GABAA Receptor-associated Protein Research Articles

Identification of Clathrin Heavy Chain as a Direct Interaction Partner for the γ-Aminobutyric Acid Type A Receptor Associated Protein

Gamma-aminobutyric acid type A receptors (GABAA receptors) are the major sites of GABA-mediated fast synaptic inhibition in the central nervous system. Variation of the cell surface receptor count is postulated to be of importance in modulating inhibitory synaptic transmission. The GABAA receptor associated protein (GABARAP) is a ubiquitin-like modifier, implicated in GABAA receptor clustering, trafficking, and turnover. GABARAP pull-down experiments with brain lysate identified clathrin heavy chain to be GABARAP-associated. Phage display screening of a randomized peptide library for GABARAP ligands yielded a sequence motif which characterizes the peptide binding specificity of GABARAP. Sequence database searches with this motif revealed clathrin heavy chain as a protein containing the identified sequence motif within its residues 510-522, supporting the result of the pull-down experiments. Calreticulin, which was identified recently as a GABARAP ligand, contains a very similar sequence motif. We demonstrate that calreticulin indeed competes with clathrin heavy chain for GABARAP binding. Finally, employing nuclear magnetic resonance spectroscopy, we mapped the GABARAP residues responsible for binding to clathrin. The hereby mapped GABARAP regions overlap very well with the homologue residues in yeast Atg8 that were recently shown to be important for autophagy. Together with the knowledge that GABARAP and clathrin are known to be involved in GABAA receptor trafficking within the cell, this strongly suggests a clear physiological relevance of the direct interaction of GABARAP with clathrin heavy chain.

Identification of calreticulin as a ligand of GABARAP by phage display screening of a peptide library

4-Aminobutyrate type A (GABA(A)) receptor-associated protein (GABARAP) is a ubiquitin-like modifier implicated in the intracellular trafficking of GABA(A) receptors, and belongs to a family of proteins involved in intracellular vesicular transport processes, such as autophagy and intra-Golgi transport. In this article, it is demonstrated that calreticulin is a high affinity ligand of GABARAP. Calreticulin, although best known for its functions as a Ca(2+) -dependent chaperone and a Ca(2+) -buffering protein in the endoplasmic reticulum, is also localized to the cytosol and exerts a variety of extra-endoplasmic reticulum functions. By phage display screening of a randomized peptide library, peptides that specifically bind GABARAP were identified. Their amino acid sequences allowed us to identify calreticulin as a potential GABARAP binding protein. GABARAP binding to calreticulin was confirmed by pull-down experiments with brain lysate and colocalization studies in N2a cells. Calreticulin and GABARAP interact with a dissociation constant K(d) = 64 nm and a mean lifetime of the complex of 20 min. Thus, the interaction between GABARAP and calreticulin is the strongest so far reported for each protein.

C-Terminal Modification Is Required for GABARAP-Mediated GABAA Receptor Trafficking

We investigated the ubiquitin-like modification of GABA(A) receptor-associated protein (GABARAP) and its function. A fusion protein of GABARAP with v5 in the N terminus and myc in the C terminus was expressed in rat cultured hippocampal neurons and PC12 cells. Western blotting with antibodies to v5 and myc revealed that the C terminus of GABARAP was cleaved off. Cleavage was blocked by mutating the C-terminal Gly116 to Ala, suggesting that G116 is required for the processing. Unlike ubiquitin, GABARAP was not incorporated covalently into higher-molecular-weight protein complexes. Nor was GABARAP degraded by ubiquitinylation through the proteasome, although GABARAP formed noncovalent dimers. Immunofluorescent confocal microscopy demonstrated that recombinantly expressed GABARAP was diffusely localized in PC12 cells. However, prevention of C-terminal processing in the mutant GABARAP(G116A) resulted in redistribution to the Golgi. In neurons, punctate cytoplasmic staining of GABARAP was seen in soma and processes, whereas GABARAP(G116A) was limited to soma. Compared with wild-type GABARAP, the colocalization and interaction of GABARAP(G116A) with GABA(A) receptors were significantly reduced, resulting in a reduction in expression of receptors in the plasma membrane. When alpha1beta2gamma2S-containing GABA(A) receptors were expressed in oocytes, the increased surface expression of GABA(A) receptors, as shown by increased GABA currents and surface-accessible GABA(A) receptor subunit polypeptides resulting from GABARAP coexpression, was prevented by mutation G116A. In addition, the distribution of NSF (N-ethylmaleimide-sensitive factor) was affected in GABARAP(G116A)-expressing neurons. These results suggest that glycine 116 is required for C-terminal processing of GABARAP and that processing is essential for the localization of GABARAP and its functions as a trafficking protein.

