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Identification of subunits mediating clustering of GABAA receptors by rapsyn
Neurochemistry International 34 (1999) 453±463
Identi®cation of subunits mediating clustering of GABAA receptors by rapsyn V. Ebert, P. Scholze, K. Fuchs, W. Sieghart* Section of Biochemical Psychiatry, University Clinic for Psychiatry, WaÈhringer GuÈrtel 18±20, A-1090, Vienna, Austria Received 1 March 1999; accepted 4 March 1999
Abstract Human embryonic kidney 293 cells transfected with a1b1g2, a1b2g2, a1b3g2, a1b1, a1b2, a1b3, b3g2, or b3 subunits formed gaminobutyric acidA receptors on the cell surface that could be clustered by rapsyn. In contrast, a1, b1, b2, or g2 subunits, or a1g2 subunit combinations could not be detected on the surface of transfected cells and could not be clustered by rapsyn. Experiments investigating the ability of rapsyn to cluster chimeras consisting of the N-terminus of the b3 subunit and the remaining part of the a1, b2 or g2 subunits indicated that the intracellular domains of b1, b2, b3, or g2 subunits, but not those of a1 subunits are able to form sites mediating clustering by rapsyn. These results demonstrate that rapsyn has the potential to cluster the majority of GABAA receptor subtypes via b or g2 subunits. Further experiments will have to clarify the physiological importance of this observation. # 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction Gamma-Aminobutyric acid type A (GABAA) receptors are ligand gated chloride ion channels and the site of action of a variety of clinically important drugs (Sieghart, 1995). These receptors are composed of ®ve subunits (Nayeem et al., 1994; Tretter et al., 1997). Each subunit consists of an N-terminal extracellular region, followed by four transmembrane segments (TM) and a large intracellular loop between TM3 and TM4 (Scho®eld et al., 1987). At least 18 dierent GABAA receptor subunits (a1±6, b1±4, g1-3, d, E, r1±3) so far have been identi®ed in vertebrate brain, that give rise to a large variety of GABAA receptor subtypes with distinct subunit composition (Barnard et al., 1998; Macdonald and Olsen, 1994; Sieghart, 1995). Depending on their subunit composition, these receptors exhibit distinct electrophysiological and pharmacological properties. Expression studies have indicated that a, b and g subunits have to combine to form * Corresponding author. Tel.: +43-1-40400-3572; fax: +43-140400-3629. E-mail address: [email protected] (W. Sieghart)
GABAA receptors with a pharmacology resembling that of native receptors (Sieghart, 1995). The precise localization of receptors in the postsynaptic membrane opposite transmitter release sites is crucial for synaptic function. In addition, the number and density of receptors in the postsynaptic membrane is one of the major determinants of the ecacy of neuronal transmission. GABAA receptors, therefore, are selectively clustered opposite nerve terminals releasing GABA (Craig et al., 1994; Todd et al., 1996). Several dierent proteins have been identi®ed that mediate clustering of transmitter receptors at synapses (Colledge and Froehner, 1998). For instance, the peripheral membrane protein rapsyn (43 kD protein) plays an essential role in clustering of the nicotinic acetylcholine (nACh) receptor of the muscle (Colledge and Froehner 1998; Phillips, 1995; Apel and Merlie, 1995). Gephyrin, a 93 kD protein, mediates clustering of the glycine receptor (Kirsch and Betz, 1995), and recently, at least two dierent proteins have been identi®ed that might be responsible for clustering of GABAA receptors. One of these proteins is the microtubule-associated protein 1B (MAP-1B) that clusters GABAC receptors containing r1 subunits (Hanley et
0197-0186/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 1 9 7 - 0 1 8 6 ( 9 9 ) 0 0 0 3 9 - X
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al., 1999). Since r subunits are structurally part of the family of GABAA receptor subunits, it was recommended that r-containing receptors should be classi®ed as a specialized set of the GABAA receptors (Barnard et al., 1998). The other protein (GABARAP) can interact with GABAA receptor g2 subunits and shows similarities to chain-3 of MAP-1A and 1B (Wang et al., 1999). Another study has indicated that gephyrine in addition to clustering glycine receptors is required for postsynaptic clustering of major GABAA receptor subtypes (Essrich et al., 1998). Gephyrin, however, copuri®ed with glycine but not with GABAA receptors and did not signi®cantly colocalize with GABAA receptors in heterologous expression systems (Kirsch et al., 1995; Meyer et al., 1995). Other results indicated that the degree of colocalization of gephyrin with GABAA receptors varied in dierent tissues (Craig et al., 1996; Todd et al., 1995; SassoeÁ-Pognetto et al., 1995). In agreement with the identi®cation of other proteins involved in clustering of GABAA receptors (Hanley et al., 1999; Wang et al., 1999) these results indicate that gephyrin might be involved in clustering of some but not all GABAA receptors. Interestingly, not only gephyrine but also rapsyn, another protein involved in clustering of a member of this receptor superfamily, induces clustering of a GABAA receptor subtype (Yang et al., 1997). In the present study, this observation was con®rmed and extended by demonstrating that rapsyn is able to cluster most, if not all recombinant GABAA receptor subtypes. In addition, GABAA receptor subunits mediating clustering by rapsyn were identi®ed using suitable subunit combinations or chimeric subunits. This information can now be used to identify the amino acid sequences mediating clustering by rapsyn and to identify proteins interacting with the respective sequences of native receptors. 2. Experimental procedures 2.1. Cloning of expression constructs The cDNA of a1, b1, b2, b3, or g2 subunits were subcloned into the mammalian expression vector pCDM8 (Invitrogen, San Diego, CA) as described previously (Fuchs et al., 1995; Jechlinger et al., 1998). The chimeric receptor subunits b3-a1 and b3-g2 with the subunit boundary immediately before TM1 were generated as described (Tretter et al., 1997). For construction of the b3-b2 chimera, the N-terminal fragment of the b3 subunit was generated from Hind III/Bgl II digestion of a b3 subunit cloned into the mammalian expression vector pcDNAI/Amp (Invitrogen), into which a Bgl II site had been introduced at the 5 ' end of TM 1
(Tretter et al., 1997). The b2-fragment spanning the region from TM1 to the C-terminus was ampli®ed by PCR using primers into which restriction sites had been introduced (Bgl II for the 5 ' end, Not I for the 3 ' end. Both fragments were ligated into a Hind III/ Not I digested expression vector pcDNAI/Amp (Invitrogen). The correct construction of the ®nal chimera was controlled by restriction enzyme cleavage and by sequencing across the chimera boundary. Mouse rapsyn cDNA was obtained from Stanley C Froehner (Department of Physiology, University of North Carolina) and was subcloned into the mammalian expression vector pCI (Promega Corporation, Madison, WI). The rapsyn with a myc-tag on its C-terminus cloned into the mammalian expression vector pRc/RSV (Invitrogen) was obtained from Jim Yeadon (Skirball Institute for Biomolecular Medicine, NYU Medical Center, New York). Large scale puri®cation for transfections was performed with the Quiagen plasmid puri®cation procedure (Qiagen, BRD). 2.2. Antibodies Antibodies a1(1±9) (Zezula et al., 1991) and b3(1±13) (Tretter et al., 1997) were generated as described. Whereas the a1(1±9) antibody was speci®c for the a1subunit (Jechlinger et al., 1998), b3(1±13) antibodies identi®ed b3 as well as b2, but no other GABAA receptor subunits. g2(1±33) antibodies were prepared using the fusion protein strategy described previously (Mossier et al., 1994). These antibodies were able to identify all g2-subunit containing recombinant GABAA receptors but in contrast to the a1(1±9) or b3(1±13) antibodies did not label a1b3-transfected cells (experiments not shown). The monoclonal antibody bd17, recognizing b2/3 subunits (Ewert et al., 1990) and the c-myc (9E10)-tag antibodies were purchased from Boehringer Mannheim (BRD). 2.3. Cell culture and transfection Human embryonic kidney 293 (HEK) cells (CRL 1573) from the American Type culture collection (Rockville, MD) were maintained in Dulbecco's modi®ed Eagle Medium (DMEM; Life Technologies, Grand Island, NY) supplemented with 50 mM b-mercaptoethanol, 10% fetal calf serum (Boehringer Ingelheim, BRD), 2 mM glutamine, 110 U/ml penicillin G, 100 mg/ml streptomycin and non essential amino acids (Life Technologies). NT-2 cells (Stratagene, La Jolla, CA) were maintained in Opti-Minimal Essential Medium with Glutamax-1 (Life Technologies) supplemented with 10% fetal calf serum (Boehringer), 110 U/ml penicillin G, 100 mg/ml streptomycin (Life Technologies). Cells were transfected with GABAA receptor subunit 2 rapsyn cDNAs by calcium phos-
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Fig. 1. Rapsyn induced clustering of a1b3g2 receptors. HEK cells were cotransfected with a1b3g2 subunits without (A) or with rapsyn (B). Receptors on the cell surface were detected by immuno¯uorescence and confocal laser microscopy after labeling with rabbit a1(1±9) and anti-rabbit IgG Bodipy FL antibodies (single sections). Essentially the same results were obtained when rabbit b3(1±13) or g2(1±33) antibodies were used for labeling of GABAA receptors. The experiment was performed seven times with similar results.
phate coprecipitation (Chen and Okayama, 1988) as described previously (Tretter et al., 1997), with a total of 21 mg DNA per 75 cm2 petri dish. Equal amounts of DNA of the dierent expression constructs were used for cotransfection experiments. For experiments performed in the absence of rapsyn DNA, a corresponding amount of empty vector DNA was added. 2.4. Immuno¯uorescence For immuno¯uorescence, cells were ®xed with 2% paraformaldehyde in phosphate-buered saline (PBS; 16 mM KH2PO4, 4 mM Na2HPO4, 150 mM NaCl; pH 7.4) 30±42 h after transfection, followed by a 10 min wash in 50 mM NH4Cl in PBS. Washes between incubation steps were performed in PBS. For detection of intracellular receptors or of the myctagged rapsyn, cells were permeabilized with 0.1% Triton X-100 for 5 min. Blocking was performed in 5% bovine serum albumin (BSA) in PBS for 10 min, followed by an incubation with primary antibody in 1% BSA in PBS. Primary antibodies generated in rabbits were detected with Goat Anti-Rabbit IgG(H+L) Bodipy FL (Molecular Probes, Eugene, OR), while monoclonal mouse antibodies were detected using Donkey Anti-Mouse IgG (H+L)Cy3 (Amersham International plc, UK) in 1% BSA in PBS. Labeling was visualized using a Zeiss Axiovert 135 M microscope attached to a confocal laser system (Carl Zeiss LSM 410, BRD), equipped with an argon laser and a
helium-neon laser and suitable ®lter sets. To verify that the labeling of cells without permeabilization was restricted to the cell surface, parallel samples were stained with antibodies directed against the intracellular loop of GABAA receptor subunits (experiments not shown). These antibodies detected GABAA receptor subunits only after permeabilization of transfected cells. Double labeling experiments for rapsyn and GABAA receptor subunits in most cases were performed after permeabilization of cells. Results obtained were compared with single labeling of parallel samples to demonstrate that the labeling pattern in the double labeling experiments was not caused by crossbleeding artifacts (experiments not shown). Similar results were obtained when the cells were ®rst labeled with the receptor speci®c antibody, were then washed, permeabilized, blocked again, labeled with the mycantibody, followed by a detection with a mixture of both secondary antibodies (experiments not shown).
