A three amino acid tail following the TM4 region of the N-methyl-D-aspartate receptor (NR) 2 subunits is sufficient to overcome endoplasmic reticulum retention of NR1-1a subunit

The cytoplasmic C-terminal domains of NR2 subunits have been proposed to modulate the assembly and trafficking of NMDA receptors. However, questions remain concerning which domains in the C terminus of NR2 subunits control the assembly of receptor complexes and how the assembled complexes are selectively trafficked through the various cellular compartments such as endoplasmic reticulum (ER) to the cell surface. In the present study, we found that the three amino acid tail after the TM4 region of NR2 subunits is necessary for surface expression of functional NMDA receptors, while truncations with only two amino acids following the TM4 region (NR2Delta2) completely eliminated surface expression of the NMDA receptor on co-expression with NR1-1a in HEK293 cells. FRET (fluorescence resonance energy transfer) analysis showed that these NR2Delta2 truncations are able to form homomers and heteromers on co-expression with NR1-1a. Furthermore, when NR2Delta2 subunits were cotransfected with either the NR1-4a or NR1-1a(AAA) mutant, lacking the ER retention motif (RRR), functional NMDA receptors were detected in the transfected HEK293 cells. Unexpectedly, we found that the replacement of five residues after TM4 with alanines gave results indistinguishable from those of NR2BDelta5 (EHLFY), demonstrating the short tail following the TM4 of NR2 subunits is not sequence-specific-dependent. Taken together, our results show that the C terminus of the NR2 subunits is not necessary for the assembly of NMDA receptor complexes, whereas a three amino acid long cytoplasmic tail following the TM4 of NR2 subunits is sufficient to overcome the ER retention existing in the C terminus of NR1, allowing the assembled NMDA receptors to reach the cell surface.     

Rescue of internalization-defective platelet-activating factor receptor function by EBP50/NHERF1

Platelet-activating factor (PAF) is a potent phospholipid mediator involved in specific disease states such as allergic asthma, atherosclerosis and psoriasis. The human PAF receptor (PAFR) is a member of the G protein-coupled receptor (GPCR) family. Following PAF stimulation, cells become rapidly desensitized; this refractory state can be maintained for hours and is dependent on PAFR phosphorylation, internalization and trafficking. EBP50/NHERF1 has been found to interact with a variety of proteins and these interactions are involved in a growing range of functions including the assembly of signalling complexes, receptor recycling and transport of proteins to the cell surface. Crucial roles of EBP50 in GPCR physiology include its involvement in internalization, recycling, and downregulation. We were interested in identifying the role of EBP50 in PAFR trafficking. Our results showed that EBP50 binds the PAFR in its basal state, while stimulation decreased the ratio of interaction between the two proteins. We also demonstrated that EBP50 could bind PAFR via its PDZ 2 domain. In addition, we studied the role of EBP50 in various functions of the PAFR such as PAF-induced inositol phosphate accumulation and receptor internalization: EBP50 decreased the WT PAFR response and rescued the function of internalization-deficient mutant receptors, as previously described for the arrestins and the GRKs. These results suggest new roles for EBP50, some of which could help understanding the complex formation after receptor activation.     

Phosphorylation-dependent C-terminal binding of 14-3-3 proteins promotes cell surface expression of HIV co-receptor GPR15

Membrane trafficking is dictated by dynamic molecular interactions involving discrete determinants in the cargo proteins and the intracellular transport machineries. We have previously reported that cell surface expression of GPR15, a G protein-coupled receptor (GPCR) that serves as a co-receptor for HIV, is correlated with the mode III binding of 14-3-3 proteins to the receptor C terminus. Here we provide a mechanistic basis for the role of 14-3-3 in promoting the cell surface expression of GPR15. The Ala mutation of penultimate phospho-Ser (S359A) that abolishes 14-3-3 binding resulted in substantially reduced O-glycosylation and the cell surface expression of GPR15. The surface membrane protein CD8 fused with the C-terminal tail of GPR15(S359A) mutant was re-localized in the endoplasmic reticulum (ER). In the context of S359A mutation, the additional mutations in the upstream stretch of basic residues (RXR motif) restored O-glycosylation and the cell surface expression. The RXR motif was responsible for the interaction with coatomer protein I (COPI), which was inversely correlated with the 14-3-3 binding and cell surface expression. These results suggest that 14-3-3 binding promotes cell surface expression of GPR15 by releasing the receptor from ER retrieval/retention pathway that is mediated by the interaction of RXR motif and COPI. Moreover, 14-3-3 binding substantially increased the stability of GPR15 protein. Thus 14-3-3 proteins play multiple roles in biogenesis and trafficking of an HIV co-receptor GPR15 to control its cell surface density in response to the phosphorylation signal.     

