GABAA Receptor-associated Protein Research Articles

Subunit specificity and interaction domain between GABA(A) receptor-associated protein (GABARAP) and GABA(A) receptors.

GABARAP (GABA(A) receptor-associated protein) interacts with both microtubules and GABA(A) receptors in vitro and in vivo and is capable of modulating receptor channel kinetics. In this study, we use the intracellular loop of 15 GABA(A) receptor subunits to show that the interaction between GABARAP and GABA(A) receptor is specific for the gamma subunits. Pharmacological characterization of proteins purified by GABARAP affinity column indicates that native GABA(A) receptors interact with GABARAP. Quantitative yeast two-hybrid assays were used to identify the interaction domain in the gamma2 subunit for GABARAP binding, and to identify the interaction domain in GABARAP for GABA(A) receptor binding. A peptide corresponding to the GABARAP interaction domain in the gamma2 subunit was used to inhibit the interaction between GABARAP and the gamma2 subunit. In addition, the ability of GABARAP to promote cluster formation of recombinant receptors expressed in QT-6 fibroblasts was inhibited by a membrane-permeable form of this peptide in a time-dependent manner. The establishment of a model for GABARAP-induced clustering of GABA(A) receptors in living cells and the identification of subunit specificity and interaction domains in the interaction between GABARAP and GABA(A) receptors is a step in dissecting the function of GABARAP in GABA(A) receptor clustering and/or targeting.

The X-ray Crystal Structure and Putative Ligand-derived Peptide Binding Properties of γ-Aminobutyric Acid Receptor Type A Receptor-associated Protein

The gamma-aminobutyric acid receptor type A (GABA(A)) receptor-associated protein (GABARAP) has been reported to mediate the interaction between the GABA(A) receptor and microtubules. We present the three-dimensional structure of GABARAP obtained by x-ray diffraction at 1.75 A resolution. The structure was determined by molecular replacement using the structure of the homologous protein GATE-16. NMR spectroscopy of isotope-labeled GABARAP showed the structure in solution to be compatible with the overall fold but showed evidence of conformation heterogeneity that is not apparent in the crystal structure. We assessed the binding of GABARAP to peptides derived from reported binding partner proteins, including the M3-M4 loop of the gamma2 subunit of the GABA(A) receptor and the acidic carboxyl-terminal tails of human alpha- and beta-tubulin. There is a small area of concentrated positive charge on one surface of GABARAP, which we found interacts weakly with all peptides tested, but we found no evidence for specific binding to the proposed physiological target peptides. These results are compatible with a more general role in membrane targeting and transportation for the GABARAP family of proteins.

Crystal structure of the GABAA‐receptor‐associated protein, GABARAP

The 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/or recycling of GABA(A) receptors. GABARAP is localized on intracellular membranes such as the trans-Golgi network, binds to the gamma 2 subunit of GABA(A) receptors and interacts with microtubules and the N-ethylmaleimide-sensitive factor. We report the X-ray crystal structure of mammalian GABARAP at 2.0 A resolution. GABARAP consists of an N-terminal basic helical region, which has been implicated in tubulin binding, and a core structure with a conserved ubiquitin-like fold. Consistent with the high extent of sequence conservation among GABARAP homologues from plants to mammals, one face of the core structure is absolutely conserved while the opposite face shows considerable divergence. These features are in agreement with the conserved surface mediating protein-protein interactions shared by all members of the family, whereas the non-conserved surface region may play specific roles, such as docking to particular membrane receptors.

The analysis of protein-protein interaction with special reference to PRIP-1

Analysis of protein-protein interaction is one of the powerful methods for elucidating the new functions of functionally unknown proteins. Using this approach, we isolated two proteins interacting with PRIP-1, which was isolated as a new Ins(1,4,5) P3 binding protein from brain. One was protein phosphatase 1 catalytic subunit (PP1c) and the other was GABARAP (GABAA-receptor-associated protein). The region of PRIP-1 responsible for their interaction was the site preceding to the pleckstrin homology domain of PRIP-1 for PP1c and the EF-hand motifs of PRIP-1 for GABARAP, which were determined by beta-galactosidase assay of yeast two-hybrid system. The association between PRIP-1 and PP1c was confirmed in vitro by a pull-down assay, a far-western assay, an immunoprecipitation analysis and a surface plasmon resonance analysis. The interaction of PRIP-1 with PP1c resulted in inhibition of the catalytic activity of PP1c in a PRIP-1 concentration-dependent manner. The association between PRIP-1 and GABARAP was also confirmed by a pull-down assay, and we found that PRIP-1 competitively inhibited the binding of the gamma 2 subunit of the GABAA receptor to GABARAP in vitro. Our electrophysiological and behavioral analysis of PRIP-1 knockout mice revealed that PRIP-1 is essential for the function of GABAA receptors, especially in response to the agents acting on the gamma 2 subunit.