3. Results 3.1. Clustering of a1b3g2-receptors Recently, it was demonstrated that a1, b3 and g2 subunits of GABAA receptors can assemble to pentameric receptors composed of all three subunits after cotransfection into HEK 293 cells (Tretter et al., 1997). As shown in Fig. 1A, these receptors appear
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Fig. 2. Colocalization of g2 subunits and rapsyn in the same clusters. HEK cells were cotransfected with a1b3g2 subunits and a myc-tagged rapsyn. Double labeling of permeabilized cells was performed using rabbit g2(1±33) antibodies (A) and a monoclonal mouse antibody against the myc-tag of rapsyn (B) followed by anti-rabbit IgG Bodipy FL and anti-mouse IgG Cy3. Receptors and rapsyn were detected in the same cell by immuno¯uorescence and confocal laser microscopy (single sections). The experiment was performed three times with similar results.
uniformly distributed on the cell surface of HEK cells upon detection with a1(1±9) antibodies. The small, brighter areas in Fig. 1A were interpreted as microvilli protruding from the cells, that due to their higher membrane density exhibit an apparently higher receptor density. Similar results were obtained when b3(1± 13) or g2(1±33) antibodies were used (experiments not shown). Only a low-level nonspeci®c signal was observed in non-transfected neighbouring cells, indicating that the antibodies used speci®cally recognized only GABAA receptor subunits and did not crossreact with other HEK cell proteins. When a1, b3 and g2 subunits were coexpressed with rapsyn, approximately 60% of the cells that were labeled by subunit-speci®c antibodies exhibited clustering of cell surface receptors (Fig. 1B). This is in agreement with previous observations indicating that rapsyn induces clustering in only a fraction of cells transfected with the nACh receptor (Yu and Hall, 1994). For double labeling experiments, GABAA receptor subunits were cotransfected with a myc-tagged rapsyn instead of the untagged version. This allowed simultaneous labeling of GABAA receptor subunits with antibodies generated in rabbits and labeling of rapsyn with the mouse monoclonal anti-myc-antibody. In agreement with results from a previous study (Yoshihara and Hall, 1993), essentially the same results were obtained when the tagged or the untagged rapsyn was used for cotransfection with GABAA receptor subunits (experiments not shown), indicating that the tag-
sequence fused to the C-terminus of rapsyn did not interfere with its clustering activity. Double labeling for g2 subunits and the myc-tagged rapsyn demonstrated a colocalization of GABAA receptors with rapsyn in the clusters (Fig. 2). Since rapsyn is a peripheral membrane protein associated with the inner plasma membrane, cells had to be permeabilized for rapsyn detection. Interestingly, the percentage of cells stained by the anti-myc-antibody was much smaller than that stained by g2(1±33) antibodies, although strongly clustered receptors could be identi®ed in many cells that were not stained by the antimyc-antibody (experiments not shown). This indicated, that detection via the myc-tag required a relatively high expression level of rapsyn in the cells. Coclustering was also visible, when a1(1±9) or b3(1±13) antibodies were used to detect GABAA receptors in combination with the anti-myc-antibody (experiments not shown). Furthermore, a colocalization of GABAA receptor subunits and rapsyn in the same clusters was also demonstrated when intact cells were ®rst labeled for GABAA receptor subunits, then permeabilized and stained for rapsyn (experiments not shown). Other double labeling experiments using a1(1±9) or g2(1±33) antibodies in combination with the monoclonal antibody bd 17, that speci®cally recognizes b2/3 subunits (Ewert et al., 1990), demonstrated that a1 and b3, or b3 and g2 subunits colocalized in the same clusters. Taken together, these results clearly demonstrated that rapsyn can induce clustering of a1b3g2 receptors in
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Fig. 3. Rapsyn is able to cluster a1b3 and b3g2, but not a1g2 subunit combinations. HEK cells were cotransfected with myc-tagged rapsyn and a1b3 (A±C), b3g2 (D±F), or a1g2 (G±I) subunits. a1 subunits were labeled on the cell surface (A,G) or in permeabilized cells (B,H) using rabbit a1(1±9) antibodies; g2 subunits were labeled on the cell surface (D) or in permeabilized cells (E) using rabbit g2(1±33) antibodies. Rabbit antibodies were detected using anti-rabbit IgG Bodipy FL antibodies. Rapsyn was detected using a mouse anti-myc antibody followed by anti-mouse IgG Cy3 (C,F,I). Immuno¯uorescence was investigated by confocal laser microscopy (single sections). The experiments were performed three times with similar results.
HEK cells and that all three GABAA receptor subunits colocalized with rapsyn in these clusters. 3.2. Subunit requirements for clusteringÐcoexpression of two subunits To investigate which subunits can be eliminated without disturbing the ability of rapsyn to cluster GABAA receptors, receptors composed of two dierent subunits were expressed. Receptors formed after transfection with a1 and b3 subunits were uniformly distributed on the cell surface (experiments not shown). When a1 and b3 subunits were cotransfected with rapsyn, clustering of receptors was induced (Fig. 3A). Double labeling for the a1 subunit and the myctagged rapsyn demonstrated the colocalization of these proteins in the same clusters (Fig. 3B,C). Essentially the same results were obtained using b3(1±13) anti-
bodies for detection of a1b3 receptors (experiments not shown). These data demonstrated that the g2 subunit is not essential for the clustering eect of rapsyn. When HEK cells were transfected with b3 and g2 subunits, again both subunits could be detected on the cell surface (experiments not shown). Upon cotransfection of b3 and g2 subunits with the myc-tagged rapsyn, clustering of receptors was induced (Fig. 3D). As expected, g2 subunits (Fig. 3E) or b3 subunits (experiments not shown) colocalized with rapsyn (Fig. 3F) in the same clusters. These results thus indicated that the a1 subunit, too, is not required for the clustering eect of rapsyn. Upon cotransfection of a1 and g2 subunits in the absence (experiments not shown) or presence (Fig. 3G) of rapsyn, no GABAA receptors could be detected on the cell surface. After permeabilization of the cells, however, both subunits were detected in intracellular
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Fig. 4. Rapsyn is able to cluster b3 subunits, but not a1 or g2 subunits transfected into HEK cells. HEK cells were cotransfected with myc-tagged rapsyn and b3 (A,B), a1 (C,D), or g2 (E,F) subunits. GABAA receptor subunits and the myc-tagged rapsyn were detected in permeabilized cells; b3, a1, or g2 subunits (A,C, or E) were labeled with rabbit b3(1±13), a1(1±9), or g2(1±33) antibodies, respectively, and were detected using anti-rabbit IgG Bodipy FL antibodies. Rapsyn was detected using mouse anti-myc antibody and anti-mouse IgG Cy3. Detection of GABAA receptor subunits and rapsyn was performed in the same cells in A and B, or C and D by immuno¯uorescence and confocal laser microscopy (single sections). Since the g2 signal was much weaker than the rapsyn signal in g2 transfected cells, in this case double labeling could not be performed in the same cell due to crossbleeding of the red rapsyn signal into the green g2 channel. Therefore, in Fig. 4E and F dierent cells are shown. The experiments were performed three times with similar results.
compartments (Fig. 3H, g2 subunits not shown). The same subcellular distribution of a1 and g2 subunits was observed in the absence or presence of rapsyn, indicating that coexpression with rapsyn did not change the subcellular distribution of subunits or redirect the subunits to the plasma membrane. In such cells, the myctagged rapsyn formed clusters (Fig. 3I) that were not colocalized with intracellular a1 (Fig. 3H) or g2 subunits (experiments not shown). 3.3. Subunit requirements for clusteringÐsingle subunits Recently, it has been demonstrated that b3 subunits can form homo-oligomeric receptors that are trans-
Fig. 5. Clustering of chimeric GABAA receptor subunits by rapsyn. (A) Structure of b3-a1, b3-g2, or b3-b2 chimera. Sequences derived from the b3 subunit are shown by a broken line; sequences derived from other subunits are shown grey (TM domains) and by continuous lines. (B±E) Cell surface labeling of the chimeric subunits b3-a1 (B,C) or b3-g2 (D,E) in HEK cells transfected with these chimeras without (B,D) or with rapsyn (C,E). Chimeras were detected by immuno¯uorescence and confocal laser microscopy using b3(1±13) antibodies and anti-rabbit IgG Bodipy FL (single sections). The experiment was performed ®ve times with similar results.
ported to the cell surface in the absence of any other GABAA receptor subunit (Slany et al., 1995; Connolly et al., 1996a). Upon transfection of HEK cells with b3 subunits, receptors formed were homogenously distributed on the cell surface (experiments not shown). Cotransfection with rapsyn induced clusters of b3 receptors (Fig. 4A), which colocalized with those formed by rapsyn (Fig. 4B). This indicates, that the b3 subunit contains a site that mediates clustering by rapsyn. Although the clustering site of the b3 subunit might have been responsible for clustering of a1b3g2, a1b3 and b3g2 receptors, additional sites on a1 or g2 subunits could exist that also mediate clustering by rapsyn. However, transfection of HEK cells with either a1 or g2 subunits in the absence or presence of rapsyn did not result in detectable cell surface receptors (exper-
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Fig. 6. Clustering of a1b1 or a1b2 receptors by rapsyn. HEK cells were cotransfected with rapsyn and a1b1 (A) or a1b2 (B) subunit combinations. a1 subunits were then detected on the surface of transfected cells by immuno¯uorescence and confocal laser microscopy using a1(1±9) and antirabbit IgG Bodipy FL antibodies (single sections). The experiment was performed three times with similar results.
