Receptor Intracellular Trafficking Research Articles

International Union of Basic and Clinical Pharmacology. CXVIII. Update on the Nomenclature for Atypical Chemokine Receptors including ACKR5.

Chemokines signal through classical G protein-coupled receptors (GPCRs) to induce cell migration during development, immune homeostasis and multiple diseases. Over the last decade a subfamily of atypical chemokine receptors (ACKRs) was delineated from GPCRs based on their inability to trigger conventional G protein signaling or mediate cell migration in response to chemokines. These receptors nevertheless play an important role within the chemokine system by sequestering, transporting or internalizing chemokines thereby regulating their availability and shaping their gradients. GPR182, the recently deorphanized chemokine receptor, shares about 30% of sequence similarity with its closest relative ACKR3. GPR182 is mainly expressed on endothelial cells and was proposed to act as a scavenger regulating the availability of a large set of chemokines from the CXC, CC and XC families and to act cooperatively with ACKR3 and ACKR4. Unlike other ACKRs, GPR182 was shown to have a strong constitutive interaction with β-arrestins that is required for intracellular receptor trafficking and chemokine scavenging. Chemokine ligation of GPR182 has no additional detectable impact on β-arrestin recruitment. Genetic ablation of GPR182 affects spleen size, myelopoiesis, and serum chemokine levels, indicating its role in chemokine homeostasis and immune regulation. GPR182 was also reported to regulate immune responses to bloodborne antigens and tumorigenesis. Taken together, compelling cumulative evidence indicates that GPR182 does not trigger G protein-mediated signaling but acts as a scavenger for chemokines in vitro and in vivo strongly supporting its inclusion as ACKR5 in the systematic nomenclature of chemokine receptors. Significance Statement The summarized presented findings strongly support the designation of GPR182 as ACKR5 and its formal inclusion in the family of atypical chemokine receptors.

Plasticity-induced actin polymerization in the dendritic shaft regulates intracellular AMPA receptor trafficking.

AMPA-type receptors (AMPARs) are rapidly inserted into synapses undergoing plasticity to increase synaptic transmission, but it is not fully understood if and how AMPAR-containing vesicles are selectively trafficked to these synapses. Here, we developed a strategy to label AMPAR GluA1 subunits expressed from their endogenous loci in cultured rat hippocampal neurons and characterized the motion of GluA1-containing vesicles using single-particle tracking and mathematical modeling. We find that GluA1-containing vesicles are confined and concentrated near sites of stimulation-induced structural plasticity. We show that confinement is mediated by actin polymerization, which hinders the active transport of GluA1-containing vesicles along the length of the dendritic shaft by modulating the rheological properties of the cytoplasm. Actin polymerization also facilitates myosin-mediated transport of GluA1-containing vesicles to exocytic sites. We conclude that neurons utilize F-actin to increase vesicular GluA1 reservoirs and promote exocytosis proximal to the sites of synaptic activity.

Mitotic genome-bookmarking by nuclear hormone receptors: A novel dimension in epigenetic reprogramming and disease assessment

Arrival of multi-colored fluorescent proteins and advances in live cell imaging has immensely contributed to our understanding of intracellular trafficking of nuclear receptors and their roles in gene regulatory functions. These regulatory events need to be faithfully propagated from progenitor to progeny cells. This is corroborated by multiple converging mechanisms that include histone modifications and lately, the phenomenon of ‘mitotic genome-bookmarking’ by specific transcription factors. This phenomenon refers to the retention and feed-forward transmission of progenitor's architectural blueprint of active transcription status which is silenced and preserved during mitosis. Upon mitotic exit, this phenomenon ensures accurate reactivation of transcriptome, proteome, cellular traits and phenotypes in the progeny cells. In addition to diverse modes of genome-bookmarking by nuclear receptors, a correlation between disease-associated receptor polymorphism and disruption of this phenomenon is apparent. However, breakthrough technologies shall reveal finer details of this phenomenon to help achieve normalcy in receptor-specific diseases.