Sustained Structural Change of GABAA Receptor-Associated Protein Underlies Long-Term Potentiation at Inhibitory Synapses on a Cerebellar Purkinje Neuron

Fast inhibitory synaptic transmission is predominantly mediated by GABA(A) receptor (GABA(A)R) in the CNS. Although several types of neuronal activity-dependent plasticity at GABAergic synapses have been reported, the detailed mechanism is elusive. Here we show that binding of structurally altered GABA(A)R-associated protein (GABARAP) to GABA(A)R gamma2 subunit and to tubulin is critical for long-term potentiation [called rebound potentiation (RP)] at inhibitory synapses on a cerebellar Purkinje neuron (PN). Either inhibition of GABARAP association with GABA(A)Rgamma2 or deletion of tubulin binding region of GABARAP impaired RP. Inhibition of tubulin polymerization also suppressed RP. Thus, precise regulation of GABA(A)Rgamma2-GABARAP-microtubule interaction is critical for RP. Furthermore, competitive inhibition of GABARAP binding to GABA(A)Rgamma2 after the RP establishment attenuated the potentiated response, suggesting that GABARAP is critical not only for the induction but also for the maintenance of RP. Fluorescence resonance energy transfer analysis revealed that GABARAP underwent sustained structural alteration after brief depolarization of a PN depending on the activity of Ca2+/calmodulin-dependent protein kinase II (CaMKII), which is required for the RP induction. The susceptibility of GABARAP to undergo structural alteration was abolished by an amino acid replacement in GABARAP. Furthermore, RP was impaired by expression of the mutant GABARAP with the replacement. Together, we conclude that GABA(A)R association with structurally altered GABARAP downstream of CaMKII activation is essential for RP.

Phosholipase C-Related Inactive Protein Is Involved in Trafficking of γ2 Subunit-Containing GABAAReceptors to the Cell Surface

The subunit composition of GABA(A) receptors is known to be associated with distinct physiological and pharmacological properties. Previous studies that used phospholipase C-related inactive protein type 1 knock-out (PRIP-1 KO) mice revealed that PRIP-1 is involved in the assembly and/or the trafficking of gamma2 subunit-containing GABA(A) receptors. There are two PRIP genes in mammals; thus the roles of PRIP-1 might be compensated partly by those of PRIP-2 in PRIP-1 KO mice. Here we used PRIP-1 and PRIP-2 double knock-out (PRIP-DKO) mice and examined the roles for PRIP in regulating the trafficking of GABA(A) receptors. Consistent with previous results, sensitivity to diazepam was reduced in electrophysiological and behavioral analyses of PRIP-DKO mice, suggesting an alteration of gamma2 subunit-containing GABA(A) receptors. The surface numbers of diazepam binding sites (alpha/gamma2 subunits) assessed by [3H]flumazenil binding were reduced in the PRIP-DKO mice as compared with those of wild-type mice, whereas the cell surface GABA binding sites (alpha/beta subunits, assessed by [3H]muscimol binding) were increased in PRIP-DKO mice. The association between GABA(A) receptors and GABA(A) receptor-associated protein (GABARAP) was reduced significantly in PRIP-DKO neurons. Disruption of the direct interaction between PRIP and GABA(A) receptor beta subunits via the use of a peptide corresponding to the PRIP-1 binding site reduced the cell surface expression of gamma2 subunit-containing GABA(A) receptors in cultured cell lines and neurons. These results suggest that PRIP is implicated in the trafficking of gamma2 subunit-containing GABA(A) receptors to the cell surface, probably by acting as a bridging molecule between GABARAP and the receptors.

