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Membrane Signalling Complexes Neuroscience Group Colloquium Organized and Edited by T. S. Sihra (Department of Pharmacology, University College London) and S. J. Moss (Department of Pharmacology, University College London). 668th Meeting held at the University of Glasgow, 7-9 April 1999.
Synaptic targeting and regulation of GABA, receptors N. J. Brandon*, F. K. Bedford*, C. N . Connolly*, A. Couve*, J. T. Kittler*, J. G. Hanley*, J. N. Jovanovic*, J. Uren*, P. Taylor*, P. Thomast, T. G. Smart.(- and S. J. Moss*' *Medical Research Council Laboratory of Molecular Cell Biology and Department of Pharmacology, University College London, London W C I E 6BT, U.K. and +Department of Pharmacology, The School of Pharmacy 29-39 Brunswick Square, London W C I N I AX, U.K.
synaptic sites. Furthermore, understanding the endogenous mechanisms used by neurons to regulate the function of these receptors may reveal novel therapeutic means of modulating the efficacy of inhibitory synaptic transmission.
Introduction Ionotropic y-aminobutyric acid type A (GABA,) and C (GABA,) receptors are the major sites of fast synaptic inhibition in the brain. GABA, receptors also represent the major sites of action in the brain for benzodiazepines, barbiturates and anaesthetic steroids based on pregnenalone. These receptors are members of the ligand-gated ion channel superfamily, which includes glycine (GlyR), nicotinic acetylcholine (AChR), and SHT, receptors [ l ] . GABA, and GABA, receptors have different pharmacological and physiological properties which reflect distinct subunit compositions [ 2-41. GABA, receptors are believed to be heteromeric or homomeric assemblies of pl-3 subunits [4,5].In contrast, GABA, receptors are more structurally diverse and can be constructed from six subunit classes with multiple members: a(1-6), p(1-3), y ( l - 4 ) , 6 , E and 72. [2,3,6,7].Localization experiments have revealed a large spatial and temporal variation in subunit expression, with many individual neurons expressing multiple numbers of subunits [2,3,8]. Given the critical importance of GABA, receptors in mediating synaptic inhibition, it is of fundamental importance to understand how these receptors are assembled and targeted to post-
Assembly and targeting of ionotropic GABA receptors to the cell surface Clearly, to understand the diversity of GABA, receptors expressed on the cell surface of neurons it is important to gain some insight into how these receptor subunits are assembled. T o this end, the assembly of recombinant receptors has been analysed. These studies have focused on receptors composed of a l , a 2 and y2 subunits in heterologous systems, as this subunit combination is widely expressed in the adult brain [2,3]. From these studies, it is evident that receptor assembly occurs within the endoplasmic reticulum (ER) and access to the cell surface is limited to receptors composed of a l p and apy2 subunits. Single subunits, and the combinations a l l y 2 , and p 2 / y 2 are retained within the ER where they are subject to rapid degradation [9, l o ] . Similar studies using viral expression, have revealed that ER retention plays a prominent role in controlling receptor assembly in neurons [ l o ] . Electrophysiological studies have established that co-expression of a and subunits produces GABA-gated channels with high zinc sensitivity, but co-expression with the y2 or y3 subunits is essential for benzodiazepine sensitivity [2,3]. However, how subunits interact selectively to produce GABA, receptors with defined subunit stoichiometry remains to be
Abbreviations used AChR, nicotinic acetylcholine receptor CAMKII, Cdcalrnodulin-dependent protein kinase, ER, endoplasrnic reticulum, GABA, y-aminobutync acid, GABARAP. GABA receptor anchoring protein, GlyR glycine receptor, MAP, rnicrotubule associated protein, PKA. protein kinase A , PKC, protein kinase C, PKG, protein kinase G 'To whom correspondence should be addressed
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established. Studies on the related AChRs and GlyRs have established that subunit interaction is controlled by ‘assembly signals’ within the Nterminus of receptor subunits [11,121. T o identify assembly signals within GABA, receptors the cell surface expression of splice variants of the a6 subunit has been examined. Cerebellar granule cells express two variants of this protein termed a6S and a6L, that differ by the absence of residues 58-67 in the N-terminus of a6S [13]. T h e a6S subunit is unable to reach the cell surface when expressed with p3, explaining the lack of functional expression seen previously with this subunit [13]. Deletion of these residues from the a1 subunit produces a similar blockade of functional cell surface expression, suggesting that residues 58-67 play a critical role in the assembly of all a subunit isoforms. In the case of the a1 subunit, deletion of residues 58-67 blocks the ability of a1 to oligomerize with 83 without affecting oligomerization with the y2 subunit. Selective mutagenesis revealed that two residues, Gln67 and an adjacent residue Ser68, were critical in mediating oligomerization of a1 and p3. Using sucrose density gradient centrifugation, mutation of these residues abolished formation of an al//?3 complex, sedimenting at 9 S, that represents functional cell surface receptors [10,14]. T o gether, these results suggest that residues Gln67 and Ser68 comprise an assembly signal conserved in all GABA, receptor a subunits that mediates specific oligomerization with receptor #? subunits. Similar studies have identified assembly signals that mediate oligomerization of receptor p and y2 subunits [15], and experiments are currently underway to identify signals controlling a/y subunit interactions. Together these studies are beginning to identify specific signals within GABA, receptor subunits that mediate specific subunitsubunit interactions. This information will be useful in placing boundary limits on GABA, receptor diversity in the brain, and is also revealing useful information on receptor structure.