Key amino acid residues within the third membrane domains of NR1 and NR2 subunits contribute to the regulation of the surface delivery of N-methyl-D-aspartate receptors

N-methyl-d-aspartate (NMDA) receptors are glutamate ionotropic receptors that play critical roles in synaptic transmission, plasticity, and excitotoxicity. The functional NMDA receptors, heterotetramers composed mainly of two NR1 and two NR2 subunits, likely pass endoplasmic reticulum quality control before they are released from the endoplasmic reticulum and trafficked to the cell surface. However, the mechanism underlying this process is not clear. Using truncated and mutated NMDA receptor subunits expressed in heterologous cells, we found that the M3 domains of both NR1 and NR2 subunits contain key amino acid residues that contribute to the regulation of the number of surface functional NMDA receptors. These key residues are critical neither for the interaction between the NR1 and NR2 subunits nor for the formation of the functional receptors, but rather they regulate the early trafficking of the receptors. We also found that the identified key amino acid residues within both NR1 and NR2 M3 domains contribute to the regulation of the surface expression of unassembled NR1 and NR2 subunits. Thus, our data identify the unique role of the membrane domains in the regulation of the number of surface NMDA receptors.     

Determinants in the β and δ Subunit Cytoplasmic Loop Regulate Golgi Trafficking and Surface Expression of the Muscle Acetylcholine Receptor

The molecular determinants that govern nicotinic acetylcholine receptor (AChR) assembly and trafficking are poorly defined, and those identified operate largely during initial receptor biogenesis in the endoplasmic reticulum. To identify determinants that regulate later trafficking steps, we performed an unbiased screen using chimeric proteins consisting of CD4 fused to the muscle AChR subunit cytoplasmic loops. In C2 mouse muscle cells, we found that CD4-β and δ subunit loops were expressed at very low levels on the cell surface, whereas the other subunit loops were robustly expressed on the plasma membrane. The low surface expression of CD4-β and δ loops was due to their pronounced retention in the Golgi apparatus and also to their rapid internalization from the plasma membrane. Both retention and recovery were mediated by the proximal 25–28 amino acids in each loop and were dependent on an ordered sequence of charged and hydrophobic residues. Indeed, βK353L and δK351L mutations increased surface trafficking of the CD4-subunit loops by >6-fold and also decreased their internalization from the plasma membrane. Similarly, combined βK353L and δK351L mutations increased the surface levels of assembled AChR expressed in HEK cells to 138% of wild-type levels. This was due to increased trafficking to the plasma membrane and not decreased AChR turnover. These findings identify novel Golgi retention signals in the β and δ subunit loops that regulate surface trafficking of assembled AChR and may help prevent surface expression of unassembled subunits. Together, these results define molecular determinants that govern a Golgi-based regulatory step in nicotinic AChR trafficking.     

Conserved intramolecular disulfide bond is critical to trafficking and fate of ATP-binding cassette (ABC) transporters ABCB6 and sulfonylurea receptor 1 (SUR1)/ABCC8