Regulation of GABAA-Receptor Surface Expression With Special Reference to the Involvement of GABARAP (GABAA Receptor-Associated Protein) and PRIP (Phospholipase C-Related, but Catalytically Inactive Protein)

GABAA receptors are heteropentameric ligand-gated chloride channels composed of a variety of subunits, including α1 – 6, β1 – 3, γ1 – 3, δ, ε, θ, and π, and play a key role in controlling inhibitory neuronal activity. Modification of the efficacy of the synaptic strength is produced by changes in both the number of neuronal surface receptors and pentameric molecular assembly, leading to differences of sensitivity to neurotransmitters and neuromimetic drugs. Therefore, it is important to understand the molecular mechanisms regulating the so-called “life cycle of GABAA receptors” including sequential pentameric assembly at the site synthesized, intracellular transport through the Golgi apparatus and the cytoplasm, insertion into the cell membrane, functional modulation at the cell surface, and finally internalization, followed by either recycling back to the surface membrane or lysosomal degradation. This review is focused on events related to the surface expression of the receptor containing the γ2 subunit and clathrin/AP2 complex–mediated phospho-regulated endocytosis of the receptor, with special reference to the function of novel GABAA receptor modulators, GABARAP (GABAA receptor-associated protein) and PRIP (phospholipase C-related, but catalytically inactive protein).

Identification of a Novel Signaling Molecule and Elucidation of Its Cellular Functions-Development of an Interface between Neuroscience and Oral Health Science-

The investigation of chemically synthesized inositol 1,4,5-trisphosphate [Ins (1,4,5)P3] analogs has led to the isolation of a novel protein with a molecular size of 130 kDa, characterized as a molecule with a domain organization similar to phospholipase C (PLC)-δ1 but lacking enzymatic activity. Two isoforms of the molecule were subsequently identified, the molecule has been named PRIP (PLC-related, but catalytically inactive protein), with the two isoforms named PRIP-1 and -2. Regarding its ability to bind Ins (1,4,5)-P3 via the pleckstrin homology domain, the involvement of PRIP-1 in Ins (1,4,5)P3-mediated Ca2+ signaling was first examined. Yeast two-hybrid screening of a brain cDNA library identified GABARAP (GABAA receptor-associated protein) and PP1 (protein phosphatase 1), which led us to examine the possible neurological involvement of PRIP, particularly in GABAA receptor signaling. PRIP-1 and -2 double knock-out (DKO) mice were analyzed for GABAA receptor function with special reference to the action of benzodiazepines whose target is the γ subunit of the receptors; sensitivity to benzodiazepine was reduced, as assessed by biochemical, electrophysiological, and behavioral analyses of DKO mice, suggesting the dysfunction of γ2 subunit-containing GABAA receptors. The mesencephalic trigeminal nucleus, which mediates perceptions from periodontal mechanoreceptors and jaw-closer muscle spindles, receives many synaptic inputs, including those from GABAA receptors, indicating that PRIP might indirectly be involved in rhythmical jaw movement. In the present article, we summarize our current research and the functional significance of PRIP.

Fine mapping of a linkage region on chromosome 17p13 reveals that GABARAP and DLG4 are associated with vulnerability to nicotine dependence in European-Americans

A two-stage association study was conducted targeting a genomic region on chromosome 17p13 that we reported likely to harbor susceptibility gene(s) for nicotine dependence (ND). Participants were 2037 subjects from 602 nuclear families of either African-American (AA) or European-American (EA) origin from our Mid-South Tobacco Family (MSTF) cohort. We first examined 10 single nucleotide polymorphisms (SNPs) in six genes within the targeted region of about 90 kb to determine which SNP/gene was associated with ND, assessed by smoking quantity (SQ), the heaviness of smoking index (HSI) and the Fagerström Test for ND (FTND). Individual SNP analysis revealed that SNPs rs17710 and rs222843 in GABA(A) receptor-associated protein (GABARAP) exhibited a significant association with at least one age- and gender-adjusted ND measure in the EA sample and rs222843 remained significant with the FTND after correction for multiple testing (P = 0.009). Although no SNP in DLG4 was significantly associated with ND, we found a G-G haplotype with a frequency of 14.2% formed by SNPs rs2242449 and rs507506 within the gene that showed significant inverse associations with all three ND measures [P = 0.003, 0.015 and 0.024, for SQ (defined as the number of cigarettes smoked per day), HSI and FTND, respectively]. We also found an A-A haplotype with a frequency of 8.8% formed by SNPs rs17710 and rs222843 in GABARAP, which revealed significant associations with all three ND measures (P = 0.006, 0.019 and 0.024, for SQ, HSI and FTND, respectively). To confirm these findings with a better coverage of GABARAP and DLG4, we conducted a second-stage association analysis by genotyping four more SNPs for GABARAP and nine more for DLG4 on the same set of samples. Our results from the second stage of individual SNP- and/or haplotype-based association analysis supported our finding of significant association of the DLG4 gene with ND. No significant association of GABARAP or DLG4 with ND was detected in the AA sample. Further, by comparing the linkage signal before and after adjustment for the SNPs of GABARAP and DLG4, we found that inclusion of the SNPs of the two genes as covariates largely reduced the linkage signal in the EA sample, but kept nearly unchanged in the AA sample. Taken together, our two-stage association analysis and linkage analysis results indicate that the GABARAP and DLG4 genes are involved in the etiology of ND in EA smokers. Further investigation of neurobiological mechanisms of the two genes in the etiology of ND is thus warranted.