iments not shown), as expected from the observation that cotransfected a1 and g2 subunits could not be detected on the cell surface (see above). When cells transfected with a1 or g2 subunits were permeabilized, a1 subunits were found in an endoplasmic reticulum (ER)-like compartment (Fig. 4C), while g2 subunits were detected in ER-like structures and intracellular vesicles (Fig. 4E). The same intracellular distribution of these subunits was observed in the absence (experiments not shown) or presence of rapsyn (Fig. 4C,E). After cotransfection with rapsyn, rapsyn formed clusters that exhibited a distribution dierent from that of GABAA receptor subunits (Fig. 4 C,D,E,F). 3.4. Subunit requirements for clusteringÐchimeric subunits Since a1 and g2 subunits were not transported to the cell surface in the absence of b3 subunits, a possible clustering site present on these subunits might not have been detected by the clustering assay. Because b3 subunits were able to reach the cell surface, it was investigated whether chimeric subunits consisting of the N-terminal part of the b3 subunit and the remaining part of the a1 or g2 subunit were also able to reach the cell surface (Fig. 5A). As can be seen in Fig. 5B, a chimera consisting of the N-terminal extracellular
domain of the b3 subunit and the remaining sequence derived from the a1 subunit could be detected on the cell surface. Thus, the N-terminus of the b3 subunit was sucient to direct the b3-a1 chimera to the plasma membrane. The amount of the b3-a1 chimera incorporated into the cell membrane, however, was lower than that of b3 subunits detected in b3 transfected cells (experiments not shown). Upon coexpression of the b3-a1 chimera with rapsyn, no clustering of the chimera (Fig. 5C), but clustering of rapsyn was observed (experiments not shown). A correct stoichiometric ratio between nACh receptor subunits and rapsyn is important for the formation of large clusters in muscle cells (Yoshihara and Hall, 1993). In order to exclude the possibility that the lack of clustering of the b3-a1 chimera was caused by the weak expression of the chimera, and thus, by a reduced stoichiometric relationship between chimera and rapsyn, cells were transfected with a ®ve fold higher amount of chimera DNA than rapsyn DNA. As expected, an elevated expression of the b3-a1 chimera did not result in the induction of clustering by rapsyn (experiments not shown). In addition, on coexpression of a1 subunits with the b3-a1 chimera, both subunits could be readily detected on the cell surface but could not be clustered by rapsyn (experiments not shown). These results indicate that the aminoacid
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sequence of the a1 subunit present in the b3-a1 chimera was unable to form a site that mediates clustering by rapsyn. When a chimeric subunit consisting of the N-terminus of the b3 subunit and the remaining part of the g2 subunit was transfected into HEK cells, these chimeras could also be detected on the cell surface (Fig. 5D). In this case, however, cotransfection with rapsyn induced clustering of the b3-g2 chimera (Fig. 5E) and the chimera and rapsyn were colocalized in the same clusters (experiments not shown). These results indicated that the g2 subunit also contains a clustering site. The clustering eect, however, was weaker than that observed in HEK cells cotransfected with b3 subunits and rapsyn. A large proportion of the b3-g2 chimera was not clustered, but diusely distributed in the plasma membrane. Upon higher magni®cation, clusters had a ring-like appearance and were not continuously stained as in experiments using b3 subunits and rapsyn (experiments not shown). A ring-like appearance of rapsyn induced clusters has also been observed with the nACh receptor under certain conditions (Krikorian and Bloch, 1992).
3.5. Subunit requirements for clusteringÐother b subunits HEK cells transfected with b1 or b2 subunits did not form cell surface receptors in a quantity sucient for detection by immuno¯uorescence (experiments not shown). Similarly, HEK cells transfected with a1 subunits did not form cell surface receptors, as mentioned above. When b1 or b2 subunits were cotransfected with a1 subunits, diusely distributed receptors were clearly detectable on the cell surface (experiments not shown). Cotransfection of a1b1 or a1b2 subunits with rapsyn resulted in clustered receptors (Fig. 6), indirectly demonstrating that b1 and b2 subunits also contain a site mediating clustering by rapsyn. The observation that chimeric subunits consisting of the N-terminus of the b3 subunit and the remaining part of the b2 subunit were able to form receptors on the cell surface that could be clustered by rapsyn (experiments not shown) directly con®rmed the presence of a clustering site on b2 subunits. In other experiments, HEK cells were transfected with a1b1g2 or a1b2g2 subunits. Receptors formed were detected on the cell surface and as expected, could be clustered by rapsyn (experiments not shown). Finally, it was demonstrated that GABAA receptors transfected into the neuronal precursor cell line NT-2 (Pleasure and Lee, 1993) were diusely distributed on the cell surface and could be clustered on cotransfection with rapsyn (experiments not shown).
4. Discussion 4.1. Rapsyn is capable of clustering heteromeric and homomeric GABAA receptors on the surface of transfected cells In the present study, it was demonstrated that HEK cells transfected with a1b1g2, a1b2g2, a1b3g2, a1b1, a1b2, a1b3, b3g2, or b3 subunits formed GABAA receptors diusely distributed in the plasma membrane. Cotransfection with rapsyn resulted in clustering of receptors, and the respective GABAA receptor subunits and rapsyn were colocalized in the same clusters. The ability of rapsyn to cluster GABAA receptors was not only observed in HEK cells but also in the neuronal precursor cell line NT-2. These data con®rm and extend a recent investigation indicating that GABAA receptors composed of a1b1g2 subunits could be clustered by rapsyn after cotransfection into quail ®broblasts (Yang et al., 1997). The formation of functional recombinant GABAA receptors from the subunit combinations mentioned above has been demonstrated previously by measuring GABA-induced chloride ¯ux (Sieghart, 1995). These ®ndings were supported by an immunocytochemical identi®cation of receptors composed of a1b2g2, a1b1, a1b2, a1b3, or b3 subunits on the surface of transfected cells (Connolly et al., 1996a,b). In the present study, the b3g2 subunit combination, too, was clearly detectable on the surface of transfected HEK cells. This is in contrast to b2g2 or b1g2 subunit combinations that were not or only hardly detectable in the cell membrane, respectively (experiments not shown). b3 subunits, thus, seem to have a higher potential to direct intracellular g2 subunits to the cell surface than the other two b subunits. 4.2. Subunits or subunit combinations not transported to the cell surface are not clustered by rapsyn After transfection of HEK cells with a1, b1, b2, or g2 subunits, or after cotransfection with a1 and g2 subunits, these subunits could not be detected on the cell surface but were located intracellularly. Similar observations were reported previously (Connolly et al., 1996b). On cotransfection with rapsyn, no clustering of these GABAA receptor subunits was observed. Rapsyn, however, was clustered on the inner side of the cell membrane, and was not colocalized with GABAA receptor subunits. In addition, the intracellular distribution of these subunits was identical in the absence or presence of rapsyn, indicating that rapsyn was not able to change the subcellular distribution of GABAA receptor subunits. These results are consistent with previous ®ndings indicating that rapsyn formed clusters that were not colocalized with nACh receptor
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subunit combinations that could not assemble eciently (Yoshihara and Hall, 1993). 4.3. Transport to the cell surface is not sucient for clustering of subunits by rapsyn Since b3 subunits were incorporated into the plasma membrane of transfected cells, whereas a1, b1, b2, or g2 subunits were not, it was investigated whether a chimera consisting of the N-terminus of the b3 and the remaining part of the a1 subunit could be transported to the cell surface. The observation that this b3-a1 chimera was detectable in the plasma membrane, as well as the conclusion that only completely assembled receptors are incorporated into the cell membrane (Hurtley and Helenius, 1989; Verrall and Hall, 1992), suggested that the N-terminus of the b3 subunit allowed ecient assembly of the chimera and subsequent incorporation into the plasma membrane. On cotransfection with the b3-a1 chimera and rapsyn, however, no clustering of the chimera was induced. This indicates that the mere presence of receptors on the cell surface is not sucient for being clustered by rapsyn. Since rapsyn was able to cluster b3 subunits, but not b3-a1 chimeras, it can be concluded that the N-terminus is not responsible for clustering by rapsyn. This is in agreement with the assumption that rapsyn causes clustering of the nACh receptor via an interaction with intracellular domains of the receptor (Yu and Hall, 1994). The observation that rapsyn was unable to cluster b3-a1 chimeras on the surface of HEK cells thus indicates that the intracellular domains of the a1 subunit are unable to form sites that mediate clustering by rapsyn. 4.4. g2 subunits, as well as b1, b2, or b3 subunits of GABAA receptors form sites that mediate clustering by rapsyn To investigate whether g2 subunits can form sites mediating clustering by rapsyn, a b3-g2 chimera was constructed consisting of the N-terminus of the b3 and the remaining part of the g2 subunit. This b3-g2 chimera could also be identi®ed on the cell surface on transfection into HEK cells. In contrast to the b3-a1 chimera, however, the b3-g2 chimera could be clustered upon cotransfection with rapsyn, indicating that the intracellular domains of the g2 subunit can form a site that mediates clustering by rapsyn. Rapsyn-induced clustering of the b3-g2 chimera, however, was less ecient than that of the b3 subunit, suggesting that the interaction of rapsyn with the g2 subunit was weaker than that with the b3 subunit. Beta1 or b2 subunits, in contrast to b3 subunits cannot be readily detected on the cell surface after transfection into HEK cells. The observation, however, that
