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Roles of PRIP in GABAA Receptor Signaling
J. Oral Biosci. 49 （2）：105−112, 2007
Rising Sun Symposium―A New Era for Young Dental Scientists REVIEW
Roles of PRIP in GABAA Receptor Signaling Akiko Mizokami, Takashi Kanematsu§ and Masato Hirata Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental Science, and Station for Collaborative Research, Kyushu University 3−1−1, Maidashi, Higashi−ku, Fukuoka 812−8582, Japan 〔Received on November 15, 2006；Accepted on December 28, 2006〕 Key words：benzodiazepine／GABAA receptor／GABARAP／knockout mice／PRIP／trafficking Abstract：GABAA receptors are a family of ligand−gated ion channels that are pentamer composed predominantly of α, β and γ subunits. They are the major target of the endogenous inhibitory neurotransmitter （GABA, γ−aminobutyric acid）and maintain the majority of fast inhibitory ion currents in the central nervous system, in addition to being drug targets for benzodiazepines, barbiturates, alcohols, neurosteroids, and some anesthetics. Moreover, modifications in GABAA receptor function are crucial in central nervous system diseases such as anxiety disorders, sleep disturbances, and seizure disorders. Therefore it is very important to understand the molecular mechanisms underlying the maintenance of functional inhibitory synapses including GABAA receptors. We have first isolated PRIP（phospholipase C−related, but catalytically inactive protein）as a novel inositol 1,4,5−trisphosphate binding protein, and subsequently found GABARAP （GABAA receptor associated protein）as one of binding partners that binds to γ2 subunit of the receptor and thus is implicated in the clustering and trafficking of the receptor to synaptic membrane. Further studies revealed that PRIP binds β subunit of the receptors and PP1c（catalytic subunit of protein phosphatase 1）. These findings have prompted us to explore the possible involvement of PRIP in modulation of GABAA receptor signaling. Here we summarize our current understanding regarding how PRIP is involved in the modification of GABAA receptor signaling on the basis of the characteristics of these interacting molecules.
Introduction The enhancement of neuronal inhibition by GABA is one of the most powerful therapeutic strategies for the treatment of central nervous system diseases. For instance, benzodiazepines （BZ）, whose targets are GABAA receptors, have widely been used clinically for anxiety, sleep disorder, muscle stiffness and so on, the therapeutic effects of which are derived by §
Corresponding author E−mail：[email protected]−u.ac.jp
increasing the affinity of the receptor for GABA and recruiting more GABAA receptors for activation by the ligand1）. Therefore, it is very important to understand the molecular mechanisms regarding the life cycle of GABAA receptors including molecular organization, transport and insertion to the surface membrane, functional modulation on the surface membrane, and internalization followed by recycling back to the surface membrane or lysosomal degradation. Recently, a number of reports have been published regarding the molecules involved in the dynamic regulation of GABAA receptor function2）. PRIP−1, originally identified as a novel D−myo−inositol 1,4,5−
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Fig. 1 Schematic illustration of GABAA receptor structure Left：Subunit structure of GABAA receptors with four transmembrane domains and extracellular N− and C−termini. The large amino−terminal domain located extracellularly, is believed to accommodate neurotransmitter, GABA and some modulator chemicals. The intracellular domain between TM3 and TM4 is the most divergent part of individual receptor subunit and contains numerous consensus sites for the action of both serine／threonine and tyrosine protein kinases. Right：Heteropentameric GABAA receptors forming Cl− channel. TM2 is believed to form the lining of the ion channel. Addition of the ligand triggers a small rotation of the extracellular domains of the receptor subunits, which then opens the channel pore formed by the adjoining TM2 regions of the five subunits.