Cloning, Expression Patterns, and Chromosome Localization of Three Human and Two Mouse Homologues of GABAA Receptor-Associated Protein

Type A receptors of γ-aminobutyric acid (GABA), an inhibitory neurotransmitter, contain α, β, δ, γ, and ρ subunits. The γ subunit has four subtypes: γ1, γ2, γ3, andγ4. GABAA receptor-associated protein (GABARAP) was previously demonstrated to act as a linker protein between microtubules and the γ2 subunit of GABAA receptors. However, no other linker proteins have been identified as mediating the linkage of microtubules and the remaining subunits of GABAA receptors. In this study we identified three human paralogues (GABARAPL1, GABARAPL2, and GABARAPL3) and two mouse orthologues (Gabarapl1 and Gabarapl2) of human GABARAP, all of which encoded 117 amino acids, as does Gabarapl. The expression patterns of GABARAPL1, GABARAPL2, and GABARAP in 16 adult tissues showed that they were expressed ubiquitously. The expression levels of GABARAPL1 as a 2.3-kb transcript were very high in brain, heart, peripheral blood leukocytes, liver, kidney, placenta, and skeletal muscle, very low in thymus and small intestine, and moderate in other tissues tested. The unique 1.35-kb transcript of GABARAPL2 was expressed at high levels in heart, brain, testis, prostate, ovary, spleen, and skeletal muscle, at very low levels in lung, thymus, and small intestine, and moderately in other tissues tested. For GABARAP, a 1.3-kb transcript was abundantly expressed in all tested tissues with small variation. The expression patterns of Gabarapl1 and Gabarapl2 were similar to those of their counterparts in human. In addition, GABARAPL1 was localized to human chromosome 12p12.3 and GABARAPL2 to 16q22.3–q24.1 by RH mapping, while GABARAP and GABARAPL3 were found to be localized at chromosomes 17p13.2 and 15q25.1, respectively, by searching the related databases. Sequence comparison of the cDNAs and their corresponding genomic sequences shows that GABARAP, GABARAPL1, and GABARAPL2 are composed of four exons each, while GABARAPL3 is distributed only at one exon.

Interaction of the Unc-51-like kinase and microtubule-associated protein light chain 3 related proteins in the brain: possible role of vesicular transport in axonal elongation

We identified two mammalian ULK1 (Unc-51-like kinase involved in neurite extension) binding proteins by yeast two-hybrid screening. Both proteins showed high structural similarity to microtubule-associated protein (MAP) light chain 3 (LC3). One is identical to the Golgi-associated ATPase Enhancer of 16 kDa (GATE-16), an essential factor for intra-Golgi transport [39]. The other is identical to the gamma 2-subunit of GABA-A receptor associated protein (GABARAP) which has a possible role in receptor transport [46]. Using the yeast two-hybrid system and the in vitro GST pull-down assay, we found that the N-terminal proline/serine rich (PS) domain of ULK1 (amino acid 287–416) is required for ULK1–GATE-16 and ULK1–GABARAP protein interactions. However, the kinase activity of ULK1 affected neither ULK1–GATE-16 nor ULK1–GABARAP interaction. Immunohistochemical analysis using ULK1 and GABARAP antibodies showed that the ULK1 and the GABARAP proteins co-localized to many kind of neurons such as pyramidal cells of the hippocampus, mitral cells of the olfactory bulb, and Purkinje cells of the cerebellum. In HeLa cells, endogenous ULK1 and tagged GABARAP showed punctate structures in the cytosol, and were colocalized. These results suggest that the interaction of ULK1 and GABARAP is important to vesicle transport and axonal elongation in mammalian neurons.

The gamma-aminobutyric acid type A (GABAA) receptor-associated protein (GABARAP) promotes GABAA receptor clustering and modulates the channel kinetics.

A microtubule-associated protein, gamma-aminobutyric acid type A (GABA(A)) receptor-associated protein (GABARAP), was previously identified as binding to the intracellular domain of GABA(A) receptors by using the yeast two-hybrid screen. In the present work, immunofluorescent staining and green fluorescent protein-tagged receptor subunits showed that GABARAP is associated with and promotes the clustering of GABA(A) receptors in QT-6 quail fibroblasts. The tubulin-binding motif of GABARAP and the gamma2 subunit of the receptor are required. Disruption of microtubules prevents the clustering in a time-dependent manner. When green fluorescent protein-tagged alpha1 or gamma2 subunit coexpressed with beta2, gamma2L, and GABARAP was used, recordings from visually identified cells revealed that clustered GABA(A) receptor had an EC(50) of about 20 microM, vs. 5.7 microM for the diffuse receptor. Clustered receptors deactivated faster and desensitized slower than the diffuse receptors, because of decrease in the apparent affinity of GABA binding. Different properties for clustered receptors relative to unclustered receptors in heterologous cells suggest that homologous differences between extrasynaptic and synaptic clustered receptors in neurons may be due to the organization of the postsynaptic machinery.