The ATP-binding cassette (ABC) transporter ABCB6 is a mitochondrial porphyrin transporter that activates porphyrin biosynthesis. ABCB6 lacks a canonical mitochondrial targeting sequence but reportedly traffics to other cellular compartments such as the plasma membrane. How ABCB6 reaches these destinations is unknown. In this study, we show that endogenous ABCB6 is glycosylated in multiple cell types, indicating trafficking through the endoplasmic reticulum (ER), and has only one atypical site for glycosylation (NXC) in its amino terminus. ABCB6 remained glycosylated when the highly conserved cysteine (Cys-8) was substituted with serine to make a consensus site, NXS. However, this substitution blocked ER exit and produced ABCB6 degradation, which was mostly reversed by the proteasomal inhibitor MG132. The amino terminus of ABCB6 has an additional highly conserved ER luminal cysteine (Cys-26). When Cys-26 was mutated alone or in combination with Cys-8, it also resulted in instability and ER retention. Further analysis revealed that these two cysteines form a disulfide bond. We discovered that other ABC transporters with an amino terminus in the ER had similarly configured conserved cysteines. This analysis led to the discovery of a disease-causing mutation in the sulfonylurea receptor 1 (SUR1)/ABCC8 from a patient with hyperinsulinemic hypoglycemia. The mutant allele only contains a mutation in a conserved amino-terminal cysteine, producing SUR1 that fails to reach the cell surface. These results suggest that for ABC transporters the propensity to form a disulfide bond in the ER defines a unique checkpoint that determines whether a protein is ER-retained.     

Differential Regulation of Kainate Receptor Trafficking by Phosphorylation of Distinct Sites on GluR6

Kainate receptors are widely expressed in the brain, and are present at pre- and postsynaptic sites where they play a prominent role in synaptic plasticity and the regulation of network activity. Within individual neurons, kainate receptors of different subunit compositions are targeted to various locations where they serve distinct functional roles. Despite this complex targeting, relatively little is known about the molecular mechanisms regulating kainate receptor subunit trafficking. Here we investigate the role of phosphorylation in the trafficking of the GluR6 kainate receptor subunit. We identify two specific residues on the GluR6 C terminus, Ser⁸⁴⁶ and Ser⁸⁶⁸, which are phosphorylated by protein kinase C (PKC) and dramatically regulate GluR6 surface expression. By using GluR6 containing phosphomimetic and nonphosphorylatable mutations for these sites expressed in heterologous cells or in neurons lacking endogenous GluR6, we show that phosphorylation of Ser⁸⁴⁶ or Ser⁸⁶⁸ regulates receptor trafficking through the biosynthetic pathway. Additionally, Ser⁸⁴⁶ phosphorylation dynamically regulates endocytosis of GluR6 at the plasma membrane. Our findings thus demonstrate that phosphorylation of PKC sites on GluR6 regulates surface expression of GluR6 at distinct intracellular trafficking pathways, providing potential molecular mechanisms for the PKC-dependent regulation of synaptic kainate receptor function observed during various forms of synaptic plasticity.     

Dynamic Na+-H+ exchanger regulatory factor-1 association and dissociation regulate parathyroid hormone receptor trafficking at membrane microdomains

Na/H exchanger regulatory factor-1 (NHERF1) is a cytoplasmic PDZ (postsynaptic density 95/disc large/zona occludens) protein that assembles macromolecular complexes and determines the localization, trafficking, and signaling of select G protein-coupled receptors and other membrane-delimited proteins. The parathyroid hormone receptor (PTHR), which regulates mineral ion homeostasis and bone turnover, is a G protein-coupled receptor harboring a PDZ-binding motif that enables association with NHERF1 and tethering to the actin cytoskeleton. NHERF1 interactions with the PTHR modify its trafficking and signaling. Here, we characterized by live cell imaging the mechanism whereby NHERF1 coordinates the interactions of multiple proteins, as well as the fate of NHERF1 itself upon receptor activation. Upon PTHR stimulation, NHERF1 rapidly dissociates from the receptor and induces receptor aggregation in long lasting clusters that are enriched with the actin-binding protein ezrin and with clathrin. After NHERF1 dissociates from the PTHR, ezrin then directly interacts with the PTHR to stabilize the PTHR at the cell membrane. Recruitment of β-arrestins to the PTHR is delayed until NHERF1 dissociates from the receptor, which is then trafficked to clathrin for internalization. The ability of NHERF to interact dynamically with the PTHR and cognate adapter proteins regulates receptor trafficking and signaling in a spatially and temporally coordinated manner.     