trisphosphate binding protein, is one such molecule, and was found to be involved in GABAA receptor signaling3）. In this article we discuss the involvement of PRIP molecules in GABAA receptor signaling. Structure of GABAA Receptor and Its Subunit Diversity GABAA receptors are the major target of the endogenous inhibitory neurotransmitter, GABA, and mediate the bulk of fast inhibitory neurotransmission in the mammalian brain. They are heteropentamers and each subunit shares a conserved structure. A large amino−terminal domain is located extracellularly and is believed to accommodate a neurotransmitter and some modulators （Fig. 1）. The receptor subunits also contain four hydrophobic transmembrane （TM）domains and a large intracellular loop between TM domains Ⅲ and Ⅳ. This domain is the most divergent part of individual receptor subunits and many receptor−associated proteins are reported to interact with this loop. Also, this domain contains numerous consensus sites for the action of both serine／threonine and tyrosine protein kinases4）. GABAA
receptor subunits are classified by sequence identity into seven subunit classes with multiple subtypes： α1−6, β1−3, γ1−3, δ, ε, θ, and π. The receptor characteristics largely depend on the subunit composition of individual GABAA receptors5）. However, receptors containing α1, 2, 3, or 5, subunits in combination with any of the β subunits and γ2 subunits with a ratio of 2：2：1 are most prevalent in the brain2,6）. The expression pattern of individual subunits in a spatio− temporal−dependent manner generates a high diversity of GABAA receptors with different functional properties. For example, the inclusion of a γ2 subunit into the pentamer is essential to produce receptors that are sensitive to BZ−type drugs that are widely used for anxiolytic, hypnotic, anticonvulsant, and muscle−relaxing actions7）. A small population of GABAA receptors that are present mainly in the cerebellum and thalamus contain the δ subunit5）, which is known to be insensitive to BZs, in a similar way to those containing α4 or α6 subunits1,9）. There have been several reports on gene targeting experiments for some subunits using mice. Gene knockout of the α1 or β2 subunit showed neither a lethal phenotype nor epileptic seizures, although the
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number of GABAA receptors on the surface membrane was reduced to 50％8）. On the other hand, targeted disruption of the γ2 subunit gene caused 94％ reduction of the BZ sites and exhibited a perinatally lethal phenotype in the majority of mutant mice7）. Most of the β3 subunit knockout mice also died in the neonatal period10）. Therefore, the presence of these two subunits, β3 and γ2, is absolutely required for GABAA receptors to function in maintaining the animal life itself, while modulation of other subunit compositions would provide changes in the functional characteristics of GABAA receptors, thus causing the impairment of behavioral events and a pharmacological effect. PRIP as a GABAA Receptor−associated Protein A family of phospholipase C−related, but catalytically inactive protein（PRIP） , have been identified as a novel D−myo−inositol 1,4,5−trisphosphate−binding protein with a domain organization similar to phospholipase C−δ but lacking enzymatic activity11―17）. The PRIP family consists of at least two types of proteins, PRIP−1, and PRIP−218）. PRIP−1 is expressed predominantly in the central nervous system, while PRIP−2 has broad tissue distribution, including the brain19,20）. We have applied a yeast two−hybrid screening system to explore the interacting molecules with PRIP and identified two molecules；the catalytic subunit of protein phosphatase 1α （PP1α）21） and GABARAP （GABAA receptor associated protein）3）. GABARAP is a molecule that interacts specifically with the γ2 subunit of GABAA receptor and has been implicated in the clustering of GABAA receptors and trafficking of receptors to the cell surface because of its ability to interact with microtubules22） and N−ethylmaleimide sensitive factor23）. The region of GABARAP responsible for binding to the γ2 subunit intracellular loop is that comprising residues 36―68, which is shared by PRIP, indicating the competition for the γ2 2）. These findings led us to subunit 22,24）（Fig. explore the possible involvement of PRIP in GABAA receptor function. Furthermore, in the course of experiments, we have also become aware that PRIP
Fig. 2 Interaction of GABARAP with PRIP and the γ2 subunit of the GABAA receptor PRIP and the γ2 subunit bind to GABARAP in a competitive manner. Numbers indicate the residues responsible for the interaction.
