Crystal structures of autoinhibitory PDZ domain of Tamalin: implications for metabotropic glutamate receptor trafficking regulation.
ABSTRACT: Metabotropic glutamate receptors (mGluRs) function as neuronal G-protein-coupled receptors and this requires efficient membrane targeting through associations with cytoplasmic proteins. However, the molecular mechanism regulating mGluR cell-surface trafficking remains unknown. We report here that mGluR trafficking is controlled by the autoregulatory assembly of a scaffold protein Tamalin. In the absence of mGluR, Tamalin self-assembles into autoinhibited conformations, through its PDZ domain and C-terminal intrinsic ligand motif. X-ray crystallographic analyses visualized integral parts of the oligomeric self-assemblies of Tamalin, which require not only the novel hydrophobic dimerization interface but also canonical and noncanonical PDZ/ligand autoinhibitory interactions. The mGluR cytoplasmic region can competitively bind to Tamalin at a higher concentration, disrupting weak inhibitory interactions. The atomic view of mGluR association suggests that this rearrangement is dominated by electrostatic attraction and repulsion. We also observed in mammalian cells that the association liberates the intrinsic ligand toward a motor protein receptor, thereby facilitating mGluR cell-surface trafficking. Our study suggests a novel regulatory mechanism of the PDZ domain, by which Tamalin switches between the trafficking-inhibited and -active forms depending on mGluR association.
Project description:Group I metabotropic glutamate receptors (mGluRs) play important roles in various neuronal functions and have also been implicated in multiple neuropsychiatric disorders like fragile X syndrome, autism, and others. mGluR trafficking not only plays important roles in controlling the spatiotemporal localization of these receptors in the cell but also regulates the activity of these receptors. Despite this obvious significance, the cellular machineries that control the trafficking of group I metabotropic glutamate receptors in the central nervous system have not been studied in detail. The post-synaptic scaffolding protein tamalin has been shown to interact with group I mGluRs and also with many other proteins involved in protein trafficking in neurons. Using a molecular replacement approach in mouse hippocampal neurons, we show here that tamalin plays a critical role in the ligand-dependent internalization of mGluR1 and mGluR5, members of the group I mGluR family. Specifically, knockdown of endogenous tamalin inhibited the ligand-dependent internalization of these two receptors. Both N-terminal and C-terminal regions of tamalin played critical roles in mGluR1 endocytosis. Furthermore, we found that tamalin regulates mGluR1 internalization by interacting with S-SCAM, a protein that has been implicated in vesicular trafficking. Finally, we demonstrate that tamalin plays a critical role in mGluR-mediated internalization of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, a process believed to be the cellular correlate for mGluR-dependent synaptic plasticity. Taken together, these findings reveal a mechanistic role of tamalin in the trafficking of group I mGluRs and suggest its physiological implications in the brain.
Project description:In this investigation, we report identification and characterization of a 95 kDa postsynaptic density protein (PSD-95)/discs-large/ZO-1 (PDZ) domain-containing protein termed tamalin, also recently named GRP1-associated scaffold protein (GRASP), that interacts with group 1 metabotropic glutamate receptors (mGluRs). The yeast two-hybrid system and in vitro pull-down assays indicated that the PDZ domain-containing, amino-terminal half of tamalin directly binds to the class I PDZ-binding motif of group 1 mGluRs. The C-terminal half of tamalin also bound to cytohesins, the members of guanine nucleotide exchange factors (GEFs) specific for the ADP-ribosylation factor (ARF) family of small GTP-binding proteins. Tamalin mRNA is expressed predominantly in the telencephalic region and highly overlaps with the expression of group 1 mGluR mRNAs. Both tamalin and cytohesin-2 were enriched and codistributed with mGluR1a in postsynaptic membrane fractions. Importantly, recombinant and native mGluR1a/tamalin/cytohesin-2 complexes were coimmunoprecipitated from transfected COS-7 cells and rat brain tissue, respectively. Transfection of tamalin and mutant tamalin lacking a cytohesin-binding domain caused an increase and decrease in cell-surface expression of mGluR1a in COS-7 cells, respectively. Furthermore, adenovirus-mediated expression of tamalin and dominant-negative tamalin facilitated and reduced the neuritic distribution of endogenous mGluR5 in cultured hippocampal neurons, respectively. The results indicate that tamalin plays a key role in the association of group 1 mGluRs with the ARF-specific GEF proteins and contributes to intracellular trafficking and the macromolecular organization of group 1 mGluRs at synapses.