Selective Processing and Metabolism of Disease-Causing Mutant Prion Proteins

Prion diseases are fatal neurodegenerative disorders caused by aberrant metabolism of the cellular prion protein (PrP^C). In genetic forms of these diseases, mutations in the globular C-terminal domain are hypothesized to favor the spontaneous generation of misfolded PrP conformers (including the transmissible PrP^Sc form) that trigger downstream pathways leading to neuronal death. A mechanistic understanding of these diseases therefore requires knowledge of the quality control pathways that recognize and degrade aberrant PrPs. Here, we present comparative analyses of the biosynthesis, trafficking, and metabolism of a panel of genetic disease-causing prion protein mutants in the C-terminal domain. Using quantitative imaging and biochemistry, we identify a misfolded subpopulation of each mutant PrP characterized by relative detergent insolubility, inaccessibility to the cell surface, and incomplete glycan modifications. The misfolded populations of mutant PrPs were neither recognized by ER quality control pathways nor routed to ER-associated degradation despite demonstrable misfolding in the ER. Instead, mutant PrPs trafficked to the Golgi, from where the misfolded subpopulation was selectively trafficked for degradation in acidic compartments. Surprisingly, selective re-routing was dependent not only on a mutant globular domain, but on an additional lysine-based motif in the highly conserved unstructured N-terminus. These results define a specific trafficking and degradation pathway shared by many disease-causing PrP mutants. As the acidic lysosomal environment has been implicated in facilitating the conversion of PrP^C to PrP^Sc, our identification of a mutant-selective trafficking pathway to this compartment may provide a cell biological basis for spontaneous generation of PrP^Sc in familial prion disease.     

N-Linked Glycosylation of Protease-activated Receptor-1 Second Extracellular Loop: A CRITICAL DETERMINANT FOR LIGAND-INDUCED RECEPTOR ACTIVATION AND INTERNALIZATION*

Protease-activated receptor-1 (PAR1) contains five N-linked glycosylation consensus sites as follows: three residing in the N terminus and two localized on the surface of the second extracellular loop (ECL2). To study the effect of N-linked glycosylation in the regulation of PAR1 signaling and trafficking, we generated mutants in which the critical asparagines of the consensus sites were mutated. Here, we report that both the PAR1 N terminus and ECL2 serve as sites for N-linked glycosylation but have different functions in the regulation of receptor signaling and trafficking. N-Linked glycosylation of the PAR1 N terminus is important for transport to the cell surface, whereas the PAR1 mutant lacking glycosylation at ECL2 (NA ECL2) trafficked to the cell surface like the wild-type receptor. However, activated PAR1 NA ECL2 mutant internalization was impaired compared with wild-type receptor, whereas constitutive internalization of unactivated receptor remained intact. Remarkably, thrombin-activated PAR1 NA ECL2 mutant displayed an enhanced maximal signaling response compared with wild-type receptor. The increased PAR1 NA ECL2 mutant signaling was not due to defects in the ability of thrombin to cleave the receptor or signal termination mechanisms. Rather, the PAR1 NA ECL2 mutant displayed a greater efficacy in thrombin-stimulated G protein signaling. Thus, N-linked glycosylation of the PAR1 extracellular surface likely influences ligand docking interactions and the stability of the active receptor conformation. Together, these studies strongly suggest that N-linked glycosylation of PAR1 at the N terminus versus the surface of ECL2 serves distinct functions critical for proper regulation of receptor trafficking and the fidelity of thrombin signaling.     

Characterization of the Chemokine CXCL11-Heparin Interaction Suggests Two Different Affinities for Glycosaminoglycans

Chemokines orchestrate the migration of leukocytes in the context of homeostasis and inflammation. In addition to interactions of chemokines with receptors on migrating cells, these processes require interactions of chemokines with glycosaminoglycans (GAGs) for cell surface localization. Most chemokines are basic proteins with Arg/Lys/His residue clusters functioning as recognition epitopes for GAGs. In this study we characterized the GAG-binding epitopes of the chemokine I-TAC/CXCL11. Four separate clusters of basic residues were mutated to alanine and tested for their ability to bind to GAGs in vitro and to activate the receptor, CXCR3. Mutation of a set of basic residues in the C-terminal helix (the 50s cluster, ⁵⁷KSKQAR⁶²) along with Lys¹⁷, significantly impaired heparin binding in vitro, identifying these residues as components of the dominant epitope. However, this GAG mutant retained nearly wild type receptor binding affinity, and its ability to induce cell migration in vitro was only mildly perturbed. Nevertheless, the mutant was unable to induce cell migration in vivo, establishing a requirement of CXCL11 for GAG binding for in vivo function. These studies also led to some interesting findings. First, CXCL11 exhibits conformational heterogeneity, as evidenced by the doubling of peaks in its HSQC spectra. Second, it exhibits more than one affinity state for both heparin and CXCR3, which may be related to its structural plasticity. Finally, although the binding affinities of chemokines for GAGs are typically weaker than interactions with receptors, the high affinity GAG binding state of CXCL11 is comparable with typical receptor binding affinities, suggesting some unique properties of this chemokine.     