Binding of the GABA(A) receptor-associated protein (GABARAP) to microtubules and microfilaments suggests involvement of the cytoskeleton in GABARAPGABA(A) receptor interaction.

GABA(A) receptor-associated protein (GABARAP) was isolated on the basis of its interaction with the gamma2 subunit of GABA(A) receptors. It has sequence similarity to light chain 3 (LC3) of microtubule-associated proteins 1A and 1B. This suggests that GABARAP may link GABA(A) receptors to the cytoskeleton. GABARAP associates with tubulin in vitro. However, little is known about the mechanism for the interaction, and it is not clear whether the interaction occurs in vivo. Here, we report that GABARAP interacts directly with both tubulin and microtubules in a salt-sensitive manner, indicating the association is mediated by ionic interactions. GABARAP coimmunoprecipitates with tubulin and associates with both microtubules and microfilaments in intact cells. The cellular distribution is altered by treatment with taxol, nocodazole, and cytochalasin D. The tubulin binding domain was located at the N terminus of GABARAP by using synthetic peptides and deletion constructs and is marked by a specific arrangement of basic amino acids. The interaction between GABARAP and actin might be mediated by other proteins. These results demonstrate the GABARAP interacts with the cytoskeleton both in vitro and in cells and suggest a role of GABARAP in the interaction between GABA(A) receptors and the cytoskeleton. Such interactions are presumably needed for receptor trafficking, anchoring, and/or synaptic clustering. The structural arrangement of the basic amino acids present in the tubulin binding domain of GABARAP may aid in recognition of the potential of tubulin binding activity in other known proteins.

Intracellular trafficking of GABA A receptors

Some of the mechanisms that control the intracellular trafficking of GABA A receptors have recently been described. Following the synthesis of α, β, and γ subunits in the endoplasmic reticulum, ternary receptor complexes assemble slowly and are inefficiently inserted into surface membranes of heterologous cells. While β3, β4, and γ2S subunits appear to contain polypeptide sequences that alone are sufficient for surface targeting, these sequences are neither conserved nor essential for surface expression of heteromeric GABA A receptors formed from α1β or α1βγ subunits. At the neuronal surface, native GABA A receptor clustering and synaptic targeting require a γ2 subunit and the participation of gephyrin, a clustering protein for glycine receptors. A linker protein, such as the GABA A receptor associated protein (GABARAP), may be necessary for the formation of GABA A receptor aggregates containing gephyrin. A substantial fraction of surface receptors are sequestered by endocytosis, another process which apparently requires a GABA A receptor γ2 subunit. In heterologous cells, constitutive endocytosis seems to predominate while, in cortical neurons, internalization is evoked when receptors are occupied by GABA A agonists. After constitutive endocytosis, receptors are relatively stable and can be rapidly recycled to the cell surface, a process that may be regulated by protein kinase C. On the other hand, a portion of the intracellular GABA A receptors derived from ligand-dependent endocytosis is apparently degraded. The clustering of GABA A receptors at synapses and at coated pits are two mechanisms that may compete for a pool of diffusable receptors, providing a model for plasticity at inhibitory synapses.

GABA(A)-receptor-associated protein links GABA(A) receptors and the cytoskeleton.

Type-A receptors for the neurotransmitter GABA (gamma-aminobutyric acid) are ligand-gated chloride channels that mediate inhibitory neurotransmission. Each subunit of the pentameric receptor protein has ligand-binding sites in the amino-terminal extracellular domain and four membrane-spanning regions, one of which forms a wall of the ion channel. Each subunit also has a large intracellular loop that may be a target for protein kinases and be required for subcellular targeting and membrane clustering of the receptor, perhaps by anchoring the receptor to the cytoskeleton. Neurotransmitter receptors need to be positioned in high density in the cell membrane at sites postsynaptic to nerve terminals releasing that neurotransmitter. Other members of the superfamily of ligand-gated ion-channel receptors associate in postsynaptic-membrane clusters by binding to the proteins rapsyn or gephyrin. Here we identify a new cellular protein, GABA(A)-receptor-associated protein (GABARAP), which can interact with the gamma2 subunit of GABA(A) receptors. GABARAP binds to GABA(A) receptors both in vitro and in vivo, and co-localizes with the punctate staining of GABA(A) receptors on cultured cortical neurons. Sequence analysis shows similarity between GABARAP and light chain-3 of microtubule-associated proteins 1A and 1B. Moreover, the N terminus of GABARAP is highly positively charged and features a putative tubulin-binding motif. The interactions among GABA(A) receptors, GABARAP and tubulin suggest a mechanism for the targeting and clustering of GABA(A) receptors.