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Keywords:

  • GABAA receptor;
  • GABAA receptor associated proteins;
  • yeast two-hybrid assay;
  • trafficking;
  • clustering;
  • internalization

Abstract

  1. Top of page
  2. Abstract
  3. GABARAP
  4. Golgi-specific DHHC zinc finger protein (GODZ)
  5. Plic-1
  6. Radixin
  7. Huntingtin-Associated Protein 1 (HAP 1)
  8. GABAA receptor interacting factor-1 (GRIF-1)
  9. Brefeldin A-inhibited GDP/GTP exchange factor 2 (BIG2)
  10. Summary
  11. References

γ-Aminobutyric acid (GABA), an important inhibitory neurotransmitter in both vertebrates and invertebrates, acts on GABA receptors that are ubiquitously expressed in the CNS. GABAA 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 GABAA receptors plays an important role in regulating GABAA 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 GABAA receptors, for example, the interaction of GABAA receptor associated protein (GABARAP) and Golgi-specific DHHC zinc finger protein (GODZ) with γ subunits, PRIP, phospholipase C-related, catalytically inactive proteins (PRIP-1) and (PRIP-2) with GABARAP and receptor γ2 and β subunits, Plic-1 with some α and β subunits, radixin with the α5 subunit, HAP1 with the β1 subunit, GABAA receptor interacting factor-1 (GRIF-1) with the β2 subunit, and brefeldin A-inhibited GDP/GTP exchange factor 2 (BIG2) with the β3 subunit. These proteins have been shown to play important roles in modulating the activities of GABAA receptors ranging from enhancing trafficking, to stabilizing surface and internalized receptors, to regulating modification of GABAA receptors. This article reviews the current studies of GABAA receptor intracellular loop-associated proteins.

Abbreviations used
ARF

ADP-ribosylation factors

ER

Endoplasmic Reticulum

GABA

γ-Aminobutyric acid

nACh

nicotinic acetylcholine

GEF-2

Ganglioside expression factor 2; 5-HT, 5-hydroxytryptamine

GABARAP

GABAA receptor associated protein

GATE-16

Golgi-associated ATPase enhancer of 16 kDa

LC3

microtubule-associated protein light chain 3

PE

phosphatidylethanolamine

GOS28

Golgi-specific v-SNARE

NSF

N-ethylamaleimide-sensitive factor

PDZ

postsynaptic density protein, disc large, zonula occludens-1

GRIP

Glutamate receptor interacting protein 1

PRIP

phospholipase C-related, catalytically inactive protein

GODZ

Golgi-specific DHHC zinc finger protein

Plic-1

protein linking IAP and cytoskeleton

HAP1

Huntingtin-associated protein 1

GRIF-1

GABAA receptor interacting factor-1

OGT

O-glcNAc transferase

BIG2

brefeldin A-inhibited GDP/GTP exchange factor 2

PKA

protein kinase A

γ-Aminobutyric acid (GABA), an important inhibitory neurotransmitter in both vertebrates and invertebrates, acts on ionotropic GABAA receptors as well as metabotropic GABAB receptors that are ubiquitously expressed in the CNS. In vertebrates, 19 related GABAA receptor subunit genes have been detected, α1–6, β1–3, γ1–3, δ, ε, θ, π, and ρ1–3. The subunits are combined into approximately 20 different native heteropentameric isoforms (Macdonald and Olsen 1994; Sieghart and Sperk 2002). GABAA receptors are members of the ligand-gated ion channel superfamily, which includes nicotinic acetylcholine (nACh), glycine, and 5-HT3 receptors. They are composed of five subunits, which assemble to form an ion channel. Each subunit possesses a long exracellular N-terminus that in some cases carries the neurotransmitter binding site, four membrane-spanning domains, including the ion channel wall primarily in M2, and a large variable sequence intracellular loop between M3 and M4. GABAA receptors also represent a major site of action of clinically relevant CNS drugs, such as benzodiazepines, barbiturates, ethanol, and general anesthetics. Upon binding with agonist, this hetero-pentameric receptor opens a selective anion channel stabilizing the cell membrane potential near the chloride ion equilibrium level. GABAA receptors composed of any of the three ρ subunits show no sensitivity for anesthetics and are predominantly expressed in retina. ρ subunits are also classified as GABAC receptors (insensitive to bicuculline and baclofen). GABAB receptors mediate the metabotropic actions of GABA through coupling with G-proteins to membrane K+ and Ca2+ channels as well as to adenylate cyclase. Receptor activation increases K+ conductance but decreases Ca2+ channel conductance.

Because both the N-terminus and C-terminus of GABAA receptor subunits extend outside the cell membrane, the intracellular M3-M4 loop becomes the most important domain interacting with the intracellular environment, e.g. providing protein-protein interactive domains involved in regulating synaptic localization and intracellular trafficking. In consequence, studies of the function of receptor intracellular loop associated proteins become important for understanding mechanisms of regulating receptor activity. Since Fields and Song (1989) first reported the yeast two-hybrid assay for studying protein–protein interactions, several labs have used this technique to identify proteins interacting with GABAA receptors using the large intracellular loop. This paper reviews the current studies of GABAA receptor intracellular loop associated proteins found by the yeast two-hybrid approach.

GABARAP

  1. Top of page
  2. Abstract
  3. GABARAP
  4. Golgi-specific DHHC zinc finger protein (GODZ)
  5. Plic-1
  6. Radixin
  7. Huntingtin-Associated Protein 1 (HAP 1)
  8. GABAA receptor interacting factor-1 (GRIF-1)
  9. Brefeldin A-inhibited GDP/GTP exchange factor 2 (BIG2)
  10. Summary
  11. References

GABARAP-associated proteins

Among those newly identified GABA receptor associated proteins, GABARAP is the best studied. It was cloned in a yeast two-hybrid system using the intracellular loop of γ2 as bait (Wang et al. 1999). The interaction is specifically limited to γ1, γ2S, and γ2L, but not other GABAA receptor subunits (Wang et al. 1999; Nymann-Andersen et al. 2002b). GABARAP belongs to a family including GEC1 (first isolated in Guinea-pig Endometrial Cells, estrogen-induced 1.8 kb RNA coded protein) or GABARAPL1; GATE-16 (Golgi-associated ATPase enhancer of 16 kDa) or GABARAPL2 or GEF-2 (Ganglioside expression factor 2); GABARAPL3; GABARAPL4; LC3 (Microtubule-associated protein light chain 3); yeast Apg8 or Aut7p; and C. elegans LGGs (LC3, GABARAP and GATE-16) protein. Figure 1 describes the amino acid conservation among the members of this family. All the proteins in this family share some common features: first, they are evolutionally conserved in eukaryotic cells from yeast to mammals. For example, at the amino acid level, all orthologues (mouse, rat and bovine) of GABARAP present 100% identity with their human counterparts, indicating a very high level of conservation in evolution and suggesting a critical function of these proteins in mammalian cells.

image

Figure 1.  Sequence alignment of Human GABARAP, GEC1, GATE-16, LC3, Yeast Apg8/Aut7, and their homologs from C. elegans LGGs.