Transmembrane region of N-methyl-D-aspartate receptor (NMDAR) subunit is required for receptor subunit assembly

N-Methyl-D-aspartate receptors (NMDARs), one of three main classes of ionotropic glutamate receptors, play major roles in synaptic plasticity, synaptogenesis, and excitotoxicity. Unlike non-NMDA receptors, NMDARs are thought to comprise obligatory heterotetrameric complexes mainly composed of GluN1 and GluN2 subunits. When expressed alone in heterogenous cells, such as HEK293 cells, most of the NMDAR subunits can neither leave the endoplasmic reticulum (ER) nor be expressed in the cell membrane because of the ER retention signals. Only when NMDARs are heteromerically assembled can the ER retention signals be masked and NMDARs be expressed in the surface membrane. However, the mechanisms underlying NMDAR assembly remain poorly understood. To identify regions in subunits that mediate this assembly, we made a series of truncated or chimeric cDNA constructs. Using FRET measurement in living cells combined with immunostaining and coimmunoprecipitation analysis, we examined the assembly-determining domains of NMDAR subunits. Our results indicate that the transmembrane region of subunits is necessary for the assembly of NMDAR subunits, both for the homodimer and the heteromer.     

Assembly-dependent trafficking assays in the detection of receptor-receptor interactions

Assembly-dependent trafficking is a property of many multimeric membrane protein complexes; this coupling of assembly and trafficking processes provides an important cellular quality control mechanism, ensuring that only properly folded and assembled complexes are expressed on the cell surface. In all membrane protein complexes whose trafficking is known to be assembly-dependent, at least one of the subunits contains an endoplasmic reticulum (ER) retention/retrieval signal that is shielded on subunit assembly, allowing the assembled protein complex to traffic to the plasma membrane. Under these conditions, presence of the normally retained subunit on the cell surface can be used as an indirect index of protein assembly in the ER. In this article, I describe the design of two complementary approaches (trafficking enhancement and trap assays) that can be used separately or in combination to determine whether two (or more) proteins assemble in the ER, i.e., whether they constitutively oligomerize. Both of the approaches are based on the measurement of plasma membrane-expressed proteins using antibody-mediated detection of extracellularly expressed epitopes and subsequent luminometric quantification. These methods provide a straightforward and relatively inexpensive way to assess protein-protein interactions early in the synthetic pathway.     

Cell surface expression of GluR5 kainate receptors is regulated by an endoplasmic reticulum retention signal

Kainate receptors (KARs) are mediators of excitatory neurotransmission in the mammalian central nervous system, and their efficient targeting and trafficking is critical for normal synaptic function. A key step in the delivery of KARs to the neuronal plasma membrane is the exit of newly assembled receptors from the endoplasmic reticulum (ER). Here we report the identification of a novel ER retention signal in the alternatively spliced C-terminal domain of the GluR5-2b subunit, which controls receptor trafficking in both heterologous cells and neurons. The ER retention motif consists of a critical arginine (Arg-896) and surrounding amino acids, disruption of which promotes ER exit and surface expression of the receptors, as well as altering their physiological properties. The Arg-896-mediated ER retention of GluR5 is regulated by a mutation that mimics phosphorylation of Thr-898, but not by PDZ interactions. Furthermore, two positively charged residues (Arg-900 and Lys-901) in the C terminus were also found to regulate ER export of the receptors. Taken together, our results identify novel trafficking signals in the C-terminal domain of GluR5-2b and demonstrate that alternative splicing is an important mechanism regulating KAR function.     