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a1b1 or a1b2 subunit combinations can be clustered by rapsyn although no clustering site is present on a1 subunits, indicates that b1 and b2 subunits also do form sites that mediate clustering by rapsyn. This conclusion was con®rmed by other experiments indicating that a chimera consisting of the N-terminus of the b3 and the remaining part of the b2 subunit was transported to the surface of transfected cells and could be clustered by rapsyn. 4.5. Mechanism of clustering of GABAA receptors by rapsyn The present experiments do not provide information on the mechanism of clustering of GABAA receptors by rapsyn. However, little is known about the mechanism of clustering of the nACh receptor either. Although rapsyn precisely colocalizes with nACh receptor at the neuromuscular junction and is able to induce clustering of this receptor in recombinant systems, so far no biochemical interaction between rapsyn and the nACh receptor has been demonstrated. In addition, no site has been identi®ed on nACh receptor subunits that mediates clustering by rapsyn (Maimone and Merlie, 1993; Wheeler et al., 1994; Yu and Hall, 1994). And ®nally, it is still not clear whether rapsyn interacts directly with the receptor or whether other proteins are involved in this interaction. Recently, a sequence motif of rapsyn has been identi®ed that might be responsible for clustering of nACh receptors (Ramarao and Cohen, 1998). The authors propose that rapsyn interacts directly with subunits of the nACh receptor via a coiled-coil motif. It will be interesting to investigate whether the same motif of rapsyn is involved in clustering GABAA receptors. 4.6. Is rapsyn involved in clustering of native GABAA receptors? Receptors consisting of a1b2g2 or a1b3g2 subunits are the major GABAA receptors in the brain (Benke et al., 1991). An assembly with b subunits is necessary for the transport of a and g subunits to the cell membrane, as shown in this and previous studies (Connolly et al., 1996a,b). In addition, the binding site for GABA is formed at the interface of a and b subunits (Sieghart, 1995; Smith and Olsen, 1995). Beta subunits, thus, are indispensable for the formation of the majority of GABAA receptors. Only r subunits seem not to require b subunits for correct assembly (Johnston, 1996). Since b1, b2, as well as b3 subunits do contain clustering sites, it has to be concluded that most, if not all GABAA receptors can be clustered by rapsyn. So far no report has been published indicating a possible colocalization of rapsyn and native GABAA receptors. Rapsyn is predominantly located in peripheral
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tissues, where it mediates clustering of nACh receptors (Musil et al., 1989). Evidence has accumulated indicating that GABAA receptors are also expressed in peripheral tissues and are involved in the modulation of smooth muscle contraction and in a variety of other functions (ErdoÈ and Wol, 1990; Greenwood Van Meerveld and Barron, 1998; ParramoÂn et al., 1995). Further experiments will have to clarify whether GABAA receptors in these tissues are clustered by rapsyn. Recently, it has been demonstrated that rapsyn mRNA is also present in the brain (Burns et al., 1997; Yang et al., 1997). So far, however, the question whether rapsyn or a rapsyn isoform (Burns et al., 1997) mediates clustering of native GABAA receptors in the brain has not been investigated. But the ability of rapsyn to cluster the majority of GABAA receptors does not necessarily mean that the majority of native GABAA receptors are clustered by rapsyn. It is possible that GABAA receptors are clustered by rapsyn only in certain tissues or cells or at certain stages of development. Recently, it has been demonstrated that rapsyn is not essential for clustering of neuronal nACh receptor in superior cervical ganglion, although it was able to cluster both muscle and neuronal ACh receptors in heterologous expression systems (Feng et al., 1998). Thus, dierent tissues can use dierent proteins for clustering of receptors. In addition, it is possible that clusters containing rapsyn (Yoshihara and Hall, 1993) or gephyrin (Colin et al., 1998; Kirsch et al., 1993) only provide a matrix for anchoring proteins that do interact with GABAA receptors. Then the same anchoring protein might possibly be recruited by dierent cluster forming proteins in dierent tissues. In any case, the strong clustering by rapsyn of many GABAA receptor subtypes observed in the present study indicates that the amino acid sequences on b1±3 or g2 subunits mediating this eect are easily accessible for cytoplasmic proteins and might therefore also mediate clustering of native GABAA receptors. The present observation that b3 subunits of GABAA receptors as well as b3-g2 or b3-b2 chimeras can be clustered by rapsyn on the surface of transfected cells, can be used to investigate the clustering of suitable chimeras modi®ed by site directed mutagenesis. This will allow to identify amino acid sequences on bx or g2 subunits mediating clustering by rapsyn. These sequences can then be used for the identi®cation of interacting proteins that possibly are involved in clustering of native GABAA receptors. Experiments in this direction are underway in our laboratory. Acknowledgements This work was supported by project SFB F610 of
the Austrian Science Foundation. We would like to thank Stanley Froehner (Department of Physiology, University of North Carolina, NC) for providing the rapsyn clone, Jim Yeadon (Skirball Institute for Biomolecular Medicine, NYU Medical Center, NY) for providing the myc-tagged rapsyn clone, Iris Killisch (Institute for Molecular Pathology, Vienna) for advice in immuno¯uorescence and confocal microscopy, and Susanne Karall for transfection of HEK cells and the Institute for Pharmacology (University of Vienna) for the permit to use the confocal microscope. References Apel, E.D., Merlie, J.P., 1995. Assembly of the postsynaptic apparatus. Curr. Op. Neurobiol. 5, 62±67. Barnard, E.A., Skolnick, P., Olsen, R.W., Mohler, H., Sieghart, W., Biggio, G., Braestrup, C., Bateson, A.N., Langer, S.Z., 1998. International Union of Pharmacology. XV. Subtypes of g-aminobutyric acid(A) receptors: classi®cation on the basis of subunit structure and receptor function. Pharmacol. Rev. 50, 291±313. Benke, D., Mertens, S., Trzeciak, A., Gillessen, D., MoÈhler, H., 1991. GABAA receptors display association of g2-subunit with a1and b2/3 subunits. J. Biol. Chem. 266, 4478±4483. Burns, A.L., Benson, D., Howard, M.J., Margiotta, J.F., 1997. Chick ciliary ganglion neurons contain transcripts coding for acetylcholine receptor-associated protein at synapses (rapsyn). J. Neurosci. 17, 5016±5026. Chen, C.A., Okayama, H., 1988. Calcium phosphate-mediated gene transfer: a highly ecient transfection system for stably transforming cells with plasmid DNA. Biotechniques 6, 632±638. Colledge, M., Froehner, S.C., 1998. To muster a cluster: anchoring neurotransmitter receptors at synapses. Proc. Natl. Acad. Sci. USA 95, 3341±3343. Colin, I., Rostaing, P., Augustin, A., Triller, A., 1998. Localization of components of glycinergic synapses during rat spinal cord development. J. Comp. Neurol. 398, 359±372. Connolly, C.N., Krishek, B.J., McDonald, B.J., Smart, T.G., Moss, S.J., 1996a. Assembly and cell surface expression of heteromeric and homomeric g-aminobutyric acid type A receptors. J. Biol. Chem. 271, 89±96. Connolly, C.N., Wooltorton, J.R.A., Smart, T.G., Moss, S.J., 1996b. Subcellular localization of g-aminobutyric acid type A receptors is determined by receptor b subunits. Proc. Natl. Acad. Sci. USA 93, 9899±9904. Craig, A.M., Banker, G., Chang, W., McGrath, M.E., Serpinskaya, A.S., 1996. Clustering of gephyrin at GABAergic but not glutamatergic synapses in cultured rat hippocampal neurons. J. Neurosci. 16, 3166±3177. Craig, A.M., Blackstone, C.D., Huganir, R.L., Banker, G., 1994. Selective clustering of glutamate and g-aminobutyric acid receptors opposite terminals releasing the corresponding neurotransmitters. Proc. Natl. Acad. Sci. USA 91, 12,373±12,377. ErdoÈ, S.L., Wol, J.R., 1990. g-Aminobutyric acid outside the mammalian brain. J. Neurochem. 54, 363±372. Essrich, C., Lorez, M., Benson, J.A., Fritschy, J.M., LuÈscher, B., 1998. Postsynaptic clustering of major GABAA receptor subtypes requires the g2 subunit and gephyrin. Nature Neurosci. 1, 563± 571. Ewert, M., Shivers, B.D., LuÈddens, H., MoÈhler, H., Seeburg, P.H., 1990. Subunit selectivity and epitope characterization of mAbs directed against the GABAA/benzodiazepine receptor. J. Cell Biol. 110, 2043±2048.
V. Ebert et al. / Neurochemistry International 34 (1999) 453±463 Feng, G., Steinbach, J.H., Sanes, J.R., 1998. Rapsyn clusters neuronal acetylcholine receptors but is inessential for formation of an interneuronal cholinergic synapse. J. Neurosci. 18, 4166±4176. Fuchs, K., Zezula, J., Slany, A., Sieghart, W., 1995. Endogenous [3H]¯unitrazepam binding in human embryonic kidney cell line 293. Eur. J. Pharmacol. 289, 87±95. Greenwood Van Meerveld, B., Barron, K.W., 1998. Tonic GABA(A) receptor-mediated neurotransmission in the dorsal vagal complex regulates intestinal motility in rats. Eur. J. Pharmacol. 346, 197± 202. Hanley, J.G., Koulen, P., Bedford, F.K., Gordon-Weeks, P.R., Moss, S.J., 1999. The protein MAP-1B links GABAC receptors to the cytoskeleton at retinal synapses. Nature 397, 66±69. Hurtley, S.M., Helenius, A., 1989. Protein oligomerization in the endoplasmic reticulum. Ann. Rev. Cell Biol. 5, 277±307. Jechlinger, M., Pelz, R., Tretter, V., Klausberger, T., Sieghart, W., 1998. Subunit composition and quantitative importance of heterooligomeric receptors: GABAA receptors containing a6 subunits. J. Neurosci. 18, 2449±2457. Johnston, G.A.R., 1996. GABAc receptors: relatively simple transmitter-gated ion channels? Trends Pharmacol. Sci. 17, 319±323. Kirsch, J., Wolters, I., Triller, A., Betz, H., 1993. Gephyrin antisense oligonucleotides prevent glycine receptor clustering in spinal neurons. Nature 366, 745±748. Kirsch, J., Betz, H., 1995. The postsynaptic localization of the glycine receptor-associated protein gephyrin is regulated by the cytoskeleton. J. Neurosci. 15, 4148±4156. Kirsch, J., Kuhse, J., Betz, H., 1995. Targeting of glycine receptor subunits to gephyrin-rich domains in transfected human embryonic kidney cells. Mol. Cell. Neurosci. 6, 450±461. Krikorian, J.G., Bloch, R.J., 1992. Treatments that extract the 43 K protein from acetylcholine receptor clusters modify the conformation of cytoplasmic domains of all subunits of the receptor. J. Biol. Chem. 267, 9118±9128. Macdonald, R.L., Olsen, R.W., 1994. GABAA receptor channels. Ann. Rev. Neurosci. 17, 569±602. Maimone, M.M., Merlie, J.P., 1993. Interaction of the 43 kD postsynaptic protein with all subunits of the muscle nicotinic acetylcholine receptor. Neuron 11, 53±66. Meyer, G., Kirsch, J., Betz, H., Langosch, D., 1995. Identi®cation of a gephyrin binding motif on the glycine receptor b subunit. Neuron 15, 563±572. Mossier, B., ToÈgel, M., Fuchs, K., Sieghart, W., 1994. Immunoanity puri®cation of g-aminobutyric acidA (GABAA) receptors containing g1-subunits. Evidence for the presence of a single type of g-subunit in GABAA receptors. J. Biol. Chem. 269, 25,777±25,782. Musil, L.S., Frail, D.E., Merlie, J.P., 1989. The mammalian 43-kD acetylcholine receptor-associated protein (RAPsyn) is expressed in some nonmuscle cells. J. Cell Biol. 108, 1833±1840. Nayeem, N., Green, T.P., Martin, I.L., Barnard, E.A., 1994. Quaternary structure of the native GABAA receptor determined by electron microscopic image analysis. J. Neurochem. 62, 815± 818. ParramoÂn, M., GonzaÂlez, M.P., Oset-Gasque, M.J., 1995. Pharmacological modulation of adrenal medullary GABAA receptor: consistent with its subunit composition. Br. J. Pharmacol. 116, 1875±1881.