Abstract 2565: Divergentnucleocytoplasmic transport influences escape from HER2-activated DCIS-like state

Abstract HER2/ErbB2 and EGFR/ErbB1 activation incite a phenotype resembling premalignant escape from ductal carcinoma in situ (DCIS) in the 3D multicellular arrangement of breast-epithelial cells. This phenomenon tends to be infrequent, and the exact mechanism for its heterogeneous presentation has been elusive. We sought to investigate the process by which ErbB receptors facilitate a picture of incomplete penetrance in DCIS escape (DE). To identify the transcriptional signatures that prime cells towards DE, we randomly profiled 10-cell transcriptomes of single spheroids after 24 hours of ERBB activation, and frequency matched gene expression to the DE-phenotype. We uncovered a network of nucleocytoplasmic transport (NCT) regulators that alter the DE-phenotype penetrance. Of the regulators, CSE1L (an exportin) synergistically increases the DE-frequency when induced, while its knockdown reduces the phenotype, suggesting its functional role. Furthermore, we evaluated the significance of this result in vivo by perturbing Cse1l in Erbb2-amplified mouse mammary-gland tumors and found that activation of Erbb2 (mimicked by ERBB-inhibitor release) alters the macroscale organization of tumors, reminiscent of DE. Specifically, we found an elevated CSE1L inhibits an accumulation of classical nuclear-localizing cargoes that require binding with importin-α/β (KPNAs, KPNB1) complexes. Using proximity labeling with BirA*-fused with CSE1L, we identified ERBB1/ERBB2/ERBB4 as CSE1L interactors. Mechanistically, hetero-dimerized ERBB receptors interact with importin-α/β complexes and re-localize to the nucleus, corroborating previous observations of receptors’ nuclear translocation. Intriguingly, we observed that Doxycycline-induced knockdown of clathrin heavy chain (CLTC, involved in intracellular trafficking of receptors via endocytosis) synergizes with ERBB activation to increase the DE-phenotype penetrance. Using ChIP-Seq, we found that ERBB1 binds to the locus of microRNA, miR205, which negatively regulates the expression of importin-α, mainly KPNA1. These results indicate that ERBB receptors translocated to the nucleus reduce the DE-phenotype by inhibiting importins via miR205, while CSE1L induction relieves this inhibition by reducing the receptor's nuclear accumulation. Taken together, these results show that nucleocytoplasmic shuttling of receptors in ERBB-active breast epithelia generates tunable cross-inhibitory feedback through nucleocytoplasmic transport that results in long-term, heterogeneous multicellular fates. Citation Format: Lixin Wang, Bishal Paudel, Anthony McKnight, Kevin Janes. Divergentnucleocytoplasmic transport influences escape from HER2-activated DCIS-like state [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2565.

The tetraspanin TSPAN5 regulates AMPAR exocytosis by interacting with the AP4 complex.

Intracellular trafficking of AMPA receptors is a tightly regulated process which involves several adaptor proteins, and is crucial for the activity of excitatory synapses both in basal conditions and during synaptic plasticity. We found that, in rat hippocampal neurons, an intracellular pool of the tetraspanin TSPAN5 promotes exocytosis of AMPA receptors without affecting their internalisation. TSPAN5 mediates this function by interacting with the adaptor protein complex AP4 and Stargazin and possibly using recycling endosomes as a delivery route. This work highlights TSPAN5 as a new adaptor regulating AMPA receptor trafficking.

Mechanoinhibition of endocytosis sensitizes cancer cells to Fas-induced apoptosis.

Fas (CD95/APO-1) is a transmembrane death receptor that transduces apoptotic signals upon binding to its ligand and assembling into a death-inducing signaling complex (DISC). Intracellular trafficking of Fas receptors, including recycling from endosomes to the plasma membrane, plays a vital role in ligand-induced assembly of DISC. Although Fas is highly expressed in tumor cells, insufficient expression of these receptors on the cell surface makes cancer cells insensitive to the Fas-induced apoptosis. Here we show that inhibition of endocytosis increases the formation of Fas microaggregates on the plasma membrane and sensitizes cancer cells to Fas-induced apoptosis. We have identified a clinically used vasodilator, Fasudil, that slows down endocytosis by increasing plasma membrane tension. Fasudil enhanced apoptosis in cancerous cells when combined with exogenous soluble Fas ligand (FasL), whereas the synergistic effect was substantially weaker in nonmalignant cells. Additionally, the FasL and Fasudil combination prevented glioblastoma cell growth in embryonic stem cell-derived brain organoids and induced tumor regression in a xenograft U87 tumor model in nude mice. Our results demonstrate that FasL treatment has strong potential as an apoptosis-directed cancer therapy when the formation of Fas microaggregates is augmented by slowing down endocytosis dynamics.