Project description:Major histocompatibility complex class I (MHCI) proteins present antigenic peptides for immune surveillance and play critical roles in nervous system development and plasticity. Most MHCI are transmembrane proteins. The extracellular domain of MHCI interacts with immunoreceptors, peptides, and co-receptors to mediate immune signaling. While the cytoplasmic domain also plays important roles in endocytic trafficking, cross-presentation of extracellularly derived antigens, and CTL priming, the molecular mediators of cytoplasmic signaling by MHCI remain largely unknown.Here we show that the cytoplasmic domain of MHCI contains putative protein-protein interaction domains known as PDZ (PSD95/disc large/zonula occludens-1) ligands. PDZ ligands are motifs that bind to PDZ domains to organize and mediate signaling at cell-cell contacts. PDZ ligands are short, degenerate motifs, and are therefore difficult to identify via sequence homology alone, but several lines of evidence suggest that putative PDZ ligand motifs in MHCI are under positive selective pressure. Putative PDZ ligands are found in all of the 99 MHCI proteins examined from diverse species, and are enriched in the cytoplasmic domain, where PDZ interactions occur. Both the position of the PDZ ligand and the class of ligand motif are conserved across species, as well as among genes within a species. Non-synonymous substitutions, when they occur, frequently preserve the motif. Of the many specific possible PDZ ligand motifs, a handful are strikingly and selectively overrepresented in MHCI's cytoplasmic domain, but not elsewhere in the same proteins. Putative PDZ ligands in MHCI encompass conserved serine and tyrosine residues that are targets of phosphorylation, a post-translational modification that can regulate PDZ interactions. Finally, proof-of-principle in vitro interaction assays demonstrate that the cytoplasmic domains of particular MHCI proteins can bind directly and specifically to PDZ1 and PDZ4&5 of MAGI-1, and identify a conserved PDZ ligand motif in the classical MHCI H2-K that is required for this interaction.These results identify cryptic protein interaction motifs in the cytoplasmic domain of MHCI. In so doing, they suggest that the cytoplasmic domain of MHCI could participate in previously unsuspected PDZ mediated protein-protein interactions at neuronal as well as immunological synapses.
Project description:The Gi-coupled somatostatin receptor 2 (SST2) is a G protein-coupled receptor (GPCR) that mediates many of somatostatin's neuroendocrine actions. Upon stimulation, SST2 is rapidly internalized and transported to early endosomes before being recycled to the plasma membrane. However, little is known about the intracellular itinerary of SST2 after it moves to the early endosomal compartment or the cytoplasmic proteins that regulate its trafficking. As postsynaptic density protein/discs large 1/zonula occludens-1 (PDZ) domain interactions often regulate the trafficking and signaling potential of GPCRs, we examined the role of the SST2 PDZ ligand and additional C-terminal residues in controlling its intracellular trafficking. We determined that SST2 can recycle to the plasma membrane via multiple pathways, including a LAMP1/Rab7-positive late endosome to the trans-Golgi network (TGN) pathway. Trafficking from the late endosome to the TGN is often regulated by the retromer complex of endosomal coat proteins, and disrupting the retromer components sorting nexins 1/2 inhibits the budding of SST2 from late endosomes. Moreover, trafficking through the late endosomal/TGN pathway is dependent on an intact PDZ ligand and C-terminal tail, as truncating either the 3 or 10 C-terminal amino acids of SST2 alters the pathway through which it recycles to the plasma membrane. Moreover, addition of these amino acids to a heterologous receptor is sufficient to redirect it from a degradation pathway to a recycling itinerary. Our results demonstrate that endosomal trafficking of SST2 is dependent on numerous regulatory mechanisms controlled by its C terminus and the retromer machinery.
Project description:The abundance of integral membrane proteins in the plasma membrane is determined by a dynamic balance between exocytosis and endocytosis, which can often be regulated by physiological stimuli. Here, we describe a mechanism that accounts for the ability of the peptide hormone vasopressin to regulate water excretion via a phosphorylation-dependent modulation of the PDZ domain-ligand interaction involving the water channel protein aquaporin-2. We discovered that the PDZ domain-containing protein Sipa1l1 (signal-induced proliferation-associated 1 like 1) binds to the cytoplasmic PDZ-ligand motif of aquaporin-2 and accelerates its endocytosis in the absence of vasopressin. Vasopressin-induced aquaporin-2 phosphorylation within the type I PDZ-ligand motif disrupted the interaction, in association with reduced aquaporin-2 endocytosis and prolonged plasma membrane aquaporin-2 retention. This phosphorylation-dependent alteration in the PDZ domain-ligand interaction was explained by 3D structural models, which showed a hormone-regulated mechanism that controls osmotic water transport and systemic water balance in mammals.