directly binds with the β subunits of GABA A receptors25,26）and protein phosphatase 2 A（PP2A）26）. These binding partners and their function further support the notion that PRIP as a GABAA receptor−associated protein, in collaboration with the above−mentioned molecules, plays an important role in receptor signaling. GABARAP in Membrane Trafficking of GABAA Receptors Before the description of the possible roles of PRIP, we briefly summarize the roles of GABARAP in GABAA receptor trafficking. The assembly and delivery of multiple GABAA receptor subtypes in a single cell and their extracellular signal−controlled delivery to different neural surfaces requires sophisticated control steps at the level of assembly and sorting into distinct trafficking vesicles as well as regulated and directed transport to their final destination. GABARAP is a possible candidate involved in some steps of these GABAA receptor assembly, sorting and trafficking processes, because GABARAP is a member of a protein family that includes microtubule−associated , the Golgi−associprotein light chain 3（MAP−LC3） ated ATPase enhancer of 16kDa（GATE−16）, and the
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yeast orthologue Atg8, all of which are reported to participate in membrane trafficking28,29）. GABARAP was first identified as the molecule responsible for clustering of GABAA receptors at the post−synaptic membranes 22）, but the idea has been recently amended to that GABARAP plays an important role in receptor trafficking, especially the γ2 subunit−containing type, to the surface membrane30,31）. However the precise molecular mechanisms underlying GABARAP−dependent transport of γ2 subunit−containing receptors remain unclear. Gene−targeting experiments of GABARAP in mice indicated no impairment in receptor clustering, supporting the notion that GABARAP is not implicated in receptor clustering32）, and no further reports regarding other phenotypes relating to GABAA receptor signaling of these mice have appeared. Analysis of PRIP Knockout Mice To examine the possible roles of PRIP interacting with GABARAP and GABAA receptor β subunits in the regulation of the receptor function, we analyzed PRIP−1 knockout（1−KO）mice from electrophysiological and behavioral aspects in combination with the effects of BZ3）. The GABAA receptor−mediated Cl− current（IGABA）in freshly isolated hippocampal CA1 cells from wild−type（WT）and PRIP−1−KO showed little difference in terms of the dose−response relationship；however, there appeared to be some dysfunction in BZ action. The BZ agonist, diazepam, reversibly potentiated IGABA elicited by GABA in a dose−dependent manner in neurons from WT mice. In contrast, the potentiation of IGABA was markedly reduced in neurons from PRIP−1−KO mice. Behavioral analysis also supported the results obtained by electrophysiological analysis. We examined the anxiolytic effects of diazepam on WT and PRIP−1−KO mice by the elevated plus−maze test. No significant difference in the time spent in the open or closed arms of the maze was apparent between WT and PRIP−1−KO mice injected with vehicle. However, a clear difference was observed when mice were injected with diazepam；injection of WT mice resulted in a marked increase in the time spent in the
open arm, indicative of the anti−anxiety effect of this drug. In contrast, the time that PRIP−1−KO mice spent in the open arm was not affected. These results clearly showed reduced sensitivity to diazepam in PRIP−1−KO mice, probably caused by the reduced number of γ subunit−containing receptors on the surface membrane. These results were supported by binding assays using radioactive ligands specific to GABA and BZ sites. As described above, the study was initiated by the finding that PRIP−1 interacts with GABARAP in a competitive manner with the γ2 subunit of GABAA receptors. On the basis of the finding that GABARAP facilitates the membrane transport of γ2−containing receptors to the cell surface30,31）, we predicted that the gene knockout of PRIP−1, which would eliminate PRIP−dependent competition of GABARAP binding to GABAA receptors, would result in an increased cell surface expression of γ2 subunit−containing receptors；however, the results obtained were opposite as . We those described（we called it“the γ−paradox”） thought that gene compensation at the level of PRIP− 2 proteins, which is also expressed in the brain, might account for the“γ −paradox”since PRIP−2 also binds to GABARAP. PRIP−1 and −2 double knockout ；however, mice have become available recently26） they have exhibited essentially similar phenotypes as type 1−KO mice regarding GABAA receptor function, although there are several small distinct differences33）. We found the direct association of PRIP with β subunits of GABAA receptors25,26）, and recently mapped the region responsible for the interaction 26） , （amino acid residues 544―568 of rat PRIP−1） which is located remote from the binding region of GABARAP；therefore, we confirmed ternary complex formation among the receptor β subunit, PRIP− 1, and GABARAP. Furthermore, disruption of this direct interaction by the peptide mimicking the binding site inhibited cell−surface expression of γ2 subunit−containing GABAA receptors, which resembled the phenotype of PRIP−DKO mice. Considering these results, we propose that the formation of the ternary complex mentioned above would facilitate the association of GABARAP with the γ2 subunit to be transported at the right place at the right time, or an