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Phillips, W., 1995. Acetylcholine receptors and cytoskeletal connection. Clin. Exp. Pharm. Phys. 22, 961±965. Pleasure, S.J., Lee, V.M.-Y., 1993. Ntera 2 Cells: a human cell line which display characteristics expected of a human committed neuronal progenitor cell. J Neurosci. Res. 35, 585±602. Ramarao, M.K., Cohen, J.B., 1998. Mechanism of nicotinic acetylcholine receptor cluster formation by rapsyn. Proc. Natl. Acad. Sci. USA 95, 4007±4012. SassoeÁ-Pognetto, M., Kirsch, J., Grunert, U., Greferath, U., Fritschy, J.M., MoÈhler, H., Betz, H., WaÈssle, H., 1995. Colocalization of gephyrin and GABAA-receptor subunits in the rat retina. J. Comp. Neurol. 357, 1±14. Scho®eld, P.R., Darlison, M.G., Fujita, N., Burt, D.R., Stephenson, F.A., Rodriguez, H., Rhee, L.M., Ramachandran, J., Reale, V., Glencorse, T.A., Seeburg, P.H., Barnard, E.A., 1987. Sequence and functional expression of the GABAA receptor shows a ligand gated receptor superfamily. Nature 328, 221±227. Sieghart, W., 1995. Structure and pharmacology of g-aminobutyric acidA receptor subtypes. Pharmacol. Rev. 47, 181±234. Slany, A., Zezula, J., Tretter, V., Sieghart, W., 1995. Rat b3 subunits expressed in human embryonic kidney 293 cells form high anity [35S]t-butylbicyclophosphorothionate binding sites modulated by several allosteric ligands of g-aminobutyric acid type A receptors. Mol. Pharmacol. 48, 385±391. Smith, G.B., Olsen, R.W., 1995. Functional domains of GABAA receptors. Trends Pharmacol. Sci. 16, 162±168. Todd, A.J., Spike, R.C., Chong, D., Neilson, M., 1995. The relationship between glycine and gephyrin in synapses of the rat spinal cord. Eur. J. Neurosci. 7, 1±11. Todd, A.J., Watt, C., Spike, R.C., Sieghart, W., 1996. Colocalization of GABA, glycine, and their receptors at synapses in the rat spinal cord. J. Neurosci. 16, 974±982. Tretter, V., Ehya, N., Fuchs, K., Sieghart, W., 1997. Stoichiometry and assembly of a recombinant GABAA receptor subtype. J. Neurosci. 17, 2728±2737. Verrall, S., Hall, Z.W., 1992. The N-terminal domains of acetylcholine receptor subunits contain recognition signals for the initial steps of receptor assembly. Cell 68, 23±31. Wang, H., Bedford, F.K., Brandon, N.J., Moss, S.J., Olsen, R.W., 1999. GABAA-receptor-associated protein links GABAA receptors and the cytoskeleton. Nature 397, 69±72. Wheeler, S.V., Jane, S.D., Cross, K.M.L., Chad, J.E., Foreman, R.C., 1994. Membrane clustering and bungarotoxin binding by the nicotinic acetylcholine receptor: role of the b subunit. J. Neurochem. 63, 1891±1899. Yang, S.-H., Armson, P.F., Cha, J., Phillips, W.D., 1997. Clustering of GABAA receptors by rapsyn/43kD protein in vitro. Mol. Cell Neurosci. 8, 430±438. Yoshihara, C.M., Hall, Z.W., 1993. Increased expression of the 43kD protein disrupts acetylcholine receptor clustering in myotubes. J. Cell. Biol. 122, 169±179. Yu, X-M., Hall, Z.W., 1994. The role of the cytoplasmic domains of individual subunits of the acetylcholine receptor in 43 kDa protein-induced clustering in COS cells. J. Neurosci. 14, 785±795. Zezula, J., Fuchs, K., Sieghart, W., 1991. Separation of a1, a2 and a3 subunits of the GABAA-benzodiazepine receptor complex by immunoanity chromatography. Brain Res. 563, 325±328.
Identi®cation of subunits mediating clustering of GABAA receptors by rapsyn V. Ebert, P. Scholze, K. Fuchs, W. Sieghart* Section of Biochemical Psychiatry, University Clinic for Psychiatry, WaÈhringer GuÈrtel 18±20, A-1090, Vienna, Austria Received 1 March 1999; accepted 4 March 1999
Abstract Human embryonic kidney 293 cells transfected with a1b1g2, a1b2g2, a1b3g2, a1b1, a1b2, a1b3, b3g2, or b3 subunits formed gaminobutyric acidA receptors on the cell surface that could be clustered by rapsyn. In contrast, a1, b1, b2, or g2 subunits, or a1g2 subunit combinations could not be detected on the surface of transfected cells and could not be clustered by rapsyn. Experiments investigating the ability of rapsyn to cluster chimeras consisting of the N-terminus of the b3 subunit and the remaining part of the a1, b2 or g2 subunits indicated that the intracellular domains of b1, b2, b3, or g2 subunits, but not those of a1 subunits are able to form sites mediating clustering by rapsyn. These results demonstrate that rapsyn has the potential to cluster the majority of GABAA receptor subtypes via b or g2 subunits. Further experiments will have to clarify the physiological importance of this observation. # 1999 Elsevier Science Ltd. All rights reserved.