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Second, the existing studies of their three-dimensional structures showed that the proteins in this family share strong similarity to ubiquitin (Paz et al. 2000; Bavro et al. 2002; Coyle et al. 2002; Knight et al. 2002; Sugawara et al. 2004), exhibiting a compact fold consisting of four or five-stranded β-sheets with two α-helices on either side. The outer strands of the β-sheets are aligned antiparallel to the inner strands and helices α3 and α4 are located on one side of the sheet. The presence of α1 and α2 helices is a unique feature of the GABARAP family, which is lacking in ubiquitin. These helices are attached non-covalently to the ubiquitin-like core by a number of interactions, e.g. hydrophobic interactions and hydrophilic interactions including hydrogen bonds and salt-bridges. The first two α-helices (N-terminal a.a. 1–22) have been shown to form a hydrophobic patch suitable for protein–protein interfaces (Bavro et al. 2002; Coyle et al. 2002). In GABARAP and GEC1, this domain was defined as interacting with microtubules (Wang and Olsen 2000; Mansuy et al. 2004). Another hydrophobic patch found in GABARAP, composing of Ile21, Pro30, Tyr49, Leu50, Val 51, Lue55, Phe60, Leu63, and Phe-104, might be involved in dimerization or oligomerization, interaction with the γ2 subunit, and/or N-ethylamaleimide-sensitive factor (NSF) (Coyle et al. 2002; Nymann-Andersen et al. 2002a; Leil et al. 2004).

Third, the GABARAP family is not only structurally similar to ubiquitin, but also undergoes a similar post-translational cascade modification process as occurs in ubiquitinylation (Hochstrasser 2000). Yeast Apg8 was the first identified member in this family undergoing this process. During the modification, the last amino acid in the C-terminus is first cleaved by the activating cysteine protease Apg4, leaving glycine116 exposed. This glycine is conserved in all the proteins in this family. Then it is activated by Apg7 and transferred to Apg3, which are E1 and E2 enzymes, respectively (Komatsu et al. 2001; Yamazaki-Sato et al. 2003). Finally, it conjugates to another biomolecule, catalyzed by an as yet unidentified E3. Apg8 and the GABARAP family are not attached to proteins but rather to the membrane lipid phosphatidylethanolamine (PE) (Ichimura et al. 2000). The lipid PE can also be removed by Apg4, releasing the C-terminal glycine exposed for a new cycle (Hemelaar et al. 2003). The C-terminal glycine is critical for this modification because mutation of glycine to alanine in Apg8 prevents cleaving off the last amino acid (Ichimura et al. 2000). The homologues of Apg4, Apg7 and Apg3 were recently cloned in human and verified as the respective enzymes involved in the modification of mammalian counterparts of Apg8 (Tanida et al. 2001; Tanida et al. 2002; Hemelaar et al. 2003; Scherz-Shouval et al. 2003; Yamazaki-Sato et al. 2003; Tanida et al. 2004). Similar to Apg8, LC3 was conjugated to PE at the end of this process, which was also demonstrated in a [3H]PE incorporation experiment (Kabeya et al. 2004). Although the final conjugated adduct remains unknown for GATE-16 and GABARAP, several lines of evidence suggest that it is lipid-related (Kabeya et al. 2004). Accumulated evidence has shown that lipopeptides have an increased tendency to insert into membranes compared to the same peptide lacking the lipid moiety. Therefore, this modification must be one facet of the important scheme regulating membrane trafficking and cell surface stability (Casey 1995). However, no E3 enzyme has been identified for this modification. Given the fact that the members in this family also have their own unique functions, the E3 enzymes were hypothesized to be the key factor differentiating their location by conjugating them to different lipids, or different pools and locations.

Fourth, all the proteins are involved in intracellular trafficking. GATE-16 (GABARAPL2) was first purified from a bovine brain cytosol based on a cell-free intra-Golgi transport assay (Legesse-Miller et al. 1998). It directly binds NSF and GOS-28, a Golgi-specific v-SNARE, and is believed to be a component of the intra-Golgi transport machinery (Legesse-Miller et al. 1998; Sagiv et al. 2000; Muller et al. 2002). GATE-16 has been shown to enhance the ATPase activity of NSF, which, in turn, stimulates the recruitment of GATE-16 to the unpaired GOS-28 in an ATP-dependent or ATPase-independent manner. This interaction protects the labile, unpaired GOS-28 from proteolysis. Recently, it has been shown that the function of GATE-16 as a SNARE protector is essential not only for intra-Golgi transport, but also for the homotypic fusion of post-mitotic Golgi fragments (Sagiv et al. 2000; Muller et al. 2002; Elazar et al. 2003). GEC1 (GABARAPL2) was originally identified as a novel early estrogen-regulated gene (Nemos et al. 2003). The first N-teminal 22 a.a. of GEC1 show 77% identity with the tubulin-binding domain of GABARAP and was shown to provide the domain for interaction with tubulin. Morever, it promotes tubulin assembly and displays tubulin bundling activity. Like GABARAP and GATE-16, GEC1 also interacts with NSF (Chen et al. 2006). In vitro binding analysis revealed an association between GEC1 and GABAA receptor γ2 subunit and tubulin at the same time (Mansuy et al. 2004). A recent study demonstrated that GEC1 also interacts directly with the κ-opioid receptors, but not µ, δ-opioid receptors. Expression of GEC1 dramatically increased total and surface κ-opioid receptors (Chen et al. 2006). All these findings strongly suggest that GEC1 may be the second protein of the GABARAP family implicated in receptor membrane trafficking via microtubules (Mansuy et al. 2004).

LC3 was first identified as a protein that copurified with microtubule-associated protein 1A and 1B from rat brain (Mann and Hammarback 1994). The lipidated form of LC has been shown to locate in membranes and was proposed as a marker of autophagosomes (Kabeya et al. 2000). Yeast Apg8 is well accepted as an autophagic factor (Ichimura et al. 2000), but it also takes part in multiple intracellular membrane trafficking processes by interacting with different v-SNARE molecules involved in ER to Golgi transport and in vacuolar fusion (Legesse-Miller et al. 2000). C. Elegans LGG-1 has also been used as the autophagasomes marker (Melendez et al. 2003; Roudier et al. 2005; Rowland et al. 2006). Autophagosomes normally transport bulk cytoplasm to the lysosome for degradation. Autophagy is recently identified as a novel degradative pathway for GABAA receptors (Rowland et al. 2006). In C. elegans, the GABAA receptors lacking presynaptic innervation, but not acetylcholine receptors, traffic to autophagosomes after endocytic removal from the cell surface (Rowland et al. 2006). The formation of autophagasome might serve as the trafficking destination of GABAA receptor-containing endocytic vesicles, thus allowing the coordinated degradation of cytoplasmic and membrane-bound postsynaptic proteins.

Unlike ubiquitin, proteins in the GABARAP family do not form covalently bound polymers at the end of this ubiquitinylation-like modification. Instead, they might form non-covalent homo-dimers or oligomers. The crystal structure shows that GABARAP exists in two conformations: the monomeric conformation at low protein concentration and a head-to-tail oligomeric conformation in high salt conditions (Coyle et al. 2002). Nymann-Andersen et al. (2002a) reported that GABARAP self-associates and dimerizes in physiological salt concentrations. However, they found no evidence for higher order complexes larger than a dimer. By using deletion constructs of GABARAP and GABARAP-derived synthetic peptides, they limited the self interaction domain to amino acid 41–51, which overlaps with the GABAA receptor γ subunit binding domain (35–52 a.a). It is possible that dimerization regulates the availability of free GABARAP for association with trafficking ‘cargo’ proteins, like GABAA receptors. Further, if both GABARAP molecules interact via this domain with their dimer partner, higher order oligomers would be prevented.