140 (PB020) - Inhibition of Fas Receptor Endocytosis Sensitizes Cancer Cells to Fas-induced Apoptosis

Fas (CD95/APO-1) is a transmembrane death receptor that transduces apoptotic signals upon binding to its ligand and assembling into a death-inducing signaling complex (DISC). Intracellular trafficking of Fas receptors, including recycling from endosomes to the plasma membrane, plays a vital role in ligand-induced assembly of DISC. Although Fas is highly expressed in tumor cells, insufficient expression of these receptors on the cell surface makes cancer cells insensitive to the Fas-induced apoptosis.

O-GlcNAcylation regulates epidermal growth factor receptor intracellular trafficking and signaling

SignificanceEpidermal growth factor receptor (EGFR) is one of the most important membrane receptors that transduce growth signals into cells to sustain cell growth, proliferation, and survival. EGFR signal termination is initiated by EGFR internalization, followed by trafficking through endosomes, and degradation in lysosomes. How this process is regulated is still poorly understood. Here, we show that hepatocyte growth factor regulated tyrosine kinase substrate (HGS), a key protein in the EGFR trafficking pathway, is dynamically modified by a single sugar N-acetylglucosamine. This modification inhibits EGFR trafficking from endosomes to lysosomes, leading to the accumulation of EGFR and prolonged signaling. This study provides an important insight into diseases with aberrant growth factor signaling, such as cancer, obesity, and diabetes.

IGFBP-3 functions as a molecular switch that mediates mitochondrial and metabolic homeostasis.

Mitochondrial dysfunction or loss of homeostasis is a central hallmark of many human diseases. Mitochondrial homeostasis is mediated by multiple quality control mechanisms including mitophagy, a form of selective autophagy that recycles terminally ill or dysfunctional mitochondria in order to preserve mitochondrial integrity. Our prior studies have shown that members of the insulin-like growth factor (IGF) family localize to the mitochondria and may play important roles in mediating mitochondrial health in the corneal epithelium, an integral tissue that is required for the maintenance of optical transparency and vision. Importantly, the IGF-binding protein-3, IGFBP-3, is secreted by corneal epithelial cells in response to stress and functions to mediate intracellular receptor trafficking in this cell type. In this study, we demonstrate a novel role for IGFBP-3 in mitochondrial homeostasis through regulation of the short isoform (s)BNIP3L/NIX mitophagy receptor in corneal epithelial cells and extend this finding to non-ocular epithelial cells. We further show that IGFBP-3-mediated control of mitochondrial homeostasis is associated with alterations in lamellar cristae morphology and mitochondrial dynamics. Interestingly, both loss and gain of function of IGFBP-3 drive an increase in mitochondrial respiration. This increase in respiration is associated with nuclear accumulation of IGFBP-3. Taken together, these findings support a novel role for IGFBP-3 as a key mediator of mitochondrial health in mucosal epithelia through the regulation of mitophagy and mitochondrial morphology.

The Role of Intracellular Trafficking of Notch Receptors in Ligand-Independent Notch Activation.

Aberrant Notch signaling has been found in a broad range of human malignancies. Consequently, small molecule inhibitors and antibodies targeting Notch signaling in human cancers have been developed and tested; however, these have failed due to limited anti-tumor efficacy because of dose-limiting toxicities in normal tissues. Therefore, there is an unmet need to discover novel regulators of malignant Notch signaling, which do not affect Notch signaling in healthy tissues. This review provides a comprehensive overview of the current knowledge on the role of intracellular trafficking in ligand-independent Notch receptor activation, the possible mechanisms involved, and possible therapeutic opportunities for inhibitors of intracellular trafficking in Notch targeting.