1. Introduction Gamma-Aminobutyric acid type A (GABAA) receptors are ligand gated chloride ion channels and the site of action of a variety of clinically important drugs (Sieghart, 1995). These receptors are composed of ®ve subunits (Nayeem et al., 1994; Tretter et al., 1997). Each subunit consists of an N-terminal extracellular region, followed by four transmembrane segments (TM) and a large intracellular loop between TM3 and TM4 (Scho®eld et al., 1987). At least 18 dierent GABAA receptor subunits (a1±6, b1±4, g1-3, d, E, r1±3) so far have been identi®ed in vertebrate brain, that give rise to a large variety of GABAA receptor subtypes with distinct subunit composition (Barnard et al., 1998; Macdonald and Olsen, 1994; Sieghart, 1995). Depending on their subunit composition, these receptors exhibit distinct electrophysiological and pharmacological properties. Expression studies have indicated that a, b and g subunits have to combine to form * Corresponding author. Tel.: +43-1-40400-3572; fax: +43-140400-3629. E-mail address: [email protected] (W. Sieghart)
GABAA receptors with a pharmacology resembling that of native receptors (Sieghart, 1995). The precise localization of receptors in the postsynaptic membrane opposite transmitter release sites is crucial for synaptic function. In addition, the number and density of receptors in the postsynaptic membrane is one of the major determinants of the ecacy of neuronal transmission. GABAA receptors, therefore, are selectively clustered opposite nerve terminals releasing GABA (Craig et al., 1994; Todd et al., 1996). Several dierent proteins have been identi®ed that mediate clustering of transmitter receptors at synapses (Colledge and Froehner, 1998). For instance, the peripheral membrane protein rapsyn (43 kD protein) plays an essential role in clustering of the nicotinic acetylcholine (nACh) receptor of the muscle (Colledge and Froehner 1998; Phillips, 1995; Apel and Merlie, 1995). Gephyrin, a 93 kD protein, mediates clustering of the glycine receptor (Kirsch and Betz, 1995), and recently, at least two dierent proteins have been identi®ed that might be responsible for clustering of GABAA receptors. One of these proteins is the microtubule-associated protein 1B (MAP-1B) that clusters GABAC receptors containing r1 subunits (Hanley et
0197-0186/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 1 9 7 - 0 1 8 6 ( 9 9 ) 0 0 0 3 9 - X
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al., 1999). Since r subunits are structurally part of the family of GABAA receptor subunits, it was recommended that r-containing receptors should be classi®ed as a specialized set of the GABAA receptors (Barnard et al., 1998). The other protein (GABARAP) can interact with GABAA receptor g2 subunits and shows similarities to chain-3 of MAP-1A and 1B (Wang et al., 1999). Another study has indicated that gephyrine in addition to clustering glycine receptors is required for postsynaptic clustering of major GABAA receptor subtypes (Essrich et al., 1998). Gephyrin, however, copuri®ed with glycine but not with GABAA receptors and did not signi®cantly colocalize with GABAA receptors in heterologous expression systems (Kirsch et al., 1995; Meyer et al., 1995). Other results indicated that the degree of colocalization of gephyrin with GABAA receptors varied in dierent tissues (Craig et al., 1996; Todd et al., 1995; SassoeÁ-Pognetto et al., 1995). In agreement with the identi®cation of other proteins involved in clustering of GABAA receptors (Hanley et al., 1999; Wang et al., 1999) these results indicate that gephyrin might be involved in clustering of some but not all GABAA receptors. Interestingly, not only gephyrine but also rapsyn, another protein involved in clustering of a member of this receptor superfamily, induces clustering of a GABAA receptor subtype (Yang et al., 1997). In the present study, this observation was con®rmed and extended by demonstrating that rapsyn is able to cluster most, if not all recombinant GABAA receptor subtypes. In addition, GABAA receptor subunits mediating clustering by rapsyn were identi®ed using suitable subunit combinations or chimeric subunits. This information can now be used to identify the amino acid sequences mediating clustering by rapsyn and to identify proteins interacting with the respective sequences of native receptors. 2. Experimental procedures 2.1. Cloning of expression constructs The cDNA of a1, b1, b2, b3, or g2 subunits were subcloned into the mammalian expression vector pCDM8 (Invitrogen, San Diego, CA) as described previously (Fuchs et al., 1995; Jechlinger et al., 1998). The chimeric receptor subunits b3-a1 and b3-g2 with the subunit boundary immediately before TM1 were generated as described (Tretter et al., 1997). For construction of the b3-b2 chimera, the N-terminal fragment of the b3 subunit was generated from Hind III/Bgl II digestion of a b3 subunit cloned into the mammalian expression vector pcDNAI/Amp (Invitrogen), into which a Bgl II site had been introduced at the 5 ' end of TM 1
(Tretter et al., 1997). The b2-fragment spanning the region from TM1 to the C-terminus was ampli®ed by PCR using primers into which restriction sites had been introduced (Bgl II for the 5 ' end, Not I for the 3 ' end. Both fragments were ligated into a Hind III/ Not I digested expression vector pcDNAI/Amp (Invitrogen). The correct construction of the ®nal chimera was controlled by restriction enzyme cleavage and by sequencing across the chimera boundary. Mouse rapsyn cDNA was obtained from Stanley C Froehner (Department of Physiology, University of North Carolina) and was subcloned into the mammalian expression vector pCI (Promega Corporation, Madison, WI). The rapsyn with a myc-tag on its C-terminus cloned into the mammalian expression vector pRc/RSV (Invitrogen) was obtained from Jim Yeadon (Skirball Institute for Biomolecular Medicine, NYU Medical Center, New York). Large scale puri®cation for transfections was performed with the Quiagen plasmid puri®cation procedure (Qiagen, BRD). 2.2. Antibodies Antibodies a1(1±9) (Zezula et al., 1991) and b3(1±13) (Tretter et al., 1997) were generated as described. Whereas the a1(1±9) antibody was speci®c for the a1subunit (Jechlinger et al., 1998), b3(1±13) antibodies identi®ed b3 as well as b2, but no other GABAA receptor subunits. g2(1±33) antibodies were prepared using the fusion protein strategy described previously (Mossier et al., 1994). These antibodies were able to identify all g2-subunit containing recombinant GABAA receptors but in contrast to the a1(1±9) or b3(1±13) antibodies did not label a1b3-transfected cells (experiments not shown). The monoclonal antibody bd17, recognizing b2/3 subunits (Ewert et al., 1990) and the c-myc (9E10)-tag antibodies were purchased from Boehringer Mannheim (BRD). 2.3. Cell culture and transfection Human embryonic kidney 293 (HEK) cells (CRL 1573) from the American Type culture collection (Rockville, MD) were maintained in Dulbecco's modi®ed Eagle Medium (DMEM; Life Technologies, Grand Island, NY) supplemented with 50 mM b-mercaptoethanol, 10% fetal calf serum (Boehringer Ingelheim, BRD), 2 mM glutamine, 110 U/ml penicillin G, 100 mg/ml streptomycin and non essential amino acids (Life Technologies). NT-2 cells (Stratagene, La Jolla, CA) were maintained in Opti-Minimal Essential Medium with Glutamax-1 (Life Technologies) supplemented with 10% fetal calf serum (Boehringer), 110 U/ml penicillin G, 100 mg/ml streptomycin (Life Technologies). Cells were transfected with GABAA receptor subunit 2 rapsyn cDNAs by calcium phos-
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Fig. 1. Rapsyn induced clustering of a1b3g2 receptors. HEK cells were cotransfected with a1b3g2 subunits without (A) or with rapsyn (B). Receptors on the cell surface were detected by immuno¯uorescence and confocal laser microscopy after labeling with rabbit a1(1±9) and anti-rabbit IgG Bodipy FL antibodies (single sections). Essentially the same results were obtained when rabbit b3(1±13) or g2(1±33) antibodies were used for labeling of GABAA receptors. The experiment was performed seven times with similar results.
phate coprecipitation (Chen and Okayama, 1988) as described previously (Tretter et al., 1997), with a total of 21 mg DNA per 75 cm2 petri dish. Equal amounts of DNA of the dierent expression constructs were used for cotransfection experiments. For experiments performed in the absence of rapsyn DNA, a corresponding amount of empty vector DNA was added. 2.4. Immuno¯uorescence For immuno¯uorescence, cells were ®xed with 2% paraformaldehyde in phosphate-buered saline (PBS; 16 mM KH2PO4, 4 mM Na2HPO4, 150 mM NaCl; pH 7.4) 30±42 h after transfection, followed by a 10 min wash in 50 mM NH4Cl in PBS. Washes between incubation steps were performed in PBS. For detection of intracellular receptors or of the myctagged rapsyn, cells were permeabilized with 0.1% Triton X-100 for 5 min. Blocking was performed in 5% bovine serum albumin (BSA) in PBS for 10 min, followed by an incubation with primary antibody in 1% BSA in PBS. Primary antibodies generated in rabbits were detected with Goat Anti-Rabbit IgG(H+L) Bodipy FL (Molecular Probes, Eugene, OR), while monoclonal mouse antibodies were detected using Donkey Anti-Mouse IgG (H+L)Cy3 (Amersham International plc, UK) in 1% BSA in PBS. Labeling was visualized using a Zeiss Axiovert 135 M microscope attached to a confocal laser system (Carl Zeiss LSM 410, BRD), equipped with an argon laser and a
helium-neon laser and suitable ®lter sets. To verify that the labeling of cells without permeabilization was restricted to the cell surface, parallel samples were stained with antibodies directed against the intracellular loop of GABAA receptor subunits (experiments not shown). These antibodies detected GABAA receptor subunits only after permeabilization of transfected cells. Double labeling experiments for rapsyn and GABAA receptor subunits in most cases were performed after permeabilization of cells. Results obtained were compared with single labeling of parallel samples to demonstrate that the labeling pattern in the double labeling experiments was not caused by crossbleeding artifacts (experiments not shown). Similar results were obtained when the cells were ®rst labeled with the receptor speci®c antibody, were then washed, permeabilized, blocked again, labeled with the mycantibody, followed by a detection with a mixture of both secondary antibodies (experiments not shown).
3. Results 3.1. Clustering of a1b3g2-receptors Recently, it was demonstrated that a1, b3 and g2 subunits of GABAA receptors can assemble to pentameric receptors composed of all three subunits after cotransfection into HEK 293 cells (Tretter et al., 1997). As shown in Fig. 1A, these receptors appear
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Fig. 2. Colocalization of g2 subunits and rapsyn in the same clusters. HEK cells were cotransfected with a1b3g2 subunits and a myc-tagged rapsyn. Double labeling of permeabilized cells was performed using rabbit g2(1±33) antibodies (A) and a monoclonal mouse antibody against the myc-tag of rapsyn (B) followed by anti-rabbit IgG Bodipy FL and anti-mouse IgG Cy3. Receptors and rapsyn were detected in the same cell by immuno¯uorescence and confocal laser microscopy (single sections). The experiment was performed three times with similar results.
uniformly distributed on the cell surface of HEK cells upon detection with a1(1±9) antibodies. The small, brighter areas in Fig. 1A were interpreted as microvilli protruding from the cells, that due to their higher membrane density exhibit an apparently higher receptor density. Similar results were obtained when b3(1± 13) or g2(1±33) antibodies were used (experiments not shown). Only a low-level nonspeci®c signal was observed in non-transfected neighbouring cells, indicating that the antibodies used speci®cally recognized only GABAA receptor subunits and did not crossreact with other HEK cell proteins. When a1, b3 and g2 subunits were coexpressed with rapsyn, approximately 60% of the cells that were labeled by subunit-speci®c antibodies exhibited clustering of cell surface receptors (Fig. 1B). This is in agreement with previous observations indicating that rapsyn induces clustering in only a fraction of cells transfected with the nACh receptor (Yu and Hall, 1994). For double labeling experiments, GABAA receptor subunits were cotransfected with a myc-tagged rapsyn instead of the untagged version. This allowed simultaneous labeling of GABAA receptor subunits with antibodies generated in rabbits and labeling of rapsyn with the mouse monoclonal anti-myc-antibody. In agreement with results from a previous study (Yoshihara and Hall, 1993), essentially the same results were obtained when the tagged or the untagged rapsyn was used for cotransfection with GABAA receptor subunits (experiments not shown), indicating that the tag-
sequence fused to the C-terminus of rapsyn did not interfere with its clustering activity. Double labeling for g2 subunits and the myc-tagged rapsyn demonstrated a colocalization of GABAA receptors with rapsyn in the clusters (Fig. 2). Since rapsyn is a peripheral membrane protein associated with the inner plasma membrane, cells had to be permeabilized for rapsyn detection. Interestingly, the percentage of cells stained by the anti-myc-antibody was much smaller than that stained by g2(1±33) antibodies, although strongly clustered receptors could be identi®ed in many cells that were not stained by the antimyc-antibody (experiments not shown). This indicated, that detection via the myc-tag required a relatively high expression level of rapsyn in the cells. Coclustering was also visible, when a1(1±9) or b3(1±13) antibodies were used to detect GABAA receptors in combination with the anti-myc-antibody (experiments not shown). Furthermore, a colocalization of GABAA receptor subunits and rapsyn in the same clusters was also demonstrated when intact cells were ®rst labeled for GABAA receptor subunits, then permeabilized and stained for rapsyn (experiments not shown). Other double labeling experiments using a1(1±9) or g2(1±33) antibodies in combination with the monoclonal antibody bd 17, that speci®cally recognizes b2/3 subunits (Ewert et al., 1990), demonstrated that a1 and b3, or b3 and g2 subunits colocalized in the same clusters. Taken together, these results clearly demonstrated that rapsyn can induce clustering of a1b3g2 receptors in
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Fig. 3. Rapsyn is able to cluster a1b3 and b3g2, but not a1g2 subunit combinations. HEK cells were cotransfected with myc-tagged rapsyn and a1b3 (A±C), b3g2 (D±F), or a1g2 (G±I) subunits. a1 subunits were labeled on the cell surface (A,G) or in permeabilized cells (B,H) using rabbit a1(1±9) antibodies; g2 subunits were labeled on the cell surface (D) or in permeabilized cells (E) using rabbit g2(1±33) antibodies. Rabbit antibodies were detected using anti-rabbit IgG Bodipy FL antibodies. Rapsyn was detected using a mouse anti-myc antibody followed by anti-mouse IgG Cy3 (C,F,I). Immuno¯uorescence was investigated by confocal laser microscopy (single sections). The experiments were performed three times with similar results.