Role of C-terminal Membrane-proximal Basic Residues in Cell Surface Trafficking of HIV Coreceptor GPR15 Protein

Cell surface density of G protein-coupled receptors (GPCRs) is controlled by dynamic molecular interactions that often involve recognition of the distinct sequence signals on the cargo receptors. We reported previously that the RXR-type dibasic motif in the distal C-terminal tail of an HIV coreceptor GPR15 negatively regulates the cell surface expression by mediating the coatomer protein I complex-dependent retrograde transport to the endoplasmic reticulum (ER). Here we demonstrate that another pair of basic residues (Arg³¹⁰-Arg³¹¹) in the membrane-proximal region of the C-terminal tail plays a pivotal role in mediating the anterograde trafficking of GPR15. The Ala mutation of the C-terminal membrane-proximal basic residues (MPBRs) (R310/311A) abolished the O-glycosylation and cell surface expression of GPR15. The subcellular fractionation and immunocytochemistry assays indicated that the R310/311A mutant was more localized in the ER but much less in the trans-Golgi when compared with the wild-type GPR15, suggesting the positive role of Arg³¹⁰-Arg³¹¹ in the ER-to-Golgi transport of GPR15. Sequence analysis on human GPCRs showed that the basic residues are frequent in the membrane-proximal region of the C-terminal tail. Similar to GPR15, mutation of the C-terminal MPBRs resulted in a marked reduction of the cell surface expression in multiple different GPCRs. Our results suggest that the C-terminal MPBRs are critically involved in mediating the anterograde trafficking of a broad range of membrane proteins, including GPCRs.     

The integrity of the glycine co-agonist binding site of N-methyl-D-aspartate receptors is a functional quality control checkpoint for cell surface delivery

N-Methyl-D-aspartate receptors are a subclass of ligand-gated, heteromeric glutamatergic neurotransmitter receptors whose cell surface expression is regulated by quality control mechanisms. Functional quality control checkpoints are known to contribute to cell surface trafficking of non-N-methyl-D-aspartate glutamate receptors. Here we investigated if similar mechanisms operate for the surface delivery of NMDA receptors. Point mutations in the glycine binding domain of the NR1-1a subunit were generated: D732A, a mutation that results in an approximately 3 x 10(4) decrease in glycine binding affinity; D732E, a conservative change; and D723A, a residue in the same NR1-1a domain that has no effect on glycine binding affinity. Each NR1-1a subunit was co-expressed with NR2A in mammalian cells. Immunoblotting and immunoprecipitations showed that all mutants were expressed to similar levels as wild-type NR1-1a and associated with NR2A. Cell surface expression measured by an enzyme-linked immunosorbent assay found that whereas NR1-1a (D732E)/NR2A and NR1-1a (D723A)/NR2A trafficked as efficiently as NR1-1a/NR2A, there was a 90% decrease in surface expression for NR1-1a (D732A)/NR2A. This was confirmed by confocal microscopy imaging and cell surface biotinylation. Further imaging showed that NR1-1a (D732A) and co-transfected NR2A co-localized with an endoplasmic reticulum marker. Dichlorokynurenic acid, a competitive glycine site antagonist, partially rescued surface expression. Mutation of the NR1-1a ER retention motif showed that the ligand binding checkpoint is an early event preceding endoplasmic reticulum sorting mechanisms. These findings demonstrate that integrity of the glycine co-agonist binding site is a functional checkpoint requisite for efficient cell surface trafficking of assembled NMDA receptors.     

Up-regulation of GABAB Receptor Signaling by Constitutive Assembly with the K+ Channel Tetramerization Domain-containing Protein 12 (KCTD12)