cytoskeleton [16]. Gephyrin for example, binds to the intracellular domain of the GlyR receptor /? subunit linking these receptors to microtubules, enabling receptor clustering at synaptic sites [17,18]. Recently, a role for gephyrin in the clustering of GABA, receptors has been suggested : disruption of gephyrin expression using antisense oligonucleotides has been reported to partially block GABA, receptor clustering in cortical neurons [19] ; however, a direct interaction of gephyrin with GABA, receptors has yet to be demonstrated. T o search for novel molecules that interact with the intracellular domains of GABA, and GABA, receptor subunits, the yeast two-hybrid methodology has been used [20]. Using the y2 subunit intracellular domain as a bait, a novel protein GABA receptor anchoring protein (GABARAP), has been isolated that interacts specifically with the y2 subunit, but not with GABA, receptor a or p subunit isoforms [21]. This 17 kDa protein, shares homology with the light chain 3 of microtubule associated proteins (MAP) 1A and 1B [21]. GABARAP has a ubiquitous distribution and has the ability to bind to microtubules in vitro [21]. GABARAP also colocalizes in distinct clusters with GABA, receptors in cultured neurons ; furthermore, co-expression of GABARAP and heteromeric GABA, receptors containing the y2 subunit leads to the aggregation of the expressed receptors into large cell surface clusters in mouse L cells (F. K. Bedford, R. Olsen and S. J. Moss, unpublished work). Therefore, GABARAP would appear to be a good candidate molecule for controlling GABA, receptor interactions with the cytoskeleton. Further studies will be needed to establish whether GABARAP is sufficient to mediate the synaptic localization of GABA, receptors. I t will also be of interest to compare the roles of gephyrin and GABARAP in mediating the synaptic targeting/anchoring of GABA, receptors: for example, do these molecules act on differing subunit combinations of GABA, receptors or do they act in synergy to control receptor targeting and/or clustering ? By using the intracellular domain of the GABA, receptor p l subunit as a bait, a strong interaction with a 290 amino acid domain of MAP1B has been identified. This interaction also occurs between GABA, receptors containing the p l subunit and full-length MAP-1 B in retinal tissue as determined by co-immunoprecipitation [22]. Interestingly the interaction of MAP-1 B is specific to the p l subunit as GABA, receptor subunits do not co-immunoprecipitate with MAP-1 B from
Synaptic targeting of GABA receptors: identification of molecules that interact with receptor subunits For efficient synaptic transmission, it is critical that ionotropic GABA receptors are localized precisely to synaptic sites. For other members of the ligand-gated ion channel superfamily this is achieved by ‘ anchoring proteins ’ that interact with subunit intracellular domains between transmembrane domains 3 and 4, linking them to the
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retinal extracts [22]. Using in vitro binding assays the role of MAP-1B in promoting the interaction of GABA, receptors with the cytoskeleton has been examined. The intracellular domain of the p l subunit is capable of binding to both actin and tubulin in vitro in a MAP-1B-dependent manner [22]. Therefore, these results strongly suggest that MAP-1B serves as an anchor to link GABA, receptors to the cytoskeleton. Furthermore, MAP1B and GABA, receptors precisely co-localize at GABAergic axo-axonic synapses between amacrine and bipolar neurons in the retina [23,24]. Finally co-expression of MAP-1B and the p l subunit in COS cells leads to the accumulation of the p l subunit as intracellular aggregates along with MAP-1B [22]. Together these observations demonstrate a novel role for MAP-1 B in the targeting or clustering of GABA, receptors. Blocking the pl-MAP1B interaction in bipolar cell neurons using inhibitor peptides directed against the MAP-1B binding site on the p l subunit [22], should clarify further the role of MAP-1B in controlling the localization of GABA, receptors. The specificity of the interaction of MAP-1B with GABA, receptors but not GABA, is also of significance. This may explain the differential subcellular distribution of these two distinct classes of inhibitory receptors in retinal neurons [24,25].