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association between the β subunit and PRIP would promote the association between the γ2 subunit and GABARAP, and thus facilitate the cell surface expression of the γ2 subunit−containing GABAA receptors （Fig. 3）；however, the precise molecular mechanisms remain to be elucidated33）. It should be also noted that possible involvement of PRIP, relating to the interaction with PP1 and PP2A, in the phospho− regulation of GABAA receptors has been recently reported25―27）. PRIP in Epilepsy Epilepsy and seizures are closely linked to the function of GABAA receptors in the brain34,35）. PRIP−1 is distributed mainly in the brain and expressed abundantly in the cerebral cortex and hippocampus20）. These brain regions are closely linked to epilepsy and seizure34,35）. Therefore, we investigated the effects of pentylenetetrazol（PTZ）, a chemical convulsant that interacts with the GABAA receptor, in mice lacking PRIP−1. PRIP−1−KO mice did not develop spontaneous behavioral seizure；however, the KO mice had markedly shorter latencies until the first clonic convulsion and tonic extensor following PTZ administration and increased incidence of convulsion compared to WT. Furthermore, the mortality rate by PTZ in KO mice was also significantly increased in comparison with WT36）. A point mutation in the GABAA receptor γ2 subunit was found in patients suffering from childhood absence epilepsy and febrile seizures37）. Electrophysiological experiments introducing such a point mutation into the γ subunit of GABAA receptors using oocytes demonstrated that the response to GABA was not different from those observed in the control receptors. However, the mutant receptors showed little potentiation by diazepam of GABA−induced Cl− current, as those observed in PRIP−1−KO mice. These findings strongly support that mice lacking PRIP−1 were hypersensitive to PTZ−induced convulsion, and that PRIP−1 might play a role in suppressing excessive excitability in neuronal activity via interactions with the GABAA receptor. Investigation into the functional roles of PRIP may elucidate novel
Fig. 3 Trafficking of GABAA receptors to the cell surface The trafficking of GABAA receptors within the secretory pathway is facilitated by GABARAP, which binds directly to γ2 subunits regulating the delivery of γ2 subunit containing receptors to the cell surface membrane. PRIP might be involved in these processes by regulating the function of GABARAP.
pathophysiological mechanisms of epilepsy and seizure. Summary PRIP was originally identified as a novel inositol 1,4,5−trisphosphate binding protein11―17,37）. Further studies to explore binding partners besides an inositol 1,4,5−trisphosphate have unexpectedly led us to examine the possible involvement of PRIP in GABAA receptor signaling, especially in those containing the γ2 subunit3,21,25―27,33,38）. GABAA receptors are the major target of the endogenous inhibitory neurotransmitter（GABA）and have been implicated in a variety of brain functions including sedation, hypnosis, anxiety, learning and memory4―6）. In particular, the receptors containing the γ2 subunit are the target of the BZ type, which are widely used clinically as anxiolytics, hypnotics, anticonvulsants, and muscle relaxants1）.
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For efficient inhibitory synaptic transmission and drug effects, it is critical that the receptors, especially those containing the γ2 subunit, are correctly transported from their site of synthesis in the endoplasmic reticulum to the appropriate synaptic or extrasynaptic site4,6）. A large amount of work has been dedicated to understanding how these processes are precisely orchestrated by multiple receptor−associated molecules. Analysis of PRIP−DKO mice suggest that PRIP plays an important role in the membrane trafficking of γ2 subunit−containing GABAA receptors, presumably by facilitating the delivery of GABARAP to the γ2 subunit through the association with the β subunits. Our findings that PRIP is implicated in the processes for expressing γ2 subunit−containing receptors, would provide a novel aspect in the complicated system of the dynamic regulation of GABAA receptors in the inhibitory synapses. Acknowledgements This work was supported by a Grant−in−Aid for Scientific Research from the MEXT of Japan（AM, TK and MH）and by the Cooperative Study Program of National Institute for Physiological Sciences（TK and MH）. Kato Memorial Bioscience Foundation （TK）, the Naito Foundation（TK）and Takeda Science Foundation （TK）. AM is a JSPS research fellow and a recipient of the Iwadare Scholarship.
