Ubiquitination of CXCR7 Controls Receptor Trafficking

The chemokine receptor CXCR7 binds CXCL11 and CXCL12 with high affinity, chemokines that were previously thought to bind exclusively to CXCR4 and CXCR3, respectively. Expression of CXCR7 has been associated with cardiac development as well as with tumor growth and progression. Despite having all the canonical features of G protein-coupled receptors (GPCRs), the signalling pathways following CXCR7 activation remain controversial, since unlike typical chemokine receptors, CXCR7 fails to activate Gαi-proteins. CXCR7 has recently been shown to interact with β-arrestins and such interaction has been suggested to be responsible for G protein-independent signals through ERK-1/2 phosphorylation. Signal transduction by CXCR7 is controlled at the membrane by the process of GPCR trafficking. In the present study we investigated the regulatory processes triggered by CXCR7 activation as well as the molecular interactions that participate in such processes. We show that, CXCR7 internalizes and recycles back to the cell surface after agonist exposure, and that internalization is not only β-arrestin-mediated but also dependent on the Serine/Threonine residues at the C-terminus of the receptor. Furthermore we describe, for the first time, the constitutive ubiquitination of CXCR7. Such ubiquitination is a key modification responsible for the correct trafficking of CXCR7 from and to the plasma membrane. Moreover, we found that CXCR7 is reversibly de-ubiquitinated upon treatment with CXCL12. Finally, we have also identified the Lysine residues at the C-terminus of CXCR7 to be essential for receptor cell surface delivery. Together these data demonstrate the differential regulation of CXCR7 compared to the related CXCR3 and CXCR4 receptors, and highlight the importance of understanding the molecular determinants responsible for this process

SHANK3 mutations identified in autism lead to modification of dendritic spine morphology via an actin-dependent mechanism

Genetic mutations of SHANK3 have been reported in patients with intellectual disability, autism spectrum disorder (ASD) and schizophrenia. At the synapse, Shank3/ProSAP2 is a scaffolding protein that connects glutamate receptors to the actin cytoskeleton via a chain of intermediary elements. Although genetic studies have repeatedly confirmed the association of SHANK3 mutations with susceptibility to psychiatric disorders, very little is known about the neuronal consequences of these mutations. Here, we report the functional effects of two de novo mutations (STOP and Q321R) and two inherited variations (R12C and R300C) identified in patients with ASD. We show that Shank3 is located at the tip of actin filaments and enhances its polymerization. Shank3 also participates in growth cone motility in developing neurons. The truncating mutation (STOP) strongly affects the development and morphology of dendritic spines, reduces synaptic transmission in mature neurons and also inhibits the effect of Shank3 on growth cone motility. The de novo mutation in the ankyrin domain (Q321R) modifies the roles of Shank3 in spine induction and morphology, and actin accumulation in spines and affects growth cone motility. Finally, the two inherited mutations (R12C and R300C) have intermediate effects on spine density and synaptic transmission. Therefore, although inherited by healthy parents, the functional effects of these mutations strongly suggest that they could represent risk factors for ASD. Altogether, these data provide new insights into the synaptic alterations caused by SHANK3 mutations in humans and provide a robust cellular readout for the development of knowledge-based therapies

Mu-Opioid Receptors Transiently Activate the Akt-nNOS Pathway to Produce Sustained Potentiation of PKC-Mediated NMDAR-CaMKII Signaling

BACKGROUND: In periaqueductal grey (PAG) matter, cross-talk between the Mu-opioid receptor (MOR) and the glutamate N-methyl-D-Aspartate receptor (NMDAR)-CaMKII pathway supports the development of analgesic tolerance to morphine. In neurons, histidine triad nucleotide binding protein 1 (HINT1) connects the regulators of G protein signaling RGSZ1 and RGSZ2 to the C terminus of the MOR. In response to morphine, this HINT1-RGSZ complex binds PKCgamma, and afterwards, the interplay between PKCgamma, Src and Gz/Gi proteins leads to sustained potentiation of NMDAR-mediated glutamate responses. METHODOLOGY/PRINCIPAL FINDINGS: Following an intracerebroventricular (icv) injection of 10 nmol morphine, Akt was recruited to the synaptosomal membrane and activated by Thr308 and Ser473 phosphorylation. The Akt activation was immediately transferred to neural Nitric Oxide Synthase (nNOS) Ser1417. Afterwards, nitric oxide (NO)-released zinc ions recruited PKCgamma to the MOR to promote the Src-mediated phosphorylation of the Tyr1325 NMDAR2A subunit. This action increased NMDAR calcium flux and CaMKII was activated in a calcium-calmodulin dependent manner. CaMKII then acted on nNOS Ser847 to produce a sustained reduction in NO levels. The activation of the Akt-nNOS pathway was also reduced by the binding of these proteins to the MOR-HINT1 complex where they remained inactive. Tolerance to acute morphine developed as a result of phosphorylation of MOR cytosolic residues, uncoupling from the regulated G proteins which are transferred to RGSZ2 proteins. The diminished effect of morphine was prevented by LNNA, an inhibitor of nNOS function, and naltrindole, a delta-opioid receptor antagonist that also inhibits Akt. CONCLUSIONS/SIGNIFICANCE: Analysis of the regulatory phosphorylation of the proteins included in the study indicated that morphine produces a transient activation of the Akt/PKB-nNOS pathway. This activation occurs upstream of PKCgamma and Src mediated potentiation of NMDAR activity, ultimately leading to morphine tolerance. In summary, the Akt-nNOS pathway acts as a primer for morphine-triggered events which leads to the sustained potentiation of the NMDAR-CaMKII pathway and MOR inhibition