pending on the identity and location of the sites phosphorylated [26,27]. Similar broad effects of protein kinase activation have been reported on GABA, receptor function in a range of neuronal preparations [26]. In order to address how protein kinases are targeted to GABA, receptors, the intracellular domains of defined receptor subunits have been used as affinity matrices to search for interacting signalling molecules. Using the /31 subunit intracellular domain a serine/threonine protein kinase was found to interact with this protein but not with the intracellular domains of the a1 or y2 subunits in brain lysates. This kinase specifically phosphorylates Ser409 within the fil subunit intracellular domain. Phosphorylation of pl was blocked by specific peptide inhibitors of PKC, but was unaffected by phorbol esters or inhibitors of PKA and CAM KII. PKC activity could also be detected binding to both the p2 and the 83 subunit intracellular domains. Using a panel of antisera against defined PKC isoforms, PCK-PI I could be detected binding to the GABA, receptor pl subunit intracellular domain but not to comparable domains of the a1 or y2 subunits. PKC-PI1 is often targeted to substrate proteins by the receptor for activated C-kinase (RACK-1). Therefore, the interaction of RACK-1 with the GABA, receptor pl subunit intracellular domain was tested in brain extracts with specific antisera. RACK-1 bound to the pl subunit intracellular domain and interestingly also to that of the a1 subunit but not to that of the y2 subunit. The direct binding of RACK-1 to the pl subunit intracellular domain was confirmed using gel overlay assays. Using similar methodology PKC-PI1 was also found to directly bind to the pl subunit intracellular domain but not to that of a l . Using deletion analysis, the binding sites for RACK-1 and PKC-jl were found to be close together and Nterminal to the major PKC phosphorylation site within the pl subunit, Ser409 [28,29]. The interaction between PKC-p and RACK1 with whole GABA, receptors has also been analysed. Both PKC-PI1 and RACK-l co-immunoprecipitate with GABA, receptors composed of a l , pl and y2 subunits expressed in A293 cells. Similar association of RACK-1 and PKC-P could also be observed in cultures of cortical neurons by immunoprecipitation. T o analyse the effects of RACK-I and PKC-PI1 on GABA, receptor function the effects of blocking RACK- 1 binding on receptor modulation by PKC activity was analysed in A293 cells. Previous studies using
Regulation of GABAA receptors: targeting protein kinases to GABA, receptors Manipulation of GABA, receptor function has been widely exploited clinically ; however, the endogenous mechanisms used by neurons to regulate receptor function remain largely unknown. Much emphasis has been placed recently on the role of direct receptor phosphorylation. Studies using recombinant receptors have revealed that the intracellular domains of receptor pl-3 and y2 subunits are the substrates for a range of protein kinases including protein kinase A (PKA), protein kinase C (PKC), Ca/calmodulin-dependent protein kinase, (CAM KII), cGMP-dependent protein kinase G, (PKG) and the prototypic tyrosine kinase SRC [26,27]. The p subunits are of special importance as kinase substrates, because a conserved residue in all p subunit isoforms (Ser409 in Bl), is a substrate of PKA, PKC, CAM K I I and PKG both in vitro and in vivo [26,27]. The effects of phosphorylation on recombinant receptor function are diverse, ranging from enhancements to inhibitions de-
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I I Green, W. N. and Millar, N. S. (I 995) Trends Neurosci. 18, receptors composed of al//.?l subunits in this 28&287 system have shown that PKC activation using 12 Gu, Y., Carnacho, P.. Gardner, P. and Hall, Z. W. ( I99 I ) phorbol esters inhibits IGABA, via phosphorylation Neuron 6,879-887 of Ser409 in /.?1 [28,29]. Intracellular dialysis with 13 Korpi. E. R, Kuner, T., Kristo, P.. Kohler, M., Herb, A., peptides corresponding to the RACK-1 binding Luddens, H. and Seeburg, P. H. (I994) J. Neurochern 63, site within /31 subunit, reduced the inhibitory 1167-1 I70 I 4 Tretter, V., Ehya, N., Fuchs, K. and Sieghart, X. (I 997) scrambled effects of PKC activation upon ICABA; J. Neurosci. 17,2728-2737 peptides were without effect. 15 Taylor, P., Thomas, P., Gome, G. H., Smart, T. G. and Moss, Together these results identify a mechanism S. J. ( 1999) J. Neurosci. in the press for targeting PKC activity to GABA, receptors. I 6 College, G. and Froehner, S. ( 1998) Proc. Natl. Acad. Sci. Studies are currently in progress to determine U.S.A. 95, 334 1-3343 I 7 Prior, P. et al. ( 1992) Neuron 8, I I 6 I - I I70 which cell signalling pathways control the inI 8 Kirsch, J., Woken, I.,Triller, A. and Betz, H. (I 993) Nature teraction of both RACK-1 and PKC-/.?I1 with (London) 366,745-748 GABA, receptors in neurons.