GABA_B receptors are the G-protein coupled receptors (GPCRs) for GABA, the main inhibitory neurotransmitter in the central nervous system. Native GABA_B receptors comprise principle and auxiliary subunits that regulate receptor properties in distinct ways. The principle subunits GABA_B1a, GABA_B1b, and GABA_B2 form fully functional heteromeric GABA_B(1a,2) and GABA_B(1b,2) receptors. Principal subunits regulate forward trafficking of the receptors from the endoplasmic reticulum to the plasma membrane and control receptor distribution to axons and dendrites. The auxiliary subunits KCTD8, -12, -12b, and -16 are cytosolic proteins that influence agonist potency and G-protein signaling of GABA_B(1a,2) and GABA_B(1b,2) receptors. Here, we used transfected cells to study assembly, surface trafficking, and internalization of GABA_B receptors in the presence of the KCTD12 subunit. Using bimolecular fluorescence complementation and metabolic labeling, we show that GABA_B receptors associate with KCTD12 while they reside in the endoplasmic reticulum. Glycosylation experiments support that association with KCTD12 does not influence maturation of the receptor complex. Immunoprecipitation and bioluminescence resonance energy transfer experiments demonstrate that KCTD12 remains associated with the receptor during receptor activity and receptor internalization from the cell surface. We further show that KCTD12 reduces constitutive receptor internalization and thereby increases the magnitude of receptor signaling at the cell surface. Accordingly, knock-out or knockdown of KCTD12 in cultured hippocampal neurons reduces the magnitude of the GABA_B receptor-mediated K⁺ current response. In summary, our experiments support that the up-regulation of functional GABA_B receptors at the neuronal plasma membrane is an additional physiological role of the auxiliary subunit KCTD12.     

Novel role for proteinase-activated receptor 2 (PAR2) in membrane trafficking of proteinase-activated receptor 4 (PAR4)

Proteinase-activated receptors 4 (PAR(4)) is a class A G protein-coupled receptor (GPCR) recognized through the ability of serine proteases such as thrombin and trypsin to mediate receptor activation. Due to the irreversible nature of activation, a fresh supply of receptor is required to be mobilized to the cell surface for responsiveness to agonist to be sustained. Unlike other PAR subtypes, the mechanisms regulating receptor trafficking of PAR(4) remain unknown. Here, we report novel features of the intracellular trafficking of PAR(4) to the plasma membrane. PAR(4) was poorly expressed at the plasma membrane and largely retained in the endoplasmic reticulum (ER) in a complex with the COPI protein subunit β-COP1. Analysis of the PAR(4) protein sequence identified an arginine-based (RXR) ER retention sequence located within intracellular loop-2 (R(183)AR → A(183)AA), mutation of which allowed efficient membrane delivery of PAR(4). Interestingly, co-expression with PAR(2) facilitated plasma membrane delivery of PAR(4), an effect produced through disruption of β-COP1 binding and facilitation of interaction with the chaperone protein 14-3-3ζ. Intermolecular FRET studies confirmed heterodimerization between PAR(2) and PAR(4). PAR(2) also enhanced glycosylation of PAR(4) and activation of PAR(4) signaling. Our results identify a novel regulatory role for PAR(2) in the anterograde traffic of PAR(4). PAR(2) was shown to both facilitate and abrogate protein interactions with PAR(4), impacting upon receptor localization and cell signal transduction. This work is likely to impact markedly upon the understanding of the receptor pharmacology of PAR(4) in normal physiology and disease.     

Mutagenesis studies of conserved proline residues of human P2X receptors for ATP indicate that proline 272 contributes to channel function

Proline residues can play a major role in the secondary structure of proteins. In the extracellular ATP binding loop of P2X receptors there are four totally conserved proline residues (P2X1 receptor numbering; P93, P166, P228 and P272) and three less conserved residues P196 (six of seven isoforms), P174 and P225 (five of seven isoforms). We have mutated individual conserved proline residues in the human P2X1 receptor and determined their properties. Mutants were expressed in Xenopus oocytes and characterized using a two-electrode voltage clamp. Mutants P166A, P174A, P196A, P225A and P228A had no effect on ATP potency compared with wild-type and P93A had a fourfold decrease in ATP potency. The P272A, P272D and P272K receptor mutants were expressed at the cell surface; however, these mutants were non-functional. In contrast, P272I, P272G and P272F produced functional channels, with either no effect or a 2.5- or 6.5-fold increase in ATP potency, respectively. At P272F receptors the apparent affinity of the ATP analogue antagonist 2',3'-O-(2,4,6-trinitrophenyl)-ATP was increased by 12.5-fold. These results suggest that individual proline residues are not essential for normal P2X receptor function and that the receptor conformation around P272 contributes to ATP binding at the receptor.