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to the cell surface. J. Neurosci. 27：1692―1701. 34）Avoli, M.：GABA−mediated synchronous potentials and seizure generation. Epilepsia 37：1035―1042, 1996. 35）Sherwin, A. L.：Neuroactive amino acids in focally epileptic human brain：a review. Neurochem. Res. 24：1387―1395, 1999. 36）Yamaguchi, T., Kubota, T., Kanematsu, T., Nakayama, K., Hirata, M. and Yamamoto, T.：Hypersensitivity to pentylenetetrazol−induced convulsion in mice lacking the PLC−related inactive protein−1. Brain Res. 1025：237―240, 2004. 37）Wallace, R. H., Marini, C., Petrou, S., Harkin, L. A., Bowser, D. N., Panchal, R. G., Williams, D. A., Sutherland, G. R., Mulley, J. C., Scheffer, I. E. and Berkovic, S. F.：Mutant GABAA receptor gamma2−subunit in childhood absence epilepsy and febrile seizures. Nat. Genet. 28：49―52, 2001. 38）Kanematsu, T., Takeuchi, H., Terunuma, M. and Hirata, M.：PRIP, a novel Ins（1,4,5）P3 binding protein：Functional significance in Ca2＋ signaling and extension to neuroscience and beyond. Mol. Cells 20：305―314, 2005.
Rising Sun Symposium―A New Era for Young Dental Scientists REVIEW
Roles of PRIP in GABAA Receptor Signaling Akiko Mizokami, Takashi Kanematsu§ and Masato Hirata Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental Science, and Station for Collaborative Research, Kyushu University 3−1−1, Maidashi, Higashi−ku, Fukuoka 812−8582, Japan 〔Received on November 15, 2006；Accepted on December 28, 2006〕 Key words：benzodiazepine／GABAA receptor／GABARAP／knockout mice／PRIP／trafficking Abstract：GABAA receptors are a family of ligand−gated ion channels that are pentamer composed predominantly of α, β and γ subunits. They are the major target of the endogenous inhibitory neurotransmitter （GABA, γ−aminobutyric acid）and maintain the majority of fast inhibitory ion currents in the central nervous system, in addition to being drug targets for benzodiazepines, barbiturates, alcohols, neurosteroids, and some anesthetics. Moreover, modifications in GABAA receptor function are crucial in central nervous system diseases such as anxiety disorders, sleep disturbances, and seizure disorders. Therefore it is very important to understand the molecular mechanisms underlying the maintenance of functional inhibitory synapses including GABAA receptors. We have first isolated PRIP（phospholipase C−related, but catalytically inactive protein）as a novel inositol 1,4,5−trisphosphate binding protein, and subsequently found GABARAP （GABAA receptor associated protein）as one of binding partners that binds to γ2 subunit of the receptor and thus is implicated in the clustering and trafficking of the receptor to synaptic membrane. Further studies revealed that PRIP binds β subunit of the receptors and PP1c（catalytic subunit of protein phosphatase 1）. These findings have prompted us to explore the possible involvement of PRIP in modulation of GABAA receptor signaling. Here we summarize our current understanding regarding how PRIP is involved in the modification of GABAA receptor signaling on the basis of the characteristics of these interacting molecules.
Introduction The enhancement of neuronal inhibition by GABA is one of the most powerful therapeutic strategies for the treatment of central nervous system diseases. For instance, benzodiazepines （BZ）, whose targets are GABAA receptors, have widely been used clinically for anxiety, sleep disorder, muscle stiffness and so on, the therapeutic effects of which are derived by §
Corresponding author E−mail：[email protected]−u.ac.jp
increasing the affinity of the receptor for GABA and recruiting more GABAA receptors for activation by the ligand1）. Therefore, it is very important to understand the molecular mechanisms regarding the life cycle of GABAA receptors including molecular organization, transport and insertion to the surface membrane, functional modulation on the surface membrane, and internalization followed by recycling back to the surface membrane or lysosomal degradation. Recently, a number of reports have been published regarding the molecules involved in the dynamic regulation of GABAA receptor function2）. PRIP−1, originally identified as a novel D−myo−inositol 1,4,5−
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Fig. 1 Schematic illustration of GABAA receptor structure Left：Subunit structure of GABAA receptors with four transmembrane domains and extracellular N− and C−termini. The large amino−terminal domain located extracellularly, is believed to accommodate neurotransmitter, GABA and some modulator chemicals. The intracellular domain between TM3 and TM4 is the most divergent part of individual receptor subunit and contains numerous consensus sites for the action of both serine／threonine and tyrosine protein kinases. Right：Heteropentameric GABAA receptors forming Cl− channel. TM2 is believed to form the lining of the ion channel. Addition of the ligand triggers a small rotation of the extracellular domains of the receptor subunits, which then opens the channel pore formed by the adjoining TM2 regions of the five subunits.