Amino acids 35–52 of GABARAP were identified as the γ2 subunit binding domain (Nymann-Andersen et al. 2002a; Leil et al. 2004). GABARAP clusters have been shown to co-localize with the clusters of GABAA receptors in cultured neurons (Wang et al. 1999). Leil et al. (2004) further confirmed the co-localization, demonstrating that over-expressed GABARAP co-localized with the γ2 subunit in the cytoplasmic perinuclear regions of cultured hippocampal neurons. Although GABARAP did not appear to co-localize extensively with the surface GABAA receptors, some GABARAP was detected to co-localize with synaptophysin, an established marker for synaptic vesicles. GABARAP shows two aspects of effects on the γ2-containing GABAA receptors: (i) regulating the ion channel properties, possibly by promoting receptor clustering; and (ii) manipulating the cell surface expression, possibly by increasing the intracellular trafficking of receptors. In quail fibroblast QT-6 cells, a recombinant expression system, GABARAP clusters GABAA receptors; these clusters demonstrated lower apparent affinity for GABA, faster deactivation, and slower desensitization at a given GABA concentration (Chen et al. 2000). Disruption of microtubules prevents the clustering of both GABARAP and GABAA receptors (Chen et al. 2000; Wang and Olsen 2000), indicating that the clustering of GABARAP might be important for the properties of GABAA receptors. In addition, the N-terminal microtubule binding domain (a.a. 1–22) and γ2 subunit are also required for the clustering effect of GABARAP (Chen et al. 2000). The similar results were demonstrated in recombinant HEK293 cells expressing GABARAP with α1β2γ2S subunits in certain ratios. GABARAP altered desensitization, deactivation, and diazepam potentiation of GABA-mediated currents. However, the authors speculated that GABARAP does not alter receptor kinetics directly but by facilitating surface expression of γ2-containing GABAA receptors, instead of αβ pentamers lacking γ2 (Boileau et al. 2005). In L929 fibroblast cells expressing the α1β2γ2S subunits with GABARAP, the channel conductance was significantly higher than that expressing the same three subunits alone, even reaching super-high conductances observed in native receptors (Everitt et al. 2004), suggesting that GABARAP might participate in the organization of GABAA receptors on the membrane.

In cultured neurons, GABARAP mainly localized in intracellular compartments, such as endoplasmic reticulum and Golgi structures, with only a small fraction in synaptic membrane co-localizing with GABAA receptors (Kneussel et al. 2000; Kittler et al. 2001; Leil et al. 2004). Some GABARAP puncta were found to co-localize with the γ2 subunit near the surface of the soma at points of synaptic contact labeled by GAD65, a marker for GABAergic presynaptic terminals, suggesting that GABARAP might participate in transporting the γ2 subunit to the cell surface, or even to synapses (Leil et al. 2004). Electron microscope evidence reveals no anchoring role for GABARAP at GABAA receptor synaptic membranes but frequent co-localization with GABAA receptors in intracellular vesicles located near synaptic membranes (Kittler et al. 2001). Recent studies confirmed that GABARAP enhances the cell surface expression of γ2-containing GABAA receptors in both heterologous expression systems and cultured hippocampal neurons. Over-expression of GABARAP increased net surface levels of GABAA receptor in Xenopus laevis oocytes as shown by both increased GABA currents and surface-expressed protein (Chen et al. 2005). This GABARAP stimulation of GABA currents required the γ2 subunit and the microtubule-binding domain (a.a. 1–22) as well as the intact polymerization of microtubules, indicating the effect of GABARAP was based on the interaction with microtubules (Chen et al. 2005). However, GABARAP co-expression did not alter the general properties of GABAA receptors such as sensitivity to GABA or benzodiazepines in oocytes. Increased surface GABAA receptors were also detected in cultured hippocampal neurons by immunofluorescence and in COS-7 cells by flow cytometer. Mutagenesis of the key amino acids responsible for the γ2 subunit interaction blocks this enhancement effect and the interaction with NSF (Leil et al. 2004). The mechanism of this enhancement still remains unknown. It might be that GABARAP increases trafficking of receptor toward the cell membrane along microtubules, facilitates the insertion, or stabilizes the surface receptors.

Recently, the GABARAP knockout mouse has been shown to be phenotypically normal and exhibits no loss of GABAR synaptic punctate localization (O'Sullivan et al. 2005). This suggests the possibility that GABARAP is not necessary for synaptic localization. Although it traffics γ2-containing GABAA receptors to the surface, increasing surface expression, and under some conditions promotes clustering and synaptic localization, it may not be necessary, but merely accelerates or promotes this process, possibly by mass-action shifting to this state. Alternatively, it could be that the GABARAP trafficking of GABAA receptors to surface, and clustering, are needed for synaptic localization, but, the homologues, or at least one of them, probably GEC1, either also has this function normally, or assumes it as compensation in the GABARAP knockout mouse. However, the homologues GEC1 and GATE-16 did not show increased levels (O'Sullivan et al. 2005).

N-ethylmaleimide-sensitive factor (NSF)

GABARAP interacts and co-localizes with NSF (Kittler et al. 2001; Leil et al. 2004), which plays a central role in general membrane fusion events underlying intracellular trafficking. NSF uses energy from ATP hydrolysis to dissociate SNARE complexes after membrane fusion, allowing the individual SNARE proteins to be recycled for subsequent rounds of fusion. One of the main roles of NSF in intracellular trafficking is the fusion of synaptic vesicles with presynaptic membrane during neurotransmssion (Rothman 1994). In addition, NSF is also enriched in the postsynaptic density, regulating the neurotransmitter receptors in synapses (Walsh and Kuruc 1992; Hong et al. 1994; Puschel et al. 1994). The function of NSF in regulating neurotransmitter receptors is well characterized by the study of NSF and AMPA receptor interaction. NSF has been shown to regulate the surface insertion/stablization of GluR2-containing AMPA receptors and thus AMPA receptor-mediated synaptic transmission (Lin and Sheng 1998; Song et al. 1998; Braithwaite et al. 2000). It was suggested that the interaction between NSF and GluR2 does not promote receptor synthesis or passage through the endoplasmic reticulum or Golgi network, but rather facilitates the synaptic localization of AMPA receptors. GATE-16, a homologue of GABARAP, has been shown to enhance the ATPase activity of NSF and increase intra-Golgi trafficking. It is highly possible that GABARAP shares this ATPase enhancing character and functions as a chaperone helping to target GABAA receptors via intracellular vesicles to the cell surface and synapses.

Gephyrin

GABARAP binds with gephyrin in both the yeast two-hybrid system and GST pull-down experiments (Kneussel et al. 2000). Gephyrin is the first isolated GABAA receptor associated protein which is tightly co-localized with both glycine and GABAA receptors at postsynaptic sites (Kneussel and Betz 2000; Luscher and Keller 2004). It is well established that inhibition of gephyrin expression leads to a dramatic decrease in GABAA receptor clustering. Kneussel et al. (2000) reported that clusters of the major subunits of GABAA receptors, α2 and γ2, were completely absent in hippocampal cultures from gephyrin knockout mice. Anti-sense against gephyrin also resulted in a corresponding reduction in density of clusters of α2 and γ2 subunits of GABAA receptors (Essrich et al. 1998). Inhibiting gephyrin expression by siRNA did not modify the total number of GABAA receptors expressed on the neuronal cell surface but significantly decreased the number of receptor clusters. In addition, the clusters that formed in the absence of gephyrin were significantly more mobile compared with those in control neurons, suggesting that gephyrin plays a specific role in reducing the diffusion of GABAA receptors, facilitating their accumulation at inhibitory synapses (Jacob et al. 2005). The interaction between gephyrin and dynein might be the motor for gephyrin recruitment, therefore, responsible for GABAA receptor clustering (Maas et al. 2006).

Conversely, γ2 knockout mice showed significant reduction of gephyrin clusters (Essrich et al. 1998). Gephyrin clusters were also lost when the γ2 subunit gene was inactivated in mature neurons by conditional knockout in 3-week-old-mice (Schweizer et al. 2003). Thus, the functional association between γ2 subunit-containing GABAA receptors and gephyrin is not limited to development of immature neurons and synaptogenesis, under conditions where GABA can be excitatory.