HEK cells and that all three GABAA receptor subunits colocalized with rapsyn in these clusters. 3.2. Subunit requirements for clusteringÐcoexpression of two subunits To investigate which subunits can be eliminated without disturbing the ability of rapsyn to cluster GABAA receptors, receptors composed of two dierent subunits were expressed. Receptors formed after transfection with a1 and b3 subunits were uniformly distributed on the cell surface (experiments not shown). When a1 and b3 subunits were cotransfected with rapsyn, clustering of receptors was induced (Fig. 3A). Double labeling for the a1 subunit and the myctagged rapsyn demonstrated the colocalization of these proteins in the same clusters (Fig. 3B,C). Essentially the same results were obtained using b3(1±13) anti-
bodies for detection of a1b3 receptors (experiments not shown). These data demonstrated that the g2 subunit is not essential for the clustering eect of rapsyn. When HEK cells were transfected with b3 and g2 subunits, again both subunits could be detected on the cell surface (experiments not shown). Upon cotransfection of b3 and g2 subunits with the myc-tagged rapsyn, clustering of receptors was induced (Fig. 3D). As expected, g2 subunits (Fig. 3E) or b3 subunits (experiments not shown) colocalized with rapsyn (Fig. 3F) in the same clusters. These results thus indicated that the a1 subunit, too, is not required for the clustering eect of rapsyn. Upon cotransfection of a1 and g2 subunits in the absence (experiments not shown) or presence (Fig. 3G) of rapsyn, no GABAA receptors could be detected on the cell surface. After permeabilization of the cells, however, both subunits were detected in intracellular
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Fig. 4. Rapsyn is able to cluster b3 subunits, but not a1 or g2 subunits transfected into HEK cells. HEK cells were cotransfected with myc-tagged rapsyn and b3 (A,B), a1 (C,D), or g2 (E,F) subunits. GABAA receptor subunits and the myc-tagged rapsyn were detected in permeabilized cells; b3, a1, or g2 subunits (A,C, or E) were labeled with rabbit b3(1±13), a1(1±9), or g2(1±33) antibodies, respectively, and were detected using anti-rabbit IgG Bodipy FL antibodies. Rapsyn was detected using mouse anti-myc antibody and anti-mouse IgG Cy3. Detection of GABAA receptor subunits and rapsyn was performed in the same cells in A and B, or C and D by immuno¯uorescence and confocal laser microscopy (single sections). Since the g2 signal was much weaker than the rapsyn signal in g2 transfected cells, in this case double labeling could not be performed in the same cell due to crossbleeding of the red rapsyn signal into the green g2 channel. Therefore, in Fig. 4E and F dierent cells are shown. The experiments were performed three times with similar results.
compartments (Fig. 3H, g2 subunits not shown). The same subcellular distribution of a1 and g2 subunits was observed in the absence or presence of rapsyn, indicating that coexpression with rapsyn did not change the subcellular distribution of subunits or redirect the subunits to the plasma membrane. In such cells, the myctagged rapsyn formed clusters (Fig. 3I) that were not colocalized with intracellular a1 (Fig. 3H) or g2 subunits (experiments not shown). 3.3. Subunit requirements for clusteringÐsingle subunits Recently, it has been demonstrated that b3 subunits can form homo-oligomeric receptors that are trans-
Fig. 5. Clustering of chimeric GABAA receptor subunits by rapsyn. (A) Structure of b3-a1, b3-g2, or b3-b2 chimera. Sequences derived from the b3 subunit are shown by a broken line; sequences derived from other subunits are shown grey (TM domains) and by continuous lines. (B±E) Cell surface labeling of the chimeric subunits b3-a1 (B,C) or b3-g2 (D,E) in HEK cells transfected with these chimeras without (B,D) or with rapsyn (C,E). Chimeras were detected by immuno¯uorescence and confocal laser microscopy using b3(1±13) antibodies and anti-rabbit IgG Bodipy FL (single sections). The experiment was performed ®ve times with similar results.
ported to the cell surface in the absence of any other GABAA receptor subunit (Slany et al., 1995; Connolly et al., 1996a). Upon transfection of HEK cells with b3 subunits, receptors formed were homogenously distributed on the cell surface (experiments not shown). Cotransfection with rapsyn induced clusters of b3 receptors (Fig. 4A), which colocalized with those formed by rapsyn (Fig. 4B). This indicates, that the b3 subunit contains a site that mediates clustering by rapsyn. Although the clustering site of the b3 subunit might have been responsible for clustering of a1b3g2, a1b3 and b3g2 receptors, additional sites on a1 or g2 subunits could exist that also mediate clustering by rapsyn. However, transfection of HEK cells with either a1 or g2 subunits in the absence or presence of rapsyn did not result in detectable cell surface receptors (exper-
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Fig. 6. Clustering of a1b1 or a1b2 receptors by rapsyn. HEK cells were cotransfected with rapsyn and a1b1 (A) or a1b2 (B) subunit combinations. a1 subunits were then detected on the surface of transfected cells by immuno¯uorescence and confocal laser microscopy using a1(1±9) and antirabbit IgG Bodipy FL antibodies (single sections). The experiment was performed three times with similar results.
iments not shown), as expected from the observation that cotransfected a1 and g2 subunits could not be detected on the cell surface (see above). When cells transfected with a1 or g2 subunits were permeabilized, a1 subunits were found in an endoplasmic reticulum (ER)-like compartment (Fig. 4C), while g2 subunits were detected in ER-like structures and intracellular vesicles (Fig. 4E). The same intracellular distribution of these subunits was observed in the absence (experiments not shown) or presence of rapsyn (Fig. 4C,E). After cotransfection with rapsyn, rapsyn formed clusters that exhibited a distribution dierent from that of GABAA receptor subunits (Fig. 4 C,D,E,F). 3.4. Subunit requirements for clusteringÐchimeric subunits Since a1 and g2 subunits were not transported to the cell surface in the absence of b3 subunits, a possible clustering site present on these subunits might not have been detected by the clustering assay. Because b3 subunits were able to reach the cell surface, it was investigated whether chimeric subunits consisting of the N-terminal part of the b3 subunit and the remaining part of the a1 or g2 subunit were also able to reach the cell surface (Fig. 5A). As can be seen in Fig. 5B, a chimera consisting of the N-terminal extracellular
domain of the b3 subunit and the remaining sequence derived from the a1 subunit could be detected on the cell surface. Thus, the N-terminus of the b3 subunit was sucient to direct the b3-a1 chimera to the plasma membrane. The amount of the b3-a1 chimera incorporated into the cell membrane, however, was lower than that of b3 subunits detected in b3 transfected cells (experiments not shown). Upon coexpression of the b3-a1 chimera with rapsyn, no clustering of the chimera (Fig. 5C), but clustering of rapsyn was observed (experiments not shown). A correct stoichiometric ratio between nACh receptor subunits and rapsyn is important for the formation of large clusters in muscle cells (Yoshihara and Hall, 1993). In order to exclude the possibility that the lack of clustering of the b3-a1 chimera was caused by the weak expression of the chimera, and thus, by a reduced stoichiometric relationship between chimera and rapsyn, cells were transfected with a ®ve fold higher amount of chimera DNA than rapsyn DNA. As expected, an elevated expression of the b3-a1 chimera did not result in the induction of clustering by rapsyn (experiments not shown). In addition, on coexpression of a1 subunits with the b3-a1 chimera, both subunits could be readily detected on the cell surface but could not be clustered by rapsyn (experiments not shown). These results indicate that the aminoacid
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sequence of the a1 subunit present in the b3-a1 chimera was unable to form a site that mediates clustering by rapsyn. When a chimeric subunit consisting of the N-terminus of the b3 subunit and the remaining part of the g2 subunit was transfected into HEK cells, these chimeras could also be detected on the cell surface (Fig. 5D). In this case, however, cotransfection with rapsyn induced clustering of the b3-g2 chimera (Fig. 5E) and the chimera and rapsyn were colocalized in the same clusters (experiments not shown). These results indicated that the g2 subunit also contains a clustering site. The clustering eect, however, was weaker than that observed in HEK cells cotransfected with b3 subunits and rapsyn. A large proportion of the b3-g2 chimera was not clustered, but diusely distributed in the plasma membrane. Upon higher magni®cation, clusters had a ring-like appearance and were not continuously stained as in experiments using b3 subunits and rapsyn (experiments not shown). A ring-like appearance of rapsyn induced clusters has also been observed with the nACh receptor under certain conditions (Krikorian and Bloch, 1992).