trisphosphate binding protein, is one such molecule, and was found to be involved in GABAA receptor signaling3）. In this article we discuss the involvement of PRIP molecules in GABAA receptor signaling. Structure of GABAA Receptor and Its Subunit Diversity GABAA receptors are the major target of the endogenous inhibitory neurotransmitter, GABA, and mediate the bulk of fast inhibitory neurotransmission in the mammalian brain. They are heteropentamers and each subunit shares a conserved structure. A large amino−terminal domain is located extracellularly and is believed to accommodate a neurotransmitter and some modulators （Fig. 1）. The receptor subunits also contain four hydrophobic transmembrane （TM）domains and a large intracellular loop between TM domains Ⅲ and Ⅳ. This domain is the most divergent part of individual receptor subunits and many receptor−associated proteins are reported to interact with this loop. Also, this domain contains numerous consensus sites for the action of both serine／threonine and tyrosine protein kinases4）. GABAA
receptor subunits are classified by sequence identity into seven subunit classes with multiple subtypes： α1−6, β1−3, γ1−3, δ, ε, θ, and π. The receptor characteristics largely depend on the subunit composition of individual GABAA receptors5）. However, receptors containing α1, 2, 3, or 5, subunits in combination with any of the β subunits and γ2 subunits with a ratio of 2：2：1 are most prevalent in the brain2,6）. The expression pattern of individual subunits in a spatio− temporal−dependent manner generates a high diversity of GABAA receptors with different functional properties. For example, the inclusion of a γ2 subunit into the pentamer is essential to produce receptors that are sensitive to BZ−type drugs that are widely used for anxiolytic, hypnotic, anticonvulsant, and muscle−relaxing actions7）. A small population of GABAA receptors that are present mainly in the cerebellum and thalamus contain the δ subunit5）, which is known to be insensitive to BZs, in a similar way to those containing α4 or α6 subunits1,9）. There have been several reports on gene targeting experiments for some subunits using mice. Gene knockout of the α1 or β2 subunit showed neither a lethal phenotype nor epileptic seizures, although the
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number of GABAA receptors on the surface membrane was reduced to 50％8）. On the other hand, targeted disruption of the γ2 subunit gene caused 94％ reduction of the BZ sites and exhibited a perinatally lethal phenotype in the majority of mutant mice7）. Most of the β3 subunit knockout mice also died in the neonatal period10）. Therefore, the presence of these two subunits, β3 and γ2, is absolutely required for GABAA receptors to function in maintaining the animal life itself, while modulation of other subunit compositions would provide changes in the functional characteristics of GABAA receptors, thus causing the impairment of behavioral events and a pharmacological effect. PRIP as a GABAA Receptor−associated Protein A family of phospholipase C−related, but catalytically inactive protein（PRIP） , have been identified as a novel D−myo−inositol 1,4,5−trisphosphate−binding protein with a domain organization similar to phospholipase C−δ but lacking enzymatic activity11―17）. The PRIP family consists of at least two types of proteins, PRIP−1, and PRIP−218）. PRIP−1 is expressed predominantly in the central nervous system, while PRIP−2 has broad tissue distribution, including the brain19,20）. We have applied a yeast two−hybrid screening system to explore the interacting molecules with PRIP and identified two molecules；the catalytic subunit of protein phosphatase 1α （PP1α）21） and GABARAP （GABAA receptor associated protein）3）. GABARAP is a molecule that interacts specifically with the γ2 subunit of GABAA receptor and has been implicated in the clustering of GABAA receptors and trafficking of receptors to the cell surface because of its ability to interact with microtubules22） and N−ethylmaleimide sensitive factor23）. The region of GABARAP responsible for binding to the γ2 subunit intracellular loop is that comprising residues 36―68, which is shared by PRIP, indicating the competition for the γ2 2）. These findings led us to subunit 22,24）（Fig. explore the possible involvement of PRIP in GABAA receptor function. Furthermore, in the course of experiments, we have also become aware that PRIP
Fig. 2 Interaction of GABARAP with PRIP and the γ2 subunit of the GABAA receptor PRIP and the γ2 subunit bind to GABARAP in a competitive manner. Numbers indicate the residues responsible for the interaction.