Spatial and temporal VEGF receptor intracellular trafficking in microvascular and macrovascular endothelial cells

The vascular endothelial growth factor receptors (VEGFRs) can shape the neovascular phenotype of vascular endothelial cells when translocated to the nucleus, however the spatial and temporal changes in the intracellular distribution and translocation of VEGFRs to the nucleus and the organelles involved in this process is unclear. This study reports the effect of exogenous VEGF on translocation of VEGFRs and organelles in micro- and macrovascular endothelial cells. We showed that VEGF is responsible for: a rapid and substantial nuclear translocation of VEGFRs; VEGFR1 and VEGFR2 exhibit distinct spatial, temporal and structural translocation characteristics both in vitro and in vivo and this determines the nuclear VEGFR1:VEGFR2 ratio which differs between microvascular and macrovascular cells; VEGFR2 nuclear translocation is associated with the endosomal pathway transporting the receptor from Golgi in microvascular endothelial cells; and an increase in the volume of intracellular organelles. In conclusion, the nuclear translocation of VEGFRs is both receptor and vessel (macro versus micro) dependent and the endosomal pathway plays a key role in the translocation of VEGFRs to the nucleus and the subsequent export to the lysosomal system. Modulating VEGF-mediated VEGFR1 and VEGFR2 intracellular transmigration pathways may offer an alternative for the development of new anti-angiogenic therapies.

Altered Intracellular Trafficking of Mutant Thyroid Hormone Receptors in Resistance to Thyroid Hormone Syndrome

Resistance to Thyroid Hormone α (RTHα), a reduced sensitivity to thyroid hormone (T3) in peripheral tissues, is caused by mutations in thyroid hormone receptor α (TRα), a nuclear receptor that mediates T3-responsive gene expression. All mutations characterized in RTHα, to date, are in the ligand binding domain (LBD), resulting in reduced affinity for T3. In addition, some mutations result in truncated proteins lacking all or part of helix 12. Previously, we have used fluorescence recovery after photobleaching (FRAP) to examine the effects of select RTHα mutations on the intracellular trafficking of TRα. After transfecting HeLa cells with expression plasmids for green fluorescent protein (GFP)-tagged wild-type TRα and each of the mutants, we first assessed their intracellular distribution and initial intranuclear mobility. Although wild-type TRα is known to shuttle between the nucleus and cytoplasm, it is primarily localized to the nucleus at a steady state. We showed that TR-E403X, Ala382ProfsX7, and F397fs406X are also primarily localized to the nucleus, and FRAP revealed that wild-type TRα and the RTHα mutants are highly dynamic within the nucleus, indicating that the receptors rapidly dissociate and reassociate with DNA binding sites and/or other nuclear binding sites, independent of T3 (1). Some studies have shown that Nuclear Receptor Corepressor 1 (NCoR1), which interacts with the hinge region of TRα (the region between the DNA-binding domain and LBD) has a higher affinity for RTHα mutants compared to wild-type TRα, supporting the hypothesis that helix 12 of the LBD also functions to disassociate NCoR1 from TRα when it is bound to T3 (2). We proposed that this increased affinity for NCoR1 alters the mobility of TRα in the nucleus, impacting its function. Here, we show that NCoR1 has slower FRAP recovery kinetics compared with TRα, and we evaluate the intranuclear dynamics of RTH TRα1 mutants ΤR-E403X, Ala382ProfsX7, and F397fs406X in response to increased levels of NCoR1. Investigation of the effects of overexpression of NCoR1 will provide further insight into the impact of altered binding affinity for NCoR1 on the intranuclear dynamics of RTHα mutants.

Getting there: Thyroid hormone receptor intracellular trafficking

A year ago, when I first contemplated writing this article, my intent was to provide a detailed review of the contributions of the diverse community of talented scientists in my lab to the nuclear receptor research field. In the throes of a deadly pandemic, political turmoil, and Black Lives Matter, however, I found myself compelled to tell a more personal story. While I will still cover milestones in our understanding of the intracellular trafficking of the thyroid hormone receptor, now these will be set against the backdrop of my path as a woman in STEM and on being intentionally inclusive. By sharing reflections on my journey, I hope to encourage young investigators to persist in their pursuit of a career in science.

Regulation of the EGFR/ErbB signalling by clathrin in response to various ligands in hepatocellular carcinoma cell lines.