3.5. Subunit requirements for clusteringÐother b subunits HEK cells transfected with b1 or b2 subunits did not form cell surface receptors in a quantity sucient for detection by immuno¯uorescence (experiments not shown). Similarly, HEK cells transfected with a1 subunits did not form cell surface receptors, as mentioned above. When b1 or b2 subunits were cotransfected with a1 subunits, diusely distributed receptors were clearly detectable on the cell surface (experiments not shown). Cotransfection of a1b1 or a1b2 subunits with rapsyn resulted in clustered receptors (Fig. 6), indirectly demonstrating that b1 and b2 subunits also contain a site mediating clustering by rapsyn. The observation that chimeric subunits consisting of the N-terminus of the b3 subunit and the remaining part of the b2 subunit were able to form receptors on the cell surface that could be clustered by rapsyn (experiments not shown) directly con®rmed the presence of a clustering site on b2 subunits. In other experiments, HEK cells were transfected with a1b1g2 or a1b2g2 subunits. Receptors formed were detected on the cell surface and as expected, could be clustered by rapsyn (experiments not shown). Finally, it was demonstrated that GABAA receptors transfected into the neuronal precursor cell line NT-2 (Pleasure and Lee, 1993) were diusely distributed on the cell surface and could be clustered on cotransfection with rapsyn (experiments not shown).
4. Discussion 4.1. Rapsyn is capable of clustering heteromeric and homomeric GABAA receptors on the surface of transfected cells In the present study, it was demonstrated that HEK cells transfected with a1b1g2, a1b2g2, a1b3g2, a1b1, a1b2, a1b3, b3g2, or b3 subunits formed GABAA receptors diusely distributed in the plasma membrane. Cotransfection with rapsyn resulted in clustering of receptors, and the respective GABAA receptor subunits and rapsyn were colocalized in the same clusters. The ability of rapsyn to cluster GABAA receptors was not only observed in HEK cells but also in the neuronal precursor cell line NT-2. These data con®rm and extend a recent investigation indicating that GABAA receptors composed of a1b1g2 subunits could be clustered by rapsyn after cotransfection into quail ®broblasts (Yang et al., 1997). The formation of functional recombinant GABAA receptors from the subunit combinations mentioned above has been demonstrated previously by measuring GABA-induced chloride ¯ux (Sieghart, 1995). These ®ndings were supported by an immunocytochemical identi®cation of receptors composed of a1b2g2, a1b1, a1b2, a1b3, or b3 subunits on the surface of transfected cells (Connolly et al., 1996a,b). In the present study, the b3g2 subunit combination, too, was clearly detectable on the surface of transfected HEK cells. This is in contrast to b2g2 or b1g2 subunit combinations that were not or only hardly detectable in the cell membrane, respectively (experiments not shown). b3 subunits, thus, seem to have a higher potential to direct intracellular g2 subunits to the cell surface than the other two b subunits. 4.2. Subunits or subunit combinations not transported to the cell surface are not clustered by rapsyn After transfection of HEK cells with a1, b1, b2, or g2 subunits, or after cotransfection with a1 and g2 subunits, these subunits could not be detected on the cell surface but were located intracellularly. Similar observations were reported previously (Connolly et al., 1996b). On cotransfection with rapsyn, no clustering of these GABAA receptor subunits was observed. Rapsyn, however, was clustered on the inner side of the cell membrane, and was not colocalized with GABAA receptor subunits. In addition, the intracellular distribution of these subunits was identical in the absence or presence of rapsyn, indicating that rapsyn was not able to change the subcellular distribution of GABAA receptor subunits. These results are consistent with previous ®ndings indicating that rapsyn formed clusters that were not colocalized with nACh receptor
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subunit combinations that could not assemble eciently (Yoshihara and Hall, 1993). 4.3. Transport to the cell surface is not sucient for clustering of subunits by rapsyn Since b3 subunits were incorporated into the plasma membrane of transfected cells, whereas a1, b1, b2, or g2 subunits were not, it was investigated whether a chimera consisting of the N-terminus of the b3 and the remaining part of the a1 subunit could be transported to the cell surface. The observation that this b3-a1 chimera was detectable in the plasma membrane, as well as the conclusion that only completely assembled receptors are incorporated into the cell membrane (Hurtley and Helenius, 1989; Verrall and Hall, 1992), suggested that the N-terminus of the b3 subunit allowed ecient assembly of the chimera and subsequent incorporation into the plasma membrane. On cotransfection with the b3-a1 chimera and rapsyn, however, no clustering of the chimera was induced. This indicates that the mere presence of receptors on the cell surface is not sucient for being clustered by rapsyn. Since rapsyn was able to cluster b3 subunits, but not b3-a1 chimeras, it can be concluded that the N-terminus is not responsible for clustering by rapsyn. This is in agreement with the assumption that rapsyn causes clustering of the nACh receptor via an interaction with intracellular domains of the receptor (Yu and Hall, 1994). The observation that rapsyn was unable to cluster b3-a1 chimeras on the surface of HEK cells thus indicates that the intracellular domains of the a1 subunit are unable to form sites that mediate clustering by rapsyn. 4.4. g2 subunits, as well as b1, b2, or b3 subunits of GABAA receptors form sites that mediate clustering by rapsyn To investigate whether g2 subunits can form sites mediating clustering by rapsyn, a b3-g2 chimera was constructed consisting of the N-terminus of the b3 and the remaining part of the g2 subunit. This b3-g2 chimera could also be identi®ed on the cell surface on transfection into HEK cells. In contrast to the b3-a1 chimera, however, the b3-g2 chimera could be clustered upon cotransfection with rapsyn, indicating that the intracellular domains of the g2 subunit can form a site that mediates clustering by rapsyn. Rapsyn-induced clustering of the b3-g2 chimera, however, was less ecient than that of the b3 subunit, suggesting that the interaction of rapsyn with the g2 subunit was weaker than that with the b3 subunit. Beta1 or b2 subunits, in contrast to b3 subunits cannot be readily detected on the cell surface after transfection into HEK cells. The observation, however, that
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a1b1 or a1b2 subunit combinations can be clustered by rapsyn although no clustering site is present on a1 subunits, indicates that b1 and b2 subunits also do form sites that mediate clustering by rapsyn. This conclusion was con®rmed by other experiments indicating that a chimera consisting of the N-terminus of the b3 and the remaining part of the b2 subunit was transported to the surface of transfected cells and could be clustered by rapsyn. 4.5. Mechanism of clustering of GABAA receptors by rapsyn The present experiments do not provide information on the mechanism of clustering of GABAA receptors by rapsyn. However, little is known about the mechanism of clustering of the nACh receptor either. Although rapsyn precisely colocalizes with nACh receptor at the neuromuscular junction and is able to induce clustering of this receptor in recombinant systems, so far no biochemical interaction between rapsyn and the nACh receptor has been demonstrated. In addition, no site has been identi®ed on nACh receptor subunits that mediates clustering by rapsyn (Maimone and Merlie, 1993; Wheeler et al., 1994; Yu and Hall, 1994). And ®nally, it is still not clear whether rapsyn interacts directly with the receptor or whether other proteins are involved in this interaction. Recently, a sequence motif of rapsyn has been identi®ed that might be responsible for clustering of nACh receptors (Ramarao and Cohen, 1998). The authors propose that rapsyn interacts directly with subunits of the nACh receptor via a coiled-coil motif. It will be interesting to investigate whether the same motif of rapsyn is involved in clustering GABAA receptors. 4.6. Is rapsyn involved in clustering of native GABAA receptors? Receptors consisting of a1b2g2 or a1b3g2 subunits are the major GABAA receptors in the brain (Benke et al., 1991). An assembly with b subunits is necessary for the transport of a and g subunits to the cell membrane, as shown in this and previous studies (Connolly et al., 1996a,b). In addition, the binding site for GABA is formed at the interface of a and b subunits (Sieghart, 1995; Smith and Olsen, 1995). Beta subunits, thus, are indispensable for the formation of the majority of GABAA receptors. Only r subunits seem not to require b subunits for correct assembly (Johnston, 1996). Since b1, b2, as well as b3 subunits do contain clustering sites, it has to be concluded that most, if not all GABAA receptors can be clustered by rapsyn. So far no report has been published indicating a possible colocalization of rapsyn and native GABAA receptors. Rapsyn is predominantly located in peripheral
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tissues, where it mediates clustering of nACh receptors (Musil et al., 1989). Evidence has accumulated indicating that GABAA receptors are also expressed in peripheral tissues and are involved in the modulation of smooth muscle contraction and in a variety of other functions (ErdoÈ and Wol, 1990; Greenwood Van Meerveld and Barron, 1998; ParramoÂn et al., 1995). Further experiments will have to clarify whether GABAA receptors in these tissues are clustered by rapsyn. Recently, it has been demonstrated that rapsyn mRNA is also present in the brain (Burns et al., 1997; Yang et al., 1997). So far, however, the question whether rapsyn or a rapsyn isoform (Burns et al., 1997) mediates clustering of native GABAA receptors in the brain has not been investigated. But the ability of rapsyn to cluster the majority of GABAA receptors does not necessarily mean that the majority of native GABAA receptors are clustered by rapsyn. It is possible that GABAA receptors are clustered by rapsyn only in certain tissues or cells or at certain stages of development. Recently, it has been demonstrated that rapsyn is not essential for clustering of neuronal nACh receptor in superior cervical ganglion, although it was able to cluster both muscle and neuronal ACh receptors in heterologous expression systems (Feng et al., 1998). Thus, dierent tissues can use dierent proteins for clustering of receptors. In addition, it is possible that clusters containing rapsyn (Yoshihara and Hall, 1993) or gephyrin (Colin et al., 1998; Kirsch et al., 1993) only provide a matrix for anchoring proteins that do interact with GABAA receptors. Then the same anchoring protein might possibly be recruited by dierent cluster forming proteins in dierent tissues. In any case, the strong clustering by rapsyn of many GABAA receptor subtypes observed in the present study indicates that the amino acid sequences on b1±3 or g2 subunits mediating this eect are easily accessible for cytoplasmic proteins and might therefore also mediate clustering of native GABAA receptors. The present observation that b3 subunits of GABAA receptors as well as b3-g2 or b3-b2 chimeras can be clustered by rapsyn on the surface of transfected cells, can be used to investigate the clustering of suitable chimeras modi®ed by site directed mutagenesis. This will allow to identify amino acid sequences on bx or g2 subunits mediating clustering by rapsyn. These sequences can then be used for the identi®cation of interacting proteins that possibly are involved in clustering of native GABAA receptors. Experiments in this direction are underway in our laboratory. Acknowledgements This work was supported by project SFB F610 of
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