directly binds with the β subunits of GABA A receptors25,26）and protein phosphatase 2 A（PP2A）26）. These binding partners and their function further support the notion that PRIP as a GABAA receptor−associated protein, in collaboration with the above−mentioned molecules, plays an important role in receptor signaling. GABARAP in Membrane Trafficking of GABAA Receptors Before the description of the possible roles of PRIP, we briefly summarize the roles of GABARAP in GABAA receptor trafficking. The assembly and delivery of multiple GABAA receptor subtypes in a single cell and their extracellular signal−controlled delivery to different neural surfaces requires sophisticated control steps at the level of assembly and sorting into distinct trafficking vesicles as well as regulated and directed transport to their final destination. GABARAP is a possible candidate involved in some steps of these GABAA receptor assembly, sorting and trafficking processes, because GABARAP is a member of a protein family that includes microtubule−associated , the Golgi−associprotein light chain 3（MAP−LC3） ated ATPase enhancer of 16kDa（GATE−16）, and the
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yeast orthologue Atg8, all of which are reported to participate in membrane trafficking28,29）. GABARAP was first identified as the molecule responsible for clustering of GABAA receptors at the post−synaptic membranes 22）, but the idea has been recently amended to that GABARAP plays an important role in receptor trafficking, especially the γ2 subunit−containing type, to the surface membrane30,31）. However the precise molecular mechanisms underlying GABARAP−dependent transport of γ2 subunit−containing receptors remain unclear. Gene−targeting experiments of GABARAP in mice indicated no impairment in receptor clustering, supporting the notion that GABARAP is not implicated in receptor clustering32）, and no further reports regarding other phenotypes relating to GABAA receptor signaling of these mice have appeared. Analysis of PRIP Knockout Mice To examine the possible roles of PRIP interacting with GABARAP and GABAA receptor β subunits in the regulation of the receptor function, we analyzed PRIP−1 knockout（1−KO）mice from electrophysiological and behavioral aspects in combination with the effects of BZ3）. The GABAA receptor−mediated Cl− current（IGABA）in freshly isolated hippocampal CA1 cells from wild−type（WT）and PRIP−1−KO showed little difference in terms of the dose−response relationship；however, there appeared to be some dysfunction in BZ action. The BZ agonist, diazepam, reversibly potentiated IGABA elicited by GABA in a dose−dependent manner in neurons from WT mice. In contrast, the potentiation of IGABA was markedly reduced in neurons from PRIP−1−KO mice. Behavioral analysis also supported the results obtained by electrophysiological analysis. We examined the anxiolytic effects of diazepam on WT and PRIP−1−KO mice by the elevated plus−maze test. No significant difference in the time spent in the open or closed arms of the maze was apparent between WT and PRIP−1−KO mice injected with vehicle. However, a clear difference was observed when mice were injected with diazepam；injection of WT mice resulted in a marked increase in the time spent in the
open arm, indicative of the anti−anxiety effect of this drug. In contrast, the time that PRIP−1−KO mice spent in the open arm was not affected. These results clearly showed reduced sensitivity to diazepam in PRIP−1−KO mice, probably caused by the reduced number of γ subunit−containing receptors on the surface membrane. These results were supported by binding assays using radioactive ligands specific to GABA and BZ sites. As described above, the study was initiated by the finding that PRIP−1 interacts with GABARAP in a competitive manner with the γ2 subunit of GABAA receptors. On the basis of the finding that GABARAP facilitates the membrane transport of γ2−containing receptors to the cell surface30,31）, we predicted that the gene knockout of PRIP−1, which would eliminate PRIP−dependent competition of GABARAP binding to GABAA receptors, would result in an increased cell surface expression of γ2 subunit−containing receptors；however, the results obtained were opposite as . We those described（we called it“the γ−paradox”） thought that gene compensation at the level of PRIP− 2 proteins, which is also expressed in the brain, might account for the“γ −paradox”since PRIP−2 also binds to GABARAP. PRIP−1 and −2 double knockout ；however, mice have become available recently26） they have exhibited essentially similar phenotypes as type 1−KO mice regarding GABAA receptor function, although there are several small distinct differences33）. We found the direct association of PRIP with β subunits of GABAA receptors25,26）, and recently mapped the region responsible for the interaction 26） , （amino acid residues 544―568 of rat PRIP−1） which is located remote from the binding region of GABARAP；therefore, we confirmed ternary complex formation among the receptor β subunit, PRIP− 1, and GABARAP. Furthermore, disruption of this direct interaction by the peptide mimicking the binding site inhibited cell−surface expression of γ2 subunit−containing GABAA receptors, which resembled the phenotype of PRIP−DKO mice. Considering these results, we propose that the formation of the ternary complex mentioned above would facilitate the association of GABARAP with the γ2 subunit to be transported at the right place at the right time, or an