Membrane receptor intracellular trafficking and signalling are frequently altered in cancers. Our aim was to investigate whether clathrin‐dependent trafficking modulates signalling of the ErbB receptor family in response to amphiregulin (AR), EGF, heparin‐binding EGF‐like growth factor (HB‐EGF) and heregulin‐1β (HRG). Experiments were performed using three hepatocellular carcinoma (HCC) cell lines, Hep3B, HepG2 and PLC/PRF/5, expressing various levels of EGFR, ErbB2 and ErbB3. Inhibition of clathrin‐mediated endocytosis (CME), by down‐regulating clathrin heavy chain expression, resulted in a cell‐ and ligand‐specific pattern of phosphorylation of the ErbB receptors and their downstream effectors. Clathrin down‐regulation significantly decreased the ratio between phosphorylated EGFR (pEGFR) and total EGFR in all cell lines when stimulated with AR, EGF, HB‐EGF or HRG, except in HRG‐stimulated Hep3B cells in which pEGFR was not detectable. The ratio between phosphorylated ErbB2 and total ErbB2 was significantly decreased in clathrin down‐regulated Hep3B cells stimulated with any of the ligands, and in HRG‐stimulated PLC/PRF/5 cells. The ratio between phosphorylated ErbB3 and total ErbB3 significantly decreased in clathrin down‐regulated cell lines upon stimulation with EGF or HB‐EGF. STAT3 phosphorylation levels significantly increased in all cell lines irrespective of stimulation, while that of AKT remained unchanged, except in AR‐stimulated Hep3B and HepG2 cells in which pAKT was significantly decreased. Finally, ERK phosphorylation was insensitive to clathrin inhibition. Altogether, our observations indicate that clathrin regulation of ErbB signalling in HCC is a complex process that likely depends on the expression of ErbB family members and on the autocrine/paracrine secretion of their ligands in the tumour environment.

GRK Regulation of Beta2 Adrenergic Receptor Mediated Gene Transcription: Impacts on Learning and Memory

BackgroundAdrenergic signaling plays critical roles in health and disease both peripherally and centrally. In the central nervous system, beta adrenergic receptors are broadly expressed and respond to norepinephrine (NE) signaling from neurons projected from the Locus Coeruleous (LC). NE is known to regulate attention, arousal, and acuity as well as fear learning in health and is implicated in neurodegenerative disease and post‐traumatic stress disorder (PTSD). Beta2 adrenergic receptors (B2ARs) are known to be involved in long term potentiation through Gs mediated activation of protein kinase A (PKA) which regulates calcium channels or through forming complexes with AMPA receptors. Recently published work from our lab has shown that functionally distinct and selectively phosphorylated subpopulations of B2ARs exist in a single neuron. These subpopulations of B2ARs are exclusively phosphorylated at either a PKA (S261/262) or g‐protein receptor kinase (GRK) (S355/356) site on the c‐terminus of the receptor and display distinct agonist‐induced receptor intracellular trafficking. Here we investigate the distinct signals associated with the selective phosphorylation of B2ARs at the GRK and PKA site and seek to elucidate the functional implications of the signals in vitro and in vivo.HypothesisWe hypothesize that stimulation of B2AR causes phosphorylation of the receptor at either PKA or GRK phosphorylation sites. Phosphorylation of the GRK site leads to receptor internalization and delivers signaling to the nucleus, which can regulate gene transcription while phosphorylation of the PKA site does not.MethodsOverexpression of wild type B2AR or receptors with serine to alanine mutations of the GRK (S355/356A) or PKA (S261/262A) phosphorylation sites in neurons as well as pharmacologic inhibitors of PKA or GRK were used to study receptor trafficking and signaling. Förster resonance energy transfer (FRET) biosensors ICUE3 expressed at the plasma membrane or the nucleus were used to detect cAMP signaling. Knockin mice expressing mutant receptor were used to examine learning and memory in the Morris water maze paradigm.ResultsStimulation of B2AR leads to distinct membrane trafficking based on phosphorylation status of the receptor. Phosphorylation of the GRK but not PKA site on B2AR is necessary for internalization of the receptor and this internalized receptor is in an active conformation. GRK phosphorylation of B2AR is also necessary for increased cAMP levels in the nucleus and transcription of cFOS, FOSB, RHOB, and OLIG2, genes associated with learning and memory. Knockin mice expressing B2AR with a S355/356A mutation showed decreased spatial learning and memory.Conclusion and SignificanceThese data demonstrate that GRK‐dependent B2AR signaling can regulate gene transcription and that loss of this internal signaling affects learning and memory in mice. These data offer new mechanistic insight into B2AR signaling in the brain that could have implications for both our understanding of learning and memory and for neurological diseases and PTSD.Support or Funding InformationThis work was funded by NIH T32GM099608 and GM GM129376