However, there are also some controversial results regarding the critical role of gephyrin in clustering. GABAergic inhibitory postsynaptic currents and the postsynaptic localization of α1 subunit-containing GABAA receptors in cultured hippocampal neurons, as well as clustering of these receptors in spinal cord sections from gephyrin knockout mice, appear to be unaffected by the loss of gephyrin (Kneussel et al. 2001). In different developmental stages (from 3 DIV to 21 DIV), some β3 and γ2 clusters associated with gephyrin were always detected in extrasynaptic sites and a proportion of synaptic gephyrin clusters were found not associating with GABAA receptor β3 or γ2 immunoreactivity, indicating that gephyrin alone is not sufficient to accumulate GABAA receptors γ2 subunits clusters at synaptic sites (Danglot et al. 2003). Levi et al. (2004) demonstrated that GABA miniature inhibitory postsynaptic currents (mIPSCs) could be recorded from cultured hippocampal neurons from gephyrin knockout mice. The mean amplitude was reduced; the frequency and kinetics remained unchanged. Furthermore, GABAA receptors could form clusters at synapses opposite to GABAergic terminals in neurons lacking gephyrin, albeit at reduced levels compared with control neurons. Cell surface labeling experiment indicated that gephyrin contributes, in part, to aggregation but not insertion or stabilization of GABAA receptor α2 and γ2 in the plasma membrane (Levi et al. 2004). Thus, a major gephyrin-independent component of hippocampal inhibitory synapse formation must exist.

There is no evidence indicating that gephyrin and GABAA receptors directly interact. GABARAP binds with both proteins directly, suggesting that GABARAP might function as the adaptor for the association of gephyrin and the γ2 subunit. Co-expression of both GABARAP and gephyrin results in a recruitment of cytoplasmic gephyrin to GABARAP-rich membrane-associated loci in PC12 cells. Since GABARAP might attach to membrane lipid, e.g., PE, any protein that it binds, such as gephyrin, will become membrane-bound. However, in gephyrin knockout mice, the immunoreactivity of GABARAP showed no significant change in number, size, or localization, suggesting that GABARAP functions prior to or independently of gephyrin (Kneussel et al. 2000).

Glutamate receptor interacting protein (GRIP)

Protein domain prediction demonstrated to us that GABARAP has a postsynaptic density protein (PDZ)-binding domain in the C-terminus (http://scansite.mit.edu/motifscan_seq.phtml). The PDZ domain (Postsynaptic density protein, Discs large, Zonula occludens-1) is a peptide motif of about 90 amino acids initially found in proteins associated with synaptic, septate and tight junctions. PDZ domains have been shown to be present either singly or as repeats in over 100 otherwise unrelated proteins and conceived as scaffolds for assembling multiprotein complexes that facilitate the integration of signaling components and pathways. Some PDZ domains interact with the intracellular C-terminus of ion channels and receptors and thus provide a mechanism for channel clustering. For example, GRIP1, a seven PDZ domain-containing protein, has been shown to interact with AMPA-type glutamate receptors and plays an essential role in their clustering and trafficking (Dong et al. 1997). However, very little evidence has shown the PDZ domain-containing proteins at inhibitory synapses. By yeast two-hybrid screening, GRIP was identified to associate with GABARAP. This association was further verified by GST-pull down and co-immunoprecipitation in COS cells (Kittler et al. 2004a). The PDZ domains 4–7 of GRIP1, corresponding to amino acids 413–794, are responsible for the interaction with GABARAP (Kittler et al. 2004a). GRIP1 has several spliced forms: GRIP1a, GRIP1b (Yamazaki et al. 2001; Yu et al. 2001) and GRIP1c (Charych et al. 2004b). GRIP1b differs from GRIP1a only in an alternate N-terminal region in which the first 18 amino acids of rat GRIP1 are replaced by a new sequence of 19 amino acids, and their downstream sequences appeared to be identical to each other (Yamazaki et al. 2001). The most notable differences between GRIP1a and GRIP1b are some cysteine residues at the N-terminus: GRIP1a contains a cysteine residue at position 8 and 10, while GRIP1b has a single cysteine at position 11. This difference makes the GRIP1b able to be palmitoylated, but GRIP1a is not (Yamazaki et al. 2001). GRIP1c lacks the PDZ domains 1–3 of GRIP1a/b, but contains PDZ domains 4–7. It also contains a specific 35 amino acid N-terminal and a 12 amino acid C-terminal peptide that differ from GRIP1a/b (Charych et al. 2004b). Therefore, GABARAP might bind to all these three isoforms of GRIP1.

GRIP is one of the few proteins present at both inhibitory and excitatory synapses although function of GRIP in the inhibitory synapses remains unknown. It has been reported that GRIP1a/b localized to both GABAergic and glutamatergic synapses in cultured hippocampal neurons (Dong et al. 1999; Wyszynski et al. 1999), but not to GABAergic synapses in the intact brain (Wyszynski et al. 1999). GRIP1c is found to be present in excitatory synapses in both cultured neurons and intact brain as demonstrated by immunofluorescence and electron microscope. Contrary to the other forms, it also localizes in GABAergic synapses, suggesting a possible role in GABAergic transmission (Charych et al. 2004b). GRIP1c does not coimmunoprecipitate with any GABAA receptors from brain extract, but with AMPA receptors (Charych et al. 2004b). As mentioned above, there is little evidence that PDZ domain-containing protein is found at inhibitory synapses. In addition, the C-terminus of GABAA receptors extends extracellularly and does not have a PDZ-binding domain. Therefore, GRIP1c might interact with GABAA receptors through GABARAP or other GABAA receptor associated proteins and participate in regulating GABAA receptor trafficking and clustering on the cell membrane. Electron microscopy demonstrated that both GRIP1 and GABARAP were found in the intracellular compartments, e.g., Golgi. The interaction between GRIP1 and GABARAP might exist at the level of intracellular compartments.

Phospholipase C-Related, catalytically Inactive Proteins (PRIP)

The PRIP family has been identified as a group of novel inositol 1,4,5-trisphosphate binding proteins with a domain organization similar to phospholipase C-δ but lacking the enzymatic activity (Kanematsu et al. 2000). PRIP-1 has been shown to regulate Ins (1,4,5) P3-mediated Ca2 + signaling by modulating type 1 inositol polyphosphate 5-phosphatase activity through binding to Ins (1,4,5) P3 (Harada et al. 2005). The family consists of at least two types of protein, PRIP-1 and PRIP-2. They have a number of binding partners, including the catalytic subunit of protein phosphatase 1α and GABARAP (Kanematsu et al. 2002; Uji et al. 2002). Both PRIP-1 and PRIP-2 compete with the γ2 subunit of the GABAA receptor for its binding site on GABARAP (Kanematsu et al. 2002). Northern blot analysis and reverse-transcription polymerase chain reaction showed that PRIP-1 was presented mainly in the brain, whereas PRIP-2 was expressed ubiquitously (Uji et al. 2002). The in situ hybridation revealed that PRIP-1 mRNA is localized in hippocampal pyramidal cells, dentate granule cells, pyramidal and granule cell layers of the cerebral cortex, and in the granule cell and Purkinje cell layers and cerebellar nuclei of the cerebellum. This regional distribution is similar to that of GABAA receptors (Kanematsu et al. 2002). PRIP-1 does not appear to be enriched at inhibitory synaptic sites and the majority of PRIP-1 and GABARAP co-localized as intracellular punctate structures in cultured cortical neurons (Kanematsu et al. 2002). Electrophysiological analysis demonstrated no overt differences in functional cell surface GABAA receptor number in the absence of PRIP-1. In agreement with the above data, binding assays indicated no marked difference in the number of GABA or BZ binding sites between wild type and PRIP-1 knockout mice. Immunoblotting results confirmed that the expression of the major GABAA receptor subunits in hippocampal and cortex neurons does not appear to be affected by the lack of PRIP-1. However, diazepam induced remarkably lower effects in neurons of PRIP-1 knockout mice, as shown by both reduced efficacy and potency. On the contrary, Zn2+ has lost its normal inhibitory effect on GABA-induced effects in hippocampal neurons from PRIP-1 knockout mice. Behaviour analysis revealed that motor coordination was impaired and intraperitoneal injection of diazepam induced markedly reduced sedative and antianxiety effects in PRIP-1 knockout mice. These results indicate that PRIP-1 is essential for the function of GABAA receptors, especially in response to the agents acting on γ2 subunit-containing isoforms (Kanematsu et al. 2002).