GABAA receptor associated proteins: a key factor regulating GABAA receptor function

gamma-Aminobutyric acid (GABA), an important inhibitory neurotransmitter in both vertebrates and invertebrates, acts on GABA receptors that are ubiquitously expressed in the CNS. GABA(A) receptors also represent a major site of action of clinically relevant drugs, such as benzodiazepines, barbiturates, ethanol, and general anesthetics. It has been shown that the intracellular M3-M4 loop of GABA(A) receptors plays an important role in regulating GABA(A) receptor function. Therefore, studies of the function of receptor intracellular loop associated proteins become important for understanding mechanisms of regulating receptor activity. Recently, several labs have used the yeast two-hybrid assay to identify proteins interacting with GABA(A) receptors, for example, the interaction of GABA(A) receptor associated protein (GABARAP) and Golgi-specific DHHC zinc finger protein (GODZ) with gamma subunits, PRIP, phospholipase C-related, catalytically inactive proteins (PRIP-1) and (PRIP-2) with GABARAP and receptor gamma2 and beta subunits, Plic-1 with some alpha and beta subunits, radixin with the alpha5 subunit, HAP1 with the beta1 subunit, GABA(A) receptor interacting factor-1 (GRIF-1) with the beta2 subunit, and brefeldin A-inhibited GDP/GTP exchange factor 2 (BIG2) with the beta3 subunit. These proteins have been shown to play important roles in modulating the activities of GABA(A) receptors ranging from enhancing trafficking, to stabilizing surface and internalized receptors, to regulating modification of GABA(A) receptors. This article reviews the current studies of GABA(A) receptor intracellular loop-associated proteins.

Autoimmunity to GABAA-receptor-associated protein in stiff-person syndrome

Stiff-person syndrome (SPS) is an autoimmune neurological disorder characterized by autoantibodies to glutamic acid decarboxylase (GAD), the enzyme responsible for the synthesis of inhibitory neurotransmitter GABA. To search for biomarkers that distinguish SPS from other neurological disorders (OND), we used surface enhanced laser desorption/ionization-time of flight (SELDI-TOF) mass spectrometry to obtain proteomic profile of sera from 25 GAD-positive SPS patients and 25 controls. A significant decrease was found in the level of a protein corresponding to GABA(A)-receptor-associated protein (GABARAP), which is responsible for the stability and surface expression of the GABA(A)-receptor. Up to 70% of the SPS sera examined, compared with 10% of the controls, immunoprecipitated GABARAP protein. Antibodies raised against GABARAP immunostained neuronal cell bodies as well as axonal and dendritic processes, as visualized by confocal microscopy. In vitro experiments demonstrated that the IgG from GABARAP antibody-positive patients, but not control IgG, significantly inhibited the surface expression of GABA(A)-receptor. We conclude that GABARAP is a new autoantigen in SPS. Because the patients' IgG inhibits the expression of GABA(A)-receptors, the circulating antibodies could impair GABAergic pathways and play a role in the clinical symptomatology of SPS patients.

Human RAB24, interestingly and predominantly distributed in the nuclei of COS-7 cells, is colocalized with cyclophilin A and GABARAP

As an atypical member of the Rab family, Rab24 has several attributes distinguishing this protein from the other members. Based on the yeast two-hybrid system, interaction between human RAB24 and two proteins, cyclophilin A (CyP-A) and gamma-aminobutyric acid type A receptor-associated protein (GABARAP), was detected and identified in COS-7 cells. Interestingly, RAB24 is predominantly localized in the nuclei of COS-7 cells, which is different from previous reports using other cell lines. RAB24 (D123I) can trigger the accumulation of intracellular inclusions, with small quantities of intranuclear inclusions in some cells. The GTPase activity of RAB24 and its two mutants was detected.