19 Essrich, C., Lorez. M., Benson, J. A., Fritschy,J-M. and Luscher, 6. ( I 998) Nature Neurosci. 7, 563-572 20 Field, S. and Song, 0.(I 989) Nature (London) 340, 245-246 21 Wang, H. W., Bedford, F. K., Brandon, N. J., Moss, S. J. and Olsen, R ( I 999) Nature (London) 397, 69-72 22 Hanley, J. G., Koulen, P., Bedford, F. K., Gordon-Weeks, P. R and Moss, S. J. ( 1999) Nature (London) 397, 66-69 23 Tachibana, M. m d Kaneko, T. ( I 987) Proc. Natl. Acad. Sci. U.S.A. 85, 350 1-3505 24 Koulen, P., Brandstatter, J. H., Enz, R and Wassle, H. ( I 998) Eur. J. Neurosci. 10, I 15- I27 25 Enz, R, Brandstatter, J.H. and Bormann, J. (I 996) J. Neurosci. 16,44794490 26 Moss, S. J. and Smart, T. G. ( I 996) Int. Rev. Neurosci. 39, I42 27 McDonald, 6. J,, Arnato, A,, Connolly, C. N., Benke, D., Moss, S. J. and Smart, T. G. (I998) Nat. Neurosci. I,23-27 28 Moss, S. J., Doherty, C. A. and Huganir, R L. (1992) J. Biol. Chern. 267, 14470- I4476 29 Krishek 6. J., Blackstone, C. D.. Huganir, R L, Moss, S. J. and Smart, T. G. ( 1994) Neuron 12, I08 I -I 095
This work was supported by the Medical Research Council (U.K.) and The Wellcorne Trust. I Unwin, N. ( I 993) Cell 72(Suppl.), 3 I 4 I 2 Macdonald, R L and Olsen, R W. ( I 994) Annu. Rev. Neurosci. 17,569-602 3 Rabow, L E.. Russek S. J. and Farb, D. H. ( 1995) Synapse 2 I, 189-274 4 Lukasiewicz, P. D. ( 1996) Mol. Neurobiol. 12, I 8 I - I94 5 Cutting, G. R, Lu, L, O'Hara, 6. F., Kasch, L. M.. MontroseRafizadeh, C., Donovan, D. M., Shirnada, S., Antonarakis, S. E., Guggino, W. 6. and Uhl , G. R (I 99 I) Proc. Natl. Acad. Sci. U.S.A. 88, 2673-2677 6 Davies, P. A., Hanna, M. C., Hales, T. G. and Kirkness, E. F. ( I 997) Nature (London) 385, 82G823 7 Hedblorn, E. and Kirkness, E. F. ( I 997) J. Biol. Chern. 272, I 5346- I 5350 8 Laurie, D. J., Wisden, W. and Seeberg, P. H. ( I 992) J. Neurosci. 12,41514172 9 Connolly, C. N., Krishek 6. J.. McDonald, 6. J., Smart, T. G. and Moss, S. J. ( 1996) J. Biol. Chern. 27 I,89-96 10 Gome, G. H.,Vallis, Y., Stephenson, A,, WhMeld, J,, Browning, B., Smart, T. G. and Moss, S. J. (1997) J. Neurosci. 17, 6587-6596
Received 9 March 1999
GABA, receptors function as heterodimers F. H. Marshall, J. White, M. Main, A. Green and A. Wise Receptor Systems, Molecular Pharmacology Unit, GlaxoWellcome Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG I 2NY U.K.
Introduction
by ligand gated chloride channels (GABA, and GABA, receptors) and by G-protein-coupled receptors (GABA, receptors). The GABA, receptor was first described by Bowery et al. [l] almost 20 years ago as a bicuculline-insensitive GABA receptor which was selectively activated by baclofen. This receptor occurs both pre-synaptically, where its major function is to regulate neurotransmitter release via blockade of voltagegated calcium channels, and post-synaptically where it mediates a long lasting hyperpolarization
y-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system. The actions of GABA are mediated both Abbreviations used: CFTR cystic fibrosis transrnernbrane regulator: CGRP, calcitonin gene-related peptide: CRLR, calcitonin receptor-like receptor: EST, expressed sequence tag; GABA, yarninobutyric acid: GIRK, G-protein-coupled inwardly rectifying K+ channel;GPCR G-protein-coupled receptor; HA, haernagglutinin; RAMP, receptor activrty modifying protein.
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