PRIP-1 also binds to the intracellular domains of all GABAA receptor β subunits and weakly with γ2 subunits directly, but not to those of the α1–6, δ or ρ1 subunits. In PRIP-1 knockout mice, forskolin caused less phosphorylation of β3 subunits than in wild-type mice (Terunuma et al. 2004). This reduction is not related to PKA because the PKA activity and the association between PKA and GABAA receptors remain the same in PRIP-1 knockout mice. Instead, the phosphatase activity of PP1α is elevated in PRIP-1 knockout mice, which is in agreement with the result that PRIP-1 is a negative modulator of phophatase (Yoshimura et al. 2001; Terunuma et al. 2004). Further experiments demonstrate that PP1α, PP2A and some unknown phosphatase all participate in dephosphorylation of the β3 subunit but with PP1α playing the predominant role, which explains how PP1α dephosphorylation of the β3 subunit can be negatively regulated in PRIP-1 knockout mice (Terunuma et al. 2004). PRIP-1 itself can be phosphorylated by PKA on T94 and S96. The former site is likely responsible for regulating the dissociation of PP1α from PRIP-1. PRIP-1 is also required for dopamine receptor modulation of GABAA receptor function. Taking this evidence together, PRIP-1 is likely to play a significant role in the phospho-dependent modulation of GABAA receptor activity regulated by endogenous neuronal signaling pathways (Terunuma et al. 2004).

Others

Recent studies indicate that GABARAP plays a more generalized function in cells rather than binding exclusively to neuronal GABAA receptors.

  • 1
    A yeast two-hybrid screen using the cytoplasmic domain of transferrin receptors identified GABARAP as the dominant interactor of transferrin receptors. The interaction between the two proteins is direct and the amino acids 36–117 of GABARAP are necessary, as shown in GST-pull down experiments, indicating that GABAA receptors and transferrin receptors are docking on either the same site or overlapping sites on GABARAP. However, GABARAP does not affect the endocytosis rate of transferrin receptors, possibly because GABARAP is sequestered in an intracellular compartment and interact with transferrin receptors in a process that is distinct from endocytosis. Immunofluorescence studies indicated that GABARAP is not primarily associated with endocytic vesicles but traffics transferrin receptors along another pathway such as the biosynthetic or degradative pathway (Green et al. 2002).
  • 2
    Unc-51-like kinases, ULK1 and ULK2, are homologues of unc-51 of C. elegans and Apg1p of yeast (Yan et al. 1998). Mouse ULK has been shown to be involved in axonal elongation (Tomoda et al. 1999). By yeast two-hybrid experiment, ULK1 was found to interact with both GATE-16 and GABARAP (Okazaki et al. 2000). This interaction suggests that these molecules might be important for vesicle transport and axonal elongation in mammalian neurons.
  • 3
    GABARAP was recently found to interact with DEAD (Asp-Glu-Ala-Asp/His) box polypeptide 47 (DDX47) (Lee et al. 2005b), an RNA helicase responsible in premRNA splicing or ribosome biogenesis (Jankowsky and Jankowsky 2000). DDX47 induces apoptosis by interaction with GABARAP.
  • 4
    The GABARAP gene was also detected as an insertion in bovine viral diarrhea virus genome from infected cows (Becher et al. 2002). The insertion of GABARAP served as a processing signal to yield NS3, critical for the cytopathogenicity of the virus. It interacts with the N-terminus of NS3, with the last amino acid cleaved off (Becher et al. 2002). Besides GABARAP, other genes were also detected to insert into some viral genomes, including ubiquitin, LC3, GATE-16 and the J domain protein Jiv.
  • 5
    By carrying out suppression substractive hybridization to detect differentially expressed genes between a breast cancer cell line and a non-tumorigenic microcell hybrid cell line, GABARAP was identified to display high expression levels in the non-tumorigenic cell line. The down-regulation of GABARAP was also seen in human breast cancer tissue. Over-expression of GABARAP in a breast cancer cell line induced a reduction of growth rate and tumorigenicity, indicating that GABARAP acts as tumor suppressor in breast cancer (Klebig et al. 2005).

Golgi-specific DHHC zinc finger protein (GODZ)

  1. Top of page
  2. Abstract
  3. GABARAP
  4. Golgi-specific DHHC zinc finger protein (GODZ)
  5. Plic-1
  6. Radixin
  7. Huntingtin-Associated Protein 1 (HAP 1)
  8. GABAA receptor interacting factor-1 (GRIF-1)
  9. Brefeldin A-inhibited GDP/GTP exchange factor 2 (BIG2)
  10. Summary
  11. References

The large intracellular loop of the γ2 subunit is rich in cysteine residues, which are absent from the equivalent domain of all the other subunits (Pritchett et al. 1989), suggesting that it might be a candidate for palmitoylation. Recent work of two labs demonstrated that the γ2 subunit is palmitoylated on all the five cysteine residues from C359 to C380 in the large intracellular loop (Keller et al. 2004; Rathenberg et al. 2004). The palmitoylation is required for controlling both GABAA receptor clustering at synaptic sites and for the cell surface stability of these proteins in neurons (Keller et al. 2004; Rathenberg et al. 2004). The mechanism underlying these phenomena might be that palmitoylation enhances the interaction of GABAA receptors with components of the subsynaptic cytoskeleton at inhibitory synapses, such as gephyrin, and thereby increases receptor clustering. Or this covalent modification may be critical for recruiting GABAA receptors into lipid rafts, facilitating their trafficking to, or stabilization at synaptic sites (Rathenberg et al. 2004).

By using a cysteine-rich 14 a.a. peptide of the intracellular loop of γ2 subunits as a bait in a yeast two-hybrid system, another γ2 subunit-interacting protein, GODZ was isolated (Keller et al. 2004). It also binds specifically with the large cytoplasmaic loop of the other two γ subunits, γ1 and γ3, but not the other GABAA receptor subunits (Keller et al. 2004). The binding domain of GODZ on γ subunits (a.a. 368–381 in γ2) is adjacent to that of GABARAP (a.a. 389–394) (Nymann-Andersen et al. 2002b). GODZ was first isolated in a yeast two-hybrid system using the carboxyl-terminal of GluR α1 as bait (Uemura et al. 2002). It contains a DHHC-CRD domain and is predicted to represent a 299-amino acid integral membrane protein with cytoplasmic N- and C-termini and four putative transmembrane regions. In COS7 cells, GODZ was specifically distributed in the Golgi-apparatus, as shown by overlapping with the trans-Golgi marker NBD C6-ceramide, suggesting it might be involved in Golgi-specific activity (Uemura et al. 2002). GODZ and GluR α1 cannot be immunoprecipitated together from COS7 cells lysate, but their immunofluorescent activity co-localized. Co-expression of GODZ recruits the GluR α1 subunit to localize to the Golgi apparatus and co-localize with GODZ (Uemura et al. 2002). This evidence suggests that over-expression of GODZ interfered with the trafficking of glutamate receptors from the Golgi apparatus.