Project description:Voltage-gated Ca(2+) channels play a key role in initiating muscle excitation-contraction coupling, neurotransmitter release, gene expression, and hormone secretion. The association of CaV1.2 with a supramolecular complex impacts trafficking, localization, turnover, and, most importantly, multifaceted regulation of its function in the heart. Several studies hint at an important role for the C terminus of the ?1C subunit as a hub for multidimensional regulation of CaV1.2 channel trafficking and function. Recent studies have demonstrated an important role for the four-residue PDZ binding motif at the C terminus of ?1C in interacting with scaffold proteins containing PDZ domains, in the subcellular localization of CaV1.2 in neurons, and in the efficient signaling to cAMP-response element-binding protein in neurons. However, the role of the ?1C PDZ ligand domain in the heart is not known. To determine whether the ?1C PDZ motif is critical for CaV1.2 trafficking and function in cardiomyocytes, we generated transgenic mice with inducible expression of an N-terminal FLAG epitope-tagged dihydropyridine-resistant ?1C with the PDZ motif deleted (?PDZ). These mice were crossed with ?-myosin heavy chain reverse transcriptional transactivator transgenic mice, and the double-transgenic mice were fed doxycycline. The ?PDZ channels expressed, trafficked to the membrane, and supported robust excitation-contraction coupling in the presence of nisoldipine, a dihydropyridine Ca(2+) channel blocker, providing functional evidence that they appropriately target to dyads. The ?PDZ Ca(2+) channels were appropriately regulated by isoproterenol and forskolin. These data indicate that the ?1C PDZ motif is not required for surface trafficking, localization to the dyad, or adrenergic stimulation of CaV1.2 in adult cardiomyocytes.
Project description: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.
Project description:Intrinsic disorder (ID) is a widespread phenomenon found especially in signaling and regulation-related eukaryotic proteins. The functional importance of flexible disordered regions often resides in their ability to allow proteins to bind different partners. The incidence and location of intrinsic disorder in 369 human single-pass transmembrane receptors with the type I topology was assessed based on both disorder predictions and amino acid physico-chemical properties. We provide evidence that ID concentrates in the receptors' cytoplasmic region. As a benchmark for this analysis, we present a structural study on the previously uncharacterized intracellular region of human Delta-4 (DLL4_IC), a single-pass transmembrane protein and a ligand of Notch receptors. DLL4_IC is required for receptor/ligand endocytosis; it undergoes regulated intramembrane proteolysis, and mediates protein-protein interactions through its C-terminal PDZ binding motif. Using a recombinant purified protein, we demonstrate using various biophysical methods that DLL4_IC is mainly disordered in solution but can form interconvertible local secondary structures in response to variations in the physico-chemical milieu. Most of these conformational changes occur in the highly conserved C-terminal segment that includes the PDZ-binding motif. On the basis of our results, we propose that global disorder, in concert with local preorganization, may play a role in Notch signaling mediated by Delta-4.
Project description:The mammalian Na(+)/H(+) exchange regulatory factor 1 (NHERF1) is a multidomain scaffolding protein essential for regulating the intracellular trafficking and macromolecular assembly of transmembrane ion channels and receptors. NHERF1 consists of tandem PDZ-1, PDZ-2 domains that interact with the cytoplasmic domains of membrane proteins and a C-terminal (CT) domain that binds the membrane-cytoskeleton linker protein ezrin. NHERF1 is held in an autoinhibited state through intramolecular interactions between PDZ2 and the CT domain that also includes a C-terminal PDZ-binding motif (-SNL). We have determined the structures of the isolated and tandem PDZ2CT domains by high resolution NMR using small angle x-ray scattering as constraints. The PDZ2CT structure shows weak intramolecular interactions between the largely disordered CT domain and the PDZ ligand binding site. The structure reveals a novel helix-turn-helix subdomain that is allosterically coupled to the putative PDZ2 domain by a network of hydrophobic interactions. This helical subdomain increases both the stability and the binding affinity of the extended PDZ structure. Using NMR and small angle neutron scattering for joint structure refinement, we demonstrate the release of intramolecular domain-domain interactions in PDZ2CT upon binding to ezrin. Based on the structural information, we show that human disease-causing mutations in PDZ2, R153Q and E225K, have significantly reduced protein stability. Loss of NHERF1 expressed in cells could result in failure to assemble membrane complexes that are important for normal physiological functions.
Project description:Hepatocyte growth factor (HGF) is a potent signaling factor that acts on epithelial cells, causing them to dissociate and scatter. This migration is coordinated by a number of small GTPases, such as ARF6 and Rac1. Active ARF6 is required for HGF-stimulated migration and intracellular levels of ARF6-GTP and Rac1-GTP increase following HGF treatment. During migration, cross talk between ARF6 and Rac1 occurs through formation of a multi-protein complex containing the ARF-GEF cytohesin-2, the scaffolding protein GRASP/Tamalin, and the Rac1-GEF Dock180. Previously, the role of ARF6 in this process was unclear. We have now found that ARF6 and ARF1 regulate trafficking of GRASP and Dock180 to the plasma membrane following HGF treatment. Trafficking of GRASP and Dock180 is impaired by blocking ARF6-mediated recycling pathways and is required for HGF-stimulated Rac1 activation. Finally, HGF treatment stimulates association of GRASP and Dock180. Inhibition of ARF6 trafficking pathways traps GRASP and Dock180 as a complex in the cell.