Mice Expressing Fluorescent Protease‐Activated Receptor‐2 as a Novel Tool to Visualize Intracellular Receptor Trafficking

Protease‐activated receptor‐2 (PAR2) is a G‐protein coupled receptor (GPCR) that plays a critical role in mediating the proinflammatory and pronociceptive actions of serine and cysteine proteases in many tissues. In common with a growing number of GPCRs, PAR2 can signal from endosomes as well as the plasma membrane, and sustained endosomal signaling underlies disease‐relevant processes. Our knowledge of the role of PAR2 in health and disease states is hampered by an inadequate understanding of its cellular and subcellular localization. Since antibodies to GPCRs often lack sensitivity and specificity, and are unsuitable for studying receptor trafficking in living cells, we used a knockin approach to generate mice expressing PAR2 fused to monomeric ultrastable GFP (muGFP). To investigate whether mouse PAR2 fused at the C‐terminus to muGFP was functional, we compared agonist‐evoked signaling and trafficking of PAR2‐muGFP and PAR2 with a C‐terminal HA11 epitope expressed in KNRK cells. Trypsin and the PAR2 selective peptide agonist 2‐Furoyl‐LIGRLO‐NH2 similarly stimulated calcium signals and PAR2 endocytosis. Thus, muGFP does not disrupt PAR2 signaling and trafficking. We then generated knockin mice expressing mouse PAR2 fused at the C‐terminus to muGFP. RT‐PCR revealed similar levels of expression of PAR2 in PAR2‐muGFP and wild‐type mice. Western blotting of colonic extracts using a GFP antibody revealed expression of PAR2‐muGFP at the protein level. We localized PAR2‐muGFP by immunofluorescence and confocal microscopy using the GFP antibody. PAR2‐muGFP was highly expressed in epithelial cells in the digestive tract and skin, and was detected at lower levels in the enteric nervous system and pancreatic islets. In the unstimulated state, PAR2‐muGFP was principally localized to the plasma membrane of colonocytes. Exposure to trypsin or 2‐Furoyl‐LIGRLO‐NH2 induced redistribution of PAR2‐muGFP from the plasma membrane to endosomes. PAR2‐muGFP mice provide a valuable tool to study the localization, regulation, and trafficking of PAR2 in intact animals in healthy and diseased conditions.Support or Funding InformationNIH

The CHORD protein CHP-1 regulates EGF receptor trafficking and signaling in C. elegans and in human cells.

The intracellular trafficking of growth factor receptors determines the activity of their downstream signaling pathways. Here, we show that the putative HSP-90 co-chaperone CHP-1 acts as a regulator of EGFR trafficking in C. elegans. Loss of chp-1 causes the retention of the EGFR in the ER and decreases MAPK signaling. CHP-1 is specifically required for EGFR trafficking, as the localization of other transmembrane receptors is unaltered in chp-1(lf) mutants, and the inhibition of hsp-90 or other co-chaperones does not affect EGFR localization. The role of the CHP-1 homolog CHORDC1 during EGFR trafficking is conserved in human cells. Analogous to C. elegans, the response of CHORDC1-deficient A431 cells to EGF stimulation is attenuated, the EGFR accumulates in the ER and ERK2 activity decreases. Although CHP-1 has been proposed to act as a co-chaperone for HSP90, our data indicate that CHP-1 plays an HSP90-independent function in controlling EGFR trafficking through the ER.