In agreement with observations in COS7 cells, overexpressed GODZ concentrated in the Golgi apparatus as a large aggregate in HEK 293T cells. Co-expression of the γ2 subunit with GODZ greatly reduced the amount of γ2 subunit that reached the plasma membrane, and γ2 subunits were trapped intracellularly and co-localized with GODZ (Keller et al. 2004). However, if GODZ was co-expressed with α2β3γ2S, the γ2 subunit could reach the plasma membrane. In other words, the ‘trapping’ effect was blocked. Some GODZ loses the co-localization with the Golgi complex, while more GODZ associates with γ2 subunits near the plasma membrane (Keller et al. 2004). In cultured neurons, GODZ immunoreactivity was found to concentrate at one face of the Golgi complex where membrane proteins are believed to be sorted into transport vesicles, suggesting that GODZ functions in the secretory pathway of γ2-containing GABAA receptors. GODZ does not co-localize with the postsynaptic marker, gephyrin, indicating that GODZ is absent from inhibitory synapses. In agreement with the interaction with GluR α1 subunits, GODZ and γ2 subunits cannot be immunoprecipitated together from cultured neurons or brain, indicating that the two proteins do not normally form a stable complex. Instead, they might interact transiently as typically observed for enzymes and substrates. It has been proposed that all DHHC zinc finger proteins might encode palmitoyltransferases that thioacylate cysteine residues in target proteins, such as Erf2 and Akr1 (Bartels et al. 1999; Roth et al. 2002). [3H]-labeled palmitoylation experiments in recombinant HEK293 cells demonstrated that the palmitoylation of the γ2 subunit requires the co-expression of GODZ, suggesting that GODZ is the palmitoyltransferase of the γ2 subunit.

Plic-1

  1. Top of page
  2. Abstract
  3. GABARAP
  4. Golgi-specific DHHC zinc finger protein (GODZ)
  5. Plic-1
  6. Radixin
  7. Huntingtin-Associated Protein 1 (HAP 1)
  8. GABAA receptor interacting factor-1 (GRIF-1)
  9. Brefeldin A-inhibited GDP/GTP exchange factor 2 (BIG2)
  10. Summary
  11. References

Plic-1 was identified as a GABAA receptor-associated protein by using the large intracellular loop of the α1 subunit as bait in a yeast two-hybrid system. It also interacts with the M3-M4 intracellular domain of α2, α3, α6, and β1–3 subunits of GABAA receptors, but not γ2L or δ subunit, or neuronal nicotinic acetylcholine receptor α7 subunit (Bedford et al. 2001). Plic-1 was originally identified through its interaction with the cytoplasmic tail of CD47, which is a ubiquitous integral membrane glycoprotein associated with integrins. CD47 also has another name, IAP (integrin-associated protein). Plic-1 has been shown to mediate interaction between IAP and vimentin-containing intermediate filaments. Therefore, it was named proteins linking IAP and cytoskeleton (Plic-1) (Wu et al. 1999). Its interaction with cytoskeleton has been shown to participate in a wide variety of cell functions, ranging from inhibition of the cell cycle to rearrangements of cytoskeleton to blocking cell migration (Wu et al. 1999; N'Diaye and Brown 2003).

Plic-1 stabilizes cell surface GABAA receptors and facilitates the insertion of GABAA receptors into the cell membranes (Bedford et al. 2001). Plic-1 is a ubiquitin-like protein sharing 33% identical sequence to ubiquitin in the N-terminus, which is responsible for the association with the GABAA receptor subunits (Bedford et al. 2001). It also has a ubiquitin-associated domain in the C-terminus (Hofmann and Bucher 1996; Jentsch and Pyrowolakis 2000). In cultured hippocampal neurons, Plic-1 exhibits an intracellular clustered distribution in neuronal processes. It co-localizes with the β2/3 subunits in intracellular structures beneath the plasma membrane. Electron microscopy demonstrates that Plic-1 is localized not only in intracellular compartments including clathrin-coated pits and the border of the Golgi apparatus but also in GABAergic synapses, suggesting that Plic-1 might be involved in the membrane trafficking of GABAA receptors. Applying an interfering peptide to block the interaction between Plic-1 and GABAA receptors in a whole-cell patch clamp and whole-cell ELISA experiment results in a loss of cell surface receptor number with no effects on receptor internalization or ion channel activity. Over-expression of Plic-1 increases total cell surface receptor number. This evidence suggests that Plic-1 participates in controlling receptor cell surface number, rather than modulating ion channel function. Plic-1 increases the half-life of intracellular GABAA receptors pools, indicating that it can stabilize the receptors, which might account for its ability to facilitate the insertion of GABAA receptors into synaptic membranes. Plic-1 might also be involved in controlling receptor biosynthesis by stabilizing unassembled subunits in the ER, which may increase subunit maturation and production of heteromeric receptors. Because Plic-1 is a ubiquitin-like protein and the GABAA receptor subunit interaction domain on Plic-1 is overlapping with its ubiquitin-associated domain, one of the possible mechanisms underlying its function might be that Plic-1 inhibits poly ubiquitination of GABAA receptors (Bedford et al. 2001).

Radixin

  1. Top of page
  2. Abstract
  3. GABARAP
  4. Golgi-specific DHHC zinc finger protein (GODZ)
  5. Plic-1
  6. Radixin
  7. Huntingtin-Associated Protein 1 (HAP 1)
  8. GABAA receptor interacting factor-1 (GRIF-1)
  9. Brefeldin A-inhibited GDP/GTP exchange factor 2 (BIG2)
  10. Summary
  11. References

Radixin, a member of the ERM (ezrin, radixin, moesin) protein family, has been identified to interact with α5 subunit, but not α1, α3, β2, and γ2 subunits (Loebrich et al. 2006). The α5 subunit assembles with β and γ subunits and is predominantly localized extrasynpatically in himppocampus, mediating tonic inhibition. Amino acids 342–257 on the α5 subunit are required for this interaction, which is not present in the extrasynaptic GABAA receptor δ subunit. Radixin can exist in both an inactive closed conformation and an active open conformation. The latter depends on Rho-family GTPase signaling-mediated phosphorylation and is required for radixin–α5 subunit interaction. In cultured hippocampal neurons, by using phospho-specific ERM antibody, active radixin has been shown to locate, cluster and co-localize with the α5 subunit at the plasma membrane. Up to 87% of radixin puncta and 90% co-clusters of radixin and α5 subunit were found at an extrasynaptic position. Radixin does not co-localize or co-immunopreciatate with gephyrin, indicating that radixin and gephyrin represent independent systems (Loebrich et al. 2006). Interfering the expression of radixin with an antisense oligonucleotide dramatically decreases the cluster number of the α5 subunit, but not α1 subunit. The total amount of α5 subunit was unchanged. In agreement with the above evidence, the cluster number of the α5 subunit was also reduced in the hippocampus of radixin-deficient mice. Similar results were observed upon interrupting the interaction between radixin and the α5 subunit. These results suggest that radixin is an essential factor for α5 subunit-containing GABAA receptors clustering (Loebrich et al. 2006).