Tandem Subunits Effectively Constrain GABAAReceptor Stoichiometry and Recapitulate Receptor Kinetics But Are Insensitive to GABAAReceptor-Associated Protein

GABAergic synapses likely contain multiple GABAA receptor subtypes, making postsynaptic currents difficult to dissect. However, even in heterologous expression systems, analysis of receptors composed of alpha, beta, and gamma subunits can be confounded by receptors expressed from alpha and beta subunits alone. To produce recombinant GABAA receptors containing fixed subunit stoichiometry, we coexpressed individual subunits with a "tandem" alpha1 subunit linked to a beta2 subunit. Cotransfection of the gamma2 subunit with alphabeta-tandem subunits in human embryonic kidney 293 cells produced currents that were similar in their macroscopic kinetics, single-channel amplitudes, and pharmacology to overexpression of the gamma subunit with nonlinked alpha1 and beta2 subunits. Similarly, expression of alpha subunits together with alphabeta-tandem subunits produced receptors having physiological and pharmacological characteristics that closely matched cotransfection of alpha with beta subunits. In this first description of tandem GABAA subunits measured with patch-clamp and rapid agonist application techniques, we conclude that incorporation of alphabeta-tandem subunits can be used to fix stoichiometry and to establish the intrinsic kinetic properties of alpha1beta2 and alpha1beta2gamma2 receptors. We used this method to test whether the accessory protein GABAA receptor-associated protein (GABARAP) alters GABAA receptor properties directly or influences subunit composition. In recombinant receptors with fixed stoichiometry, coexpression of GABARAP-enhanced green fluorescent protein (EGFP) fusion protein had no effect on desensitization, deactivation, or diazepam potentiation of GABA-mediated currents. However, in alpha1beta2gamma2S transfections in which stoichiometry was not fixed, GABARAP-EGFP altered desensitization, deactivation, and diazepam potentiation of GABA-mediated currents. The data suggest that GABARAP does not alter receptor kinetics directly but by facilitating surface expression of alphabetagamma receptors.

GABAA receptor-associated protein (GABARAP) induces apoptosis by interacting with DEAD (Asp-Glu-Ala-Asp/His) box polypeptide 47 (DDX 47)

GABA(A) receptor-associated protein (GABARAP) is a 14-kDa cytoplasmic protein initially identified as a molecular chaperone for GABA(A) receptor in cortical neurons. However, evidence indicates that the function of GABARAP is much broader than a specific role in neuronal cells. As an initial step to define the biological role of GABARAP, we searched for binding partners of GABARAP using co-immunoprecipitation coupled with LC-MS/MS analysis. As a result, DEAD (Asp-Glu-Ala-Asp/His) box polypeptide 47 (DDX 47), recently identified as RNA helicase, is identified as a binding partner of GABARAP. An interaction between GABARAP and DDX 47 was further confirmed by yeast two-hybrid systems. Further, co-transfection of GABARAP and DDX47 cDNA into a tumor cell line induces apoptosis. This result may be the first evidence showing that GABARAP and DDX 47 are involved in the apoptotic process.

GABAAReceptor-Associated Protein Regulates GABAAReceptor Cell-Surface Number inXenopus laevisOocytes

GABA(A) receptor-associated protein (GABARAP) was isolated previously in a yeast two-hybrid screen using the intracellular loop of the gamma2 subunit of the GABA(A) receptor as bait. GABARAP has been shown to participate in the membrane-clustering and intracellular-trafficking of GABA(A) receptors, including a stimulation of the surface expression of GABA(A) receptors. To assess this quantitatively, we used Xenopus laevis oocytes expressing alpha1beta2gamma2S-containing GABA(A) receptors to demonstrate that coexpression of GABARAP increased net surface levels of GABA(A) receptors as shown by both increased GABA currents and surface-expressed protein. This GABARAP stimulation of GABA currents required the receptor gamma2 subunit and full-length GABARAP: deletion of the microtubule-binding domain (amino acids 1-22) or disrupting the polymerization of microtubules abolished the enhancement, indicating that the effect of GABARAP was derived from the interaction with microtubules. GABARAP coexpression did not alter the general properties of GABA(A) receptors such as sensitivity to GABA or benzodiazepines, but it increased surface levels of receptor protein in oocytes. Rather, it seems to supplement inadequate amounts of endogenous GABARAP to support optimum trafficking and/or stabilization of surface GABA(A) receptors.

