners GOSR2, SEC22b, and Syntaxin‐5 for fusion of endoplasmic reticulum‐derived vesicles with the ER‐Golgi intermediate compartment (ERGIC) and the cis‐Golgi. Here, we report three individuals, from two families, with severe congenital muscular dystrophy (CMD) and biallelic variants in BET1 (P1 p.(Asp68His)/p.(Ala45Valfs*2); P2 and P3 homozygous p.(Ile51Ser)). Due to aberrant splicing and frameshifting, the variants in P1 result in low BET1 protein levels and impaired ER‐to‐Golgi transport. Since in silico modeling suggested that p.(Ile51Ser) interferes with binding to interaction partners other than SNARE complex subunits, we set off and identified novel BET1 interaction partners with low affinity for p.(Ile51Ser) BET1 protein compared to wild‐type, among them ERGIC‐53. The BET1/ERGIC‐53 interaction was validated by endogenous co‐immunoprecipitation with both proteins colocalizing to the ERGIC compartment. Mislocalization of ERGIC‐53 was observed in P1 and P2’s derived fibroblasts; while in the p.(Ile51Ser) P2 fibroblasts specifically, mutant BET1 was also mislocalized along with ERGIC‐53. Thus, we establish BET1 as a novel CMD/epilepsy gene and confirm the emerging role of ER/Golgi SNAREs in CMD.

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.