Huntingtin-Associated Protein 1 (HAP 1)

  1. Top of page
  2. Abstract
  3. GABARAP
  4. Golgi-specific DHHC zinc finger protein (GODZ)
  5. Plic-1
  6. Radixin
  7. Huntingtin-Associated Protein 1 (HAP 1)
  8. GABAA receptor interacting factor-1 (GRIF-1)
  9. Brefeldin A-inhibited GDP/GTP exchange factor 2 (BIG2)
  10. Summary
  11. References

Yeast two-hybrid assays demonstrated that HAP 1 interacts with the intracellular loop of the β1 subunit of GABAA receptors, but not the α1, γ2, or δ subunits or the intracellular C-terminal tail of the GABAB receptor R1 subunit (Kittler et al. 2004b). HAP1 was first identified to associate with huntingtin, which is associated with Huntington's disease patients (Li et al. 1995). HAP1 lacks conserved transmembrane domains and nuclear localization signals, suggesting that it is a cytoplasmic protein. It contains coiled-coil domains in the middle region and multiple N-myristoylation sites, which are present in a large number of proteins that are associated with membrane proteins and involved in vesicular trafficking (Li and Li 2005). HAP1 has been shown to be transported in axons and to associate with p150glued dynactin subunit, an essential component of the dynein/dynactin microtubule-based motor complex (Engelender et al. 1997; Li et al. 1998; Gauthier et al. 2004).

In cultured hippocampal neurons, HAP1 was demonstrated by an immunofluorescence study to exhibit clusters in dendrites and axons, in addition to substantial intracellular somatic staining, including large perinuclear structures consistent with the localization of this protein to intracellular compartments such as endosomes and tubulovesicular structures. A proportion of HAP1 co-localizes with GABAA receptors (Gutekunst et al. 1998; Martin et al. 1999; Kittler et al. 2004b). Suppression of HAP1 by siRNA decreases the level and activity of GABAA receptors in hypothalamus. Food intake and body weight of mice was also reduced by the HAP siRNA and in HAP1 knockout mice (Dragatsis et al. 2004; Sheng et al. 2006). Given the fact that GABA stimulates eating, this evidence suggests that HAP1 might function as a mediator for regulating the activity of hypothalamic GABAA receptors in control of feeding behaviour. Over-expression of HAP1 significantly enhances the stability of internalized GABAA receptors and facilitates their recycling to the plasma membrane in cultured cortical neurons. In addition, HAP1 also enhances GABAA receptor synaptic transmission by increasing the number of active cell surface and synaptic GABAA receptors in neurons. This result suggests that HAP1 plays an important role in controlling GABAA receptor endocytic sorting, and therefore cell surface stability (Kittler et al. 2004b). However, the mechanism underlying HAP1′s effect on GABAA receptors membrane trafficking remains to be elucidated. The HAP1 knockout mice die within the first days. Those mice that survived into adulthood show normal brain and behaviour, suggesting that HAP1 has a fundamental role in regulating brain development in the early age after birth and a non-essential role in the adult mouse (Dragatsis et al. 2004).

GABAA receptor interacting factor-1 (GRIF-1)

  1. Top of page
  2. Abstract
  3. GABARAP
  4. Golgi-specific DHHC zinc finger protein (GODZ)
  5. Plic-1
  6. Radixin
  7. Huntingtin-Associated Protein 1 (HAP 1)
  8. GABAA receptor interacting factor-1 (GRIF-1)
  9. Brefeldin A-inhibited GDP/GTP exchange factor 2 (BIG2)
  10. Summary
  11. References

GRIF-1 was cloned and identified as a GABAA receptor associated protein through the interaction with the intracellular loop of the β2 subunit (Beck et al. 2002). The binding sites for GRIF-1 and the β2 subunit are located on a.a. 124–283 on GRIF-1 and a.a. 324–394 on the β2 subunit, which includes a coiled-coil domain (a.a. 348–361). Amino acid sequence analysis predicted that GRIF-1 is a hydrophilic protein with no transmembrane domain or hydrophobic signal peptide. Structural predictions indicated α-helical content and a coiled-coil domain that is believed to be involved in homo- or heterodimerization. GRIF-1 belongs to a new protein family which is conserved in evolution, e.g. OIP106 and KIAA0549 (human). GRIF-1 is also a homologue of Huntingtin-associated protein, mentioned above, a known trafficking factor, which suggests that it is important for intracellular trafficking of receptors (Beck et al. 2002).

GRIF-1 shares 44% a.a. homologue with Milton, which was identified originally by screening Drosophila mutations searching for effects on the cell biology of the axon and synaptic terminals (Stowers et al. 2002). Without Milton, synaptic terminals and axons lack mitochondria, although mitochondria are numerous in neuronal cell bodies. In contrast, synaptic vesicles continue to be transported to and concentrated at synapses. It was believed to be associated with mitochondria and required for kinesin-mediated transport of mitochondria to nerve terminals (Stowers et al. 2002). Accumulated evidence suggests that mitochondria are required to maintain synaptic plasticity in neurons. They not only provide ATP as energy for trafficking and maintenance of membrane potential, but also help buffer local Ca2+ concentrations in some nerve terminals (Werth and Thayer 1994; Zucker 1999). Mitochondria migrate within the cell in response to changes in the local energy state and are involved in axonal transport in neurons. GRIF-1 has been shown to associate with subtypes of kinesin in several tissues and the interaction between GRIF-1 and kinesin is direct as shown in yeast two-hybrid experiments (Brickley et al. 2005), therefore was speculated to play a pivotal role in the trafficking of mitochondria distribution in concert with receptor-vesicle transportation, ensuring fidelity of synaptic function during development and synaptic activity (Brickley et al. 2005).

Brefeldin A-inhibited GDP/GTP exchange factor 2 (BIG2)

  1. Top of page
  2. Abstract
  3. GABARAP
  4. Golgi-specific DHHC zinc finger protein (GODZ)
  5. Plic-1
  6. Radixin
  7. Huntingtin-Associated Protein 1 (HAP 1)
  8. GABAA receptor interacting factor-1 (GRIF-1)
  9. Brefeldin A-inhibited GDP/GTP exchange factor 2 (BIG2)
  10. Summary
  11. References

BIG2 is a 200-kDa protein belonging to a class of high molecular weight GDP/GTP exchange factors that catalyzes GDP/GTP exchange on the small G-protein ADP-ribosylation factors (ARF) (Togawa et al. 1999). ARF are well known to play key roles in intracellular trafficking. Activation of ARF by GDP/GTP exchange is required for membrane budding in the Golgi apparatus allowing proteins to progress through the Trans-Golgi Network (TGN) and the exocytotic pathway (Moss and Vaughan 1995, 1998). BIG2 has been recently shown to be a GABAA receptor associated protein by the yeast two-hybrid system using the large intracellular loop of β3 subunit as bait (Charych et al. 2004a). BIG2 has been shown to have high binding affinity for the large intracellular loop of all the β subunits. However, it does not interact with the intracellular loop of α1, γ1, γ2S or γ2L (Charych et al. 2004a). By using truncation constructs, an 18 amino acid sequence located at the N-terminus of β3 intracellular loop was shown to be responsible for the interaction with BIG2. Both the N-terminus and C-terminus of BIG2 are involved in the interaction with the β3 subunit. In cultured neurons, BIG2 is mainly localized in the TGN in the soma, the proximal part of the dendrites, microtubules in the axon as well as some trafficking vesicles and synaptic structures. Co-expression of BIG2 and the β3 subunit promotes the loss of the β3-containing GABAA receptors from ER and/or Golgi apparatus; the interaction between BIG2 and GABAAβ subunits is transient, suggesting BIG2 plays a role in GABAA receptor intracellular trafficking. Some BIG2 is also co-localized with GABAA receptors on the plasma membrane of neurons, indicating the interaction between BIG2 and β subunits might play a role in the transport of newly assembled GABAA receptors by clathrin/AP-1-coated vesicles to the synaptic plasma membrane (Charych et al. 2004a).