GABAAReceptor-Associated Protein Traffics GABAAReceptors to the Plasma Membrane in Neurons

The trafficking of GABA(A) receptors is an important component of the pathway that regulates plasticity of inhibitory synapses. The 17 kDa GABA(A) receptor-associated protein (GABARAP) has been implicated in the trafficking of GABA(A) receptors because of its ability to interact not only with the gamma2 subunit of the receptor but also with microtubules and the N-ethylmaleimide-sensitive factor (NSF). To elucidate the role of GABARAP in the trafficking of GABA(A) receptors, we have constructed a yellow fluorescent protein (YFP) fusion protein of GABARAP and expressed it in neurons using adenovirus, so that its function may be examined. YFP-GABARAP colocalized with gamma2 subunit-containing GABA(A) receptors and NSF to the perinuclear cytoplasm in cultured hippocampal neurons and to the proximal regions of dendrites that are making synaptic contact. Expression of YFP-GABARAP in Cos7 cells and cultured hippocampal neurons was able to increase the level of GABA(A) receptors detected at the plasma membrane, even at low levels of YFP-GABARAP expression. This effect is specific to the function of GABARAP on GABA(A) receptor trafficking, because point mutations in the gamma2-binding domain of YFP-GABARAP interfered with the ability of YFP-GABARAP to increase GABA(A) receptor surface levels. These mutations also disrupted the colocalization of YFP-GABARAP with the gamma2 subunit and with NSF in hippocampal neurons. The results of this study show for the first time that GABARAP has a functional effect on the trafficking of GABA(A) receptors and provide decisive evidence for the role of GABARAP in transporting GABA(A) receptors to the plasma membrane in neurons.

The crystal structure of microtubule‐associated protein light chain 3, a mammalian homologue of Saccharomyces cerevisiae Atg8

Microtubule-associated protein light chain 3 (LC3), a mammalian homologue of yeast Atg8, plays an essential role in autophagy, which is involved in the bulk degradation of cytoplasmic components by the lysosomal system. Here, we report the crystal structure of LC3 at 2.05 A resolution with an R-factor of 21.8% and a free R-factor of 24.9%. The structure of LC3, which is similar to those of Golgi-associated ATPase enhancer of 16 kDa (GATE-16) and GABAA receptor-associated protein (GABARAP), contains a ubiquitin core with two alpha helices, alpha1 and alpha2, attached at its N-terminus. Some common and distinct features are observed among these proteins, including the conservation of residues required to form an interaction among alpha1, alpha2 and the ubiquitin core. However, the electrostatic potential surfaces of these helices differ, implicating particular roles to select specific binding partners. Hydrophobic patches on the ubiquitin core of LC3, GABARAP and GATE-16 are well conserved and are similar to the E1 binding surface of ubiquitin and NEDD8. Therefore, we propose that the hydrophobic patch is a binding surface for mammalian Atg7 similar to a ubiquitin-like conjugation system. We also propose the functional implications of the ubiquitin fold as a recognition module of target proteins.

Caldendrin but not Calmodulin Binds to Light Chain 3 of MAP1A/B: An Association with the Microtubule Cytoskeleton Highlighting Exclusive Binding Partners for Neuronal Ca 2+-sensor Proteins

Caldendrin is a neuronal Ca 2+-sensor protein (NCS), which represents the closest homologue of calmodulin (CaM) in nerve cells. It is tightly associated with the somato-dendritic cytoskeleton of neurons and highly enriched in the postsynaptic cytomatrix. Here, we report that caldendrin specifically associates with the microtubule cytoskeleton via an interaction with light chain 3 (LC3), a microtubule component with sequence homology to the GABA A receptor-associated protein (GABARAP), which is, like LC3, probably involved in cellular transport processes. Interestingly, two binding sites exist in LC3 for caldendrin from which only one exhibits a strict Ca 2+-dependency for the interaction to take place but both require the presence of the first two EF-hands of caldendrin. CaM, however, is not capable of binding to LC3 at both sites despite its high degree of primary structure similarity with caldendrin. Computer modelling suggests that this might be explained by an altered distribution of surface charges at the first two EF-hands rendering each molecule, in principle, specific for a discrete set of binding partners. These findings provide molecular evidence that NCS can transduce signals to a specific target interaction irrespective of Ca 2+-concentrations and CaM-levels.