SAP97-mediated ADAM10 trafficking from Golgi outposts depends on PKC phosphorylation

A disintegrin and metalloproteinase 10 (ADAM10) is the major α-secretase that catalyzes the amyloid precursor protein (APP) ectodomain shedding in the brain and prevents amyloid formation. Its activity depends on correct intracellular trafficking and on synaptic membrane insertion. Here, we describe that in hippocampal neurons the synapse-associated protein-97 (SAP97), an excitatory synapse scaffolding element, governs ADAM10 trafficking from dendritic Golgi outposts to synaptic membranes. This process is mediated by a previously uncharacterized protein kinase C phosphosite in SAP97 SRC homology 3 domain that modulates SAP97 association with ADAM10. Such mechanism is essential for ADAM10 trafficking from the Golgi outposts to the synapse, but does not affect ADAM10 transport from the endoplasmic reticulum. Notably, this process is altered in Alzheimer's disease brains. These results help in understanding the mechanism responsible for the modulation of ADAM10 intracellular path, and can constitute an innovative therapeutic strategy to finely tune ADAM10 shedding activity towards APP

Agonist-Specific Recruitment of Arrestin Isoforms Differentially Modify Delta Opioid Receptor Function

International audienceLigand-specific recruitment of arrestins facilitates functional selectivity of G-protein-coupled receptor signaling. Here, we describe agonist-selective recruitment of different arrestin isoforms to the delta opioid receptor in mice. A high-internalizing delta opioid receptor agonist (SNC80) preferentially recruited arrestin 2 and, in arrestin 2 knock-outs (KOs), we observed a significant increase in the potency of SNC80 to inhibit mechanical hyperalgesia and decreased acute tolerance. In contrast, the low-internalizing delta agonists (ARM390, JNJ20788560) preferentially recruited arrestin 3 with unaltered behavioral effects in arrestin 2 KOs. Surprisingly, arrestin 3 KO revealed an acute tolerance to these low-internalizing agonists, an effect never observed in wild-type animals. Furthermore, we examined delta opioid receptor-Ca(2+)channel coupling in dorsal root ganglia desensitized by ARM390 and the rate of resensitization was correspondingly decreased in arrestin 3 KOs. Live-cell imaging in HEK293 cells revealed that delta opioid receptors are in pre-engaged complexes with arrestin 3 at the cell membrane and that ARM390 strengthens this membrane interaction. The disruption of these complexes in arrestin 3 KOs likely accounts for the altered responses to low-internalizing agonists. Together, our results show agonist-selective recruitment of arrestin isoforms and reveal a novel endogenous role of arrestin 3 as a facilitator of resensitization and an inhibitor of tolerance mechanisms. SIGNIFICANCE STATEMENT: Agonists that bind to the same receptor can produce highly distinct signaling events and arrestins are a major mediator of this ligand bias. Here, we demonstrate that delta opioid receptor agonists differentially recruit arrestin isoforms. We found that the high-internalizing agonist SNC80 preferentially recruits arrestin 2 and knock-out (KO) of this protein results in increased efficacy of SNC80. In contrast, low-internalizing agonists (ARM390 and JNJ20788560) preferentially recruit arrestin 3 and, surprisingly, KO of arrestin 3 produces acute tolerance and impaired receptor resensitization to these agonists. Arrestin 3 is in pre-engaged complexes with the delta opioid receptor at the cell membrane and low-internalizing agonists promote this interaction. This study reveals a novel role for arrestin 3 as a facilitator of receptor resensitization