Summary

  1. Top of page
  2. Abstract
  3. GABARAP
  4. Golgi-specific DHHC zinc finger protein (GODZ)
  5. Plic-1
  6. Radixin
  7. Huntingtin-Associated Protein 1 (HAP 1)
  8. GABAA receptor interacting factor-1 (GRIF-1)
  9. Brefeldin A-inhibited GDP/GTP exchange factor 2 (BIG2)
  10. Summary
  11. References

The evidence to date suggests that these GABAA receptor associated proteins play important roles in regulating the activities of GABAA receptors. As summarized in Fig. 2 and Table 1, Plic-1 interacts with α2, α3, α6 and β1–3 subunits; radixin interacts with α5 subunit; HAP interacts with β1 subunit; GRIF-1 interacts with β2 subunit; BIG2 interacts with β1–3 subunits, and GODZ and GABARAP associates with γ1–3 subunits. The GABAA receptor associated proteins may or may not bind to the subunits at the same time. Accompanied by proteins, the GABAA receptors traffic from intracellular compartments to the cell membrane along microtubules; moves between synapses and extrasynaptic membrane; and internalizes from the cell membrane to the degenerative pathway.

image

Figure 2.  Newly found proteins that associate with GABAA receptor subunits. Plic-1 interacts with α2, α3,α6 and β1–3 subunits; radion interacts with α5 subunit HAP interacts with β1 subunit; GRIF-1 interacts with β2 subunit; BIG2 interacts with β1–3 subunits, and GODZ and GABARAP associates with γ1–3 subunits.

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Table 1.   Summary of the newly found GABAA receptor associated proteins
NameInteracting subunitsInteraction domain on the GABAR subunitInteraction domain on associated proteinStructure featureSubcellular localizationOther interacting proteins other than subunitsFunctionsRef.
  1. Ref.1, Bedford et al. 2001; N'Diaye and Brown 2003; Ref. 3, Li et al. 1995; Li et al. 1998; Kittler et al. 2004b; Li and Li 2005; Ref. 4, Beck et al. 2002; Iyer et al. 2003; Brickley et al. 2005; Ref. 5, Becher et al. 2002; Charych et al. 2004; Ref. 6, Kanematsu and Hirata 2002; Uji et al. 2002; Kanematsu and Hirata 2003; Terunuma et al. 2004; Yamaguchi et al. 2004; Harada et al. 2005; Ref. 7, Uemura et al. 2002; Keller et al. 2004; Ref. 8, Wang et al. 1999; Chen et al. 2000; Kneussel et al. 2000; Wang and Olsen 2000; Kittler et al. 2001; Tanida et al. 2001; Coyle et al. 2002; Kanematsu et al. 2002; Nymann-Andersen et al. 2002b; Nymann-Andersen et al. 2002a; Stangler et al. 2002; Tanida et al. 2002; Tanida et al. 2003; Everitt et al. 2004; Kittler et al. 2004; Leil et al. 2004; Sugawara et al. 2004; Tanida et al. 2004; Chen et al. 2005.

Plic-1α2, α3,α6 and β1–3N-terminusUnknown Intracellular soma and processesCD47; intermediate filamentsClathrin-coated vesicle trafficking; stabilize receptorsRef. 1
Radixinα5u.a. 342–357C-termini Plasma membraneF-actinMembrane-cytoskeletal crosslinker; GABAR clusteringRef .2
HAPβ1UnknownUnknownCoiled-coiled domains; N-myristoylation Huntingtin; p150GluedVesicular traffickingRef. 3
GRIF-1β2Coil 348–361; coil 316–339a.a. 124–283α-helix; coil-coil domain O-glcNAc transferase; kinesinO-glcNAc modificationRef. 4
BIG2β1, β2 and β3β3 a.a. 328–345N- and C-termini TGN, dendrites, axon and synapse Receptor traffickingRef. 5
PRIPβ and γ2UnknownUnknown  Ins (1,4,5) P3; phosphatase 1α; GABARAPRegulating the phosphorylation of GABA receptorsRef. 6
GODZγ1, γ2S and γ2Lγ2 a.a. 368–381 DHHC-CRD domainGolgi and cytoplasmic membrane PalmitoyltransferaseRef. 7
GABARAPγ1, γ2S and γ2Lγ2 a.a. 389–394a.a. 37-52α-helix; β-sheetMainly in Golgi and ERMicrotubules; NSF; GRIP1; PRIP-1Receptor trafficking and clusteringRef. 8

One of the important functions of these proteins is to regulate the surface numbers of GABAA receptors. GABARAP increases the surface level of γ2-containing GABAA receptor by increasing the transportation along microtubules. In addition, GABARAP can modulate the ion channel kinetics of GABAA receptors. Plic-1 stabilizes cell surface GABAA receptors and facilitates the insertion of GABAA receptor into the cell membrane. HAP1 enhances the stability of internalized GABAA receptors and facilitates their recycling to the plasma membrane. BIG2 might be involved in GABAA receptor vesicular trafficking. GODZ and GRIF-1 participate in the post-translational modification of GABAA receptors. GODZ is believed to function as the enzyme catalyzing the palmitoylation of the γ2 subunit, which is required for controlling GABAA receptor clustering and cell surface stability. Radixin is essential for α5 subunit clustering. GRIF-1 is suggested to be involved in bringing O-glcNAc transferase to GABAA receptors. PRIP-1 and PRIP-2 are involved in the regulation of GABAA receptor phosphorylation.

The ρ subunits of GABAA receptors were found to interact with microtubule-associated protein 1B and glycine transporter in a yeast two hybrid assay using the intracellular loop as bait (Billups et al. 2000; Hanley et al. 2000; Pattnaik et al. 2000). Besides these newly identified proteins mentioned above, another group of GABAA receptor associated proteins also regulates the trafficking, clustering, channel activity and functional plasticity of GABAA receptors. These proteins include clathrin adaptor protein 2 that regulates receptor endocytosis (Kittler et al. 2005), PKA, protein kinase C (PKC), calcineurin, Akt and Src that modulate receptor activity via phosphorylation and PRIP-1, PRIP-2, α kinase anchoring protein (AKAP) and receptor for activated protein kinase C (RACK-1) that function as phosphorylation-modulators (Fritschy and Brunig 2003; Luscher and Keller 2004). In addition, GABAA receptors are also found to associate with other neurotransmitter receptors, such as GABAB receptors (Balasubramanian et al. 2004) and dopamine receptor (Lee et al. 2005a).

Given the fact that the subunits assemble into pentameric receptors very soon after they are synthesized in the ER, the associated proteins might be critical in guiding the receptors to their destination, synaptic or extrasynaptic sites. Still, there are many questions arising from these studies. For example, do all the proteins associate with the assembled pentameric receptors as they traffic to the cytoplasma membrane or do they accompany the receptors on the membrane? In both cases, which protein plays a critical role? If only one or some, how do the cells control the association? Is there any cross-talk between these associated proteins? These questions can be answered by suitable functional analysis.

References

  1. Top of page
  2. Abstract
  3. GABARAP
  4. Golgi-specific DHHC zinc finger protein (GODZ)
  5. Plic-1
  6. Radixin
  7. Huntingtin-Associated Protein 1 (HAP 1)
  8. GABAA receptor interacting factor-1 (GRIF-1)
  9. Brefeldin A-inhibited GDP/GTP exchange factor 2 (BIG2)
  10. Summary
  11. References
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