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association between the β subunit and PRIP would promote the association between the γ2 subunit and GABARAP, and thus facilitate the cell surface expression of the γ2 subunit−containing GABAA receptors （Fig. 3）；however, the precise molecular mechanisms remain to be elucidated33）. It should be also noted that possible involvement of PRIP, relating to the interaction with PP1 and PP2A, in the phospho− regulation of GABAA receptors has been recently reported25―27）. PRIP in Epilepsy Epilepsy and seizures are closely linked to the function of GABAA receptors in the brain34,35）. PRIP−1 is distributed mainly in the brain and expressed abundantly in the cerebral cortex and hippocampus20）. These brain regions are closely linked to epilepsy and seizure34,35）. Therefore, we investigated the effects of pentylenetetrazol（PTZ）, a chemical convulsant that interacts with the GABAA receptor, in mice lacking PRIP−1. PRIP−1−KO mice did not develop spontaneous behavioral seizure；however, the KO mice had markedly shorter latencies until the first clonic convulsion and tonic extensor following PTZ administration and increased incidence of convulsion compared to WT. Furthermore, the mortality rate by PTZ in KO mice was also significantly increased in comparison with WT36）. A point mutation in the GABAA receptor γ2 subunit was found in patients suffering from childhood absence epilepsy and febrile seizures37）. Electrophysiological experiments introducing such a point mutation into the γ subunit of GABAA receptors using oocytes demonstrated that the response to GABA was not different from those observed in the control receptors. However, the mutant receptors showed little potentiation by diazepam of GABA−induced Cl− current, as those observed in PRIP−1−KO mice. These findings strongly support that mice lacking PRIP−1 were hypersensitive to PTZ−induced convulsion, and that PRIP−1 might play a role in suppressing excessive excitability in neuronal activity via interactions with the GABAA receptor. Investigation into the functional roles of PRIP may elucidate novel
Fig. 3 Trafficking of GABAA receptors to the cell surface The trafficking of GABAA receptors within the secretory pathway is facilitated by GABARAP, which binds directly to γ2 subunits regulating the delivery of γ2 subunit containing receptors to the cell surface membrane. PRIP might be involved in these processes by regulating the function of GABARAP.
pathophysiological mechanisms of epilepsy and seizure. Summary PRIP was originally identified as a novel inositol 1,4,5−trisphosphate binding protein11―17,37）. Further studies to explore binding partners besides an inositol 1,4,5−trisphosphate have unexpectedly led us to examine the possible involvement of PRIP in GABAA receptor signaling, especially in those containing the γ2 subunit3,21,25―27,33,38）. GABAA receptors are the major target of the endogenous inhibitory neurotransmitter（GABA）and have been implicated in a variety of brain functions including sedation, hypnosis, anxiety, learning and memory4―6）. In particular, the receptors containing the γ2 subunit are the target of the BZ type, which are widely used clinically as anxiolytics, hypnotics, anticonvulsants, and muscle relaxants1）.
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For efficient inhibitory synaptic transmission and drug effects, it is critical that the receptors, especially those containing the γ2 subunit, are correctly transported from their site of synthesis in the endoplasmic reticulum to the appropriate synaptic or extrasynaptic site4,6）. A large amount of work has been dedicated to understanding how these processes are precisely orchestrated by multiple receptor−associated molecules. Analysis of PRIP−DKO mice suggest that PRIP plays an important role in the membrane trafficking of γ2 subunit−containing GABAA receptors, presumably by facilitating the delivery of GABARAP to the γ2 subunit through the association with the β subunits. Our findings that PRIP is implicated in the processes for expressing γ2 subunit−containing receptors, would provide a novel aspect in the complicated system of the dynamic regulation of GABAA receptors in the inhibitory synapses. Acknowledgements This work was supported by a Grant−in−Aid for Scientific Research from the MEXT of Japan（AM, TK and MH）and by the Cooperative Study Program of National Institute for Physiological Sciences（TK and MH）. Kato Memorial Bioscience Foundation （TK）, the Naito Foundation（TK）and Takeda Science Foundation （TK）. AM is a JSPS research fellow and a recipient of the Iwadare Scholarship.
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