Biochemical identification of the binding domain in the GABA A receptor-associated protein (GABARAP) mediating dimer formation

The γ-aminobutyric acid receptor type A (GABA A) receptor-associated protein (GABARAP) is a member of a growing family of intracellular membrane trafficking and/or fusion proteins and has been implicated in plasma membrane targeting and clustering of GABA A receptors. GABARAP interacts with microtubules and the γ2 subunit of GABA A receptor and modulates channel kinetics. From crystal structures of GABARAP in high salt concentration it has been proposed that oligomerization of GABARAP might take place in a head-to-tail fashion. In this study, we report that GABARAP self-associates and dimerizes in physiological salt concentrations. We find no evidence for higher order complex larger than a dimer. By using deletion constructs of GABARAP we show that interaction takes place between amino acid 36 and 68. We further narrow the interacting domain by inhibiting the self-association, by adding GABARAP-derived synthetic peptides in GST pull-down assays and shows that the interaction specifically takes place in the previously identified GABARAP-GABA A receptor interaction domain from amino acid 41–51. The identification of binding domains in GABARAP allows for the study of GABARAP functions, including GABA A receptor dynamics.

Identification, Molecular Cloning, and Characterization of a Novel GABAA Receptor-associated Protein, GRIF-1

A novel 913-amino acid protein, gamma-aminobutyric acid type A (GABA(A)) receptor interacting factor-1 (GRIF-1), has been cloned and identified as a GABA(A) receptor-associated protein by virtue of its specific interaction with the GABA(A) receptor beta 2 subunit intracellular loop in a yeast two-hybrid assay. GRIF-1 has no homology with proteins of known function, but it is the rat orthologue of the human ALS2CR3/KIAA0549 gene. GRIF-1 is expressed as two alternative splice forms, GRIF-1a and a C-terminally truncated form, GRIF-1b. GRIF-1 mRNA has a wide distribution with a major transcript size of 6.2 kb. GRIF-1a protein is only expressed in excitable tissues, i.e. brain, heart, and skeletal muscle major immunoreactive bands of M(r) approximately 115 and 106 kDa and, in muscle and heart only, an additional 88-kDa species. When expressed in human embryonic kidney 293 cells, GRIF-1a yielded three immunoreactive bands with M(r) approximately 115, 106, and 98 kDa. Co-expression of GRIF-1a and alpha 1 beta 2 gamma 2 GABA(A) receptors in mammalian cells revealed some co-localization in the cell cytoplasm. Anti-FLAG-agarose specifically precipitated GRIF-1(FLAG) and GABA(A) receptor beta 2 subunits from human embryonic kidney 293 cells co-transfected with GRIF-1a(FLAG) and beta 2 subunit clones. Further, immobilized GRIF-1-(8-633) specifically precipitated in vitro GABA(A) receptor alpha 1 and beta 2 subunit immunoreactivities from detergent extracts of adult rat brain. The respective GABA(A) receptor beta 2 subunit/GRIF-1 binding domains were mapped using the yeast two-hybrid reporter gene assays. A possible role for GRIF-1 as a GABA(A) receptor beta 2 subunit trafficking factor is proposed.

Solution Structure of Human GABAA Receptor-associated Protein GABARAP: IMPLICATIONS FOR BIOLOGICAL FUNCTION AND ITS REGULATION

Control of neurotransmitter receptor expression and delivery to the postsynaptic membrane is of critical importance for neural signal transduction at synapses. The gamma-aminobutyric acid, type A (GABA(A)) receptor-associated protein GABARAP was reported to have an important role for movement and sorting of GABA(A) receptor molecules to the postsynaptic membrane. GABARAP not only binds to GABA(A) receptor gamma2-subunit but also to tubulin, gephyrin, and ULK1. We present for the first time the high resolution structure of human GABARAP determined by nuclear magnetic resonance in aqueous solution. One part of the molecule, despite being well ordered and rigid on a MHz time scale, exists in at least two different conformations that interchange with each other on a time scale slower than 25 Hz. An important feature of the solution structure is the observation that amino- and carboxyl-terminal ends of the protein directly interact with each other, which is not seen in recently reported crystal structures. The possible biological relevance of these observations for the regulation of GABARAP interactions and functions is discussed.