Administration of a novel high affinity PICK1 PDZ domain inhibitor attenuates cocaine seeking in rats

Protein interacting with C kinase-1 (PICK1) regulates intra-cellular trafficking of GluA2-containing AMPA receptors, a process known to play a critical role in cocaine-seeking behavior. This suggests that PICK1 may represent a molecular target for developing novel pharmacotherapies to treat cocaine craving-induced relapse. Emerging evidence indicates that inhibition of PICK1 attenuates the reinstatement of cocaine-seeking behavior, an animal model of relapse. Here, we show that systemic administration of TAT-P4-(DATC5)2, a novel high-affinity peptide inhibitor of the PICK1 PDZ domain, dose-dependently attenuated the reinstatement of cocaine seeking in rats at doses that did not produce operant learning deficits or suppress locomotor activity. We also show that systemic TAT-P4-(DATC5)2 penetrated the brain where it was visualized in the nucleus accumbens shell. Consistent with these effects, infusions of TAT-P4-(DATC5)2 directly into the accumbens shell reduced cocaine, but not sucrose, seeking. The effects of TAT-P4-(DATC5)2 on cocaine seeking are likely due, in part, to inhibition of PICK1 in medium spiny neurons (MSNs) of the accumbens shell as TAT-P4-(DATC5)2 was shown to accumulate in striatal neurons and bind PICK1. Taken together, these findings highlight a novel role for PICK1 in the reinstatement of cocaine seeking and support future studies examining the efficacy of peptide inhibitors of PICK1 in animal and human models of cocaine relapse.

Intra- and Extracellular Pillars of a Unifying Framework for Homeostatic Plasticity: A Crosstalk Between Metabotropic Receptors and Extracellular Matrix.

In the face of chronic changes in incoming sensory inputs, neuronal networks are capable of maintaining stable conditions of electrical activity over prolonged periods of time by adjusting synaptic strength, to amplify or dampen incoming inputs [homeostatic synaptic plasticity (HSP)], or by altering the intrinsic excitability of individual neurons [homeostatic intrinsic plasticity (HIP)]. Emerging evidence suggests a synergistic interplay between extracellular matrix (ECM) and metabotropic receptors in both forms of homeostatic plasticity. Activation of dopaminergic, serotonergic, or glutamate metabotropic receptors stimulates intracellular signaling through calmodulin-dependent protein kinase II, protein kinase A, protein kinase C, and inositol trisphosphate receptors, and induces changes in expression of ECM molecules and proteolysis of both ECM molecules (lecticans) and ECM receptors (NPR, CD44). The resulting remodeling of perisynaptic and synaptic ECM provides permissive conditions for HSP and plays an instructive role by recruiting additional signaling cascades, such as those through metabotropic glutamate receptors and integrins. The superimposition of all these signaling events determines intracellular and diffusional trafficking of ionotropic glutamate receptors, resulting in HSP and modulation of conditions for inducing Hebbian synaptic plasticity (i.e., metaplasticity). It also controls cell-surface delivery and activity of voltage- and Ca2+-gated ion channels, resulting in HIP. These mechanisms may modify epileptogenesis and become a target for therapeutic interventions.

Characterization of the interaction between β-catenin and sorting nexin 27: contribution of the type I PDZ-binding motif to Wnt signaling.

Background: Sorting Nexin 27 (SNX27) is a 62-kDa protein localized to early endosomes and known to regulate the intracellular trafficking of ion channels and receptors. In addition to a PX domain common among all of the sorting nexin family, SNX27 is the only sorting family member that contains a PDZ domain. To identify novel SNX27–PDZ binding partners, we performed a proteomic screen in mouse principal kidney cortical collecting duct cells (mpkCCD) using a GST-SNX27 fusion construct as bait. We found that the C-terminal type I PDZ binding motif (DTDL) of β-catenin, an adherens junction scaffolding protein and transcriptional co-activator, interacts directly with SNX27. Using biochemical and immunofluorescent techniques, β-catenin was identified in endosomal compartments where co-localization with SNX27 was observed. Furthermore, E-cadherin, but not Axin, GSK3 or Lef-1 was located in SNX27 protein complexes. While overexpression of wild-type β-catenin protein increased TCF-LEF dependent transcriptional activity, an enhanced transcriptional activity was not observed in cells expressing β-Catenin ΔFDTDL or diminished SNX27 expression.These results imply importance of the C-terminal PDZ binding motif for the transcriptional activity of β-catenin and propose that SNX27 might be involved in the assembly of β-catenin complexes in the endosome.