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GABAB-receptor isoforms
Life Sciences 68 (2001) 2297–2300
GABAB-receptor isoforms Molecular architecture and distribution H. Möhler*, D. Benke, J.-M. Fritschy Institute of Pharmacology, Swiss Federal Institute of Technology (ETH) and University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland
Abstract The slow component of GABAergic inhibition in the brain is mediated by the metabotropic GABABreceptors. Most if not all GABAB-receptors are heterodimers of GABABR1 (GBR1) and GABABR2 (GBR2) proteins. Distinctive receptor isoforms are based on the presence of two GBR1 splice variants termed GBR1a and GBR1b. Both were found to be associated with GBR2 suggesting that the isoforms GBR1a/GBR2 and GBR1b/GBR2 represent the vast majority of GABAB-receptors in the brain. The two isoforms differed strikingly in their pattern of expression on the regional, cellular and subcellular level. These results point to distinct funcional roles of the two receptor isoforms. © 2001 Elsevier Science Inc. All rights reserved. Keywords: GABAB-Receptor subtype; GABAB-Receptor expression; Immunohistochemistry
Functional role of GABAB-receptors in neurotransmission Inhibitory neurotransmission is mainly mediated by g-aminobutiric acid (GABA) that displays a fast and a slow component. Whereas the fast inhibitory response results from the activation of the postsynaptically localized GABAA-receptors by triggering the opening of an integral chloride channel the slow GABA action is mediated by the metabotropic GABABreceptors. GABAB-receptors are present in most regions of the mammalian brain on presynaptic terminals as well as on postsynaptic neurons, where they interact via G-proteins with a variety of effector systems [1, 2]. Activation of presynaptic GABAB-receptors located on GABA-containing nerve terminals (autoreceptors) or terminals of various other neurons (heteroreceptors) suppresses the release of neurotransmitters, whereas the stimulation of postsynaptic receptors produces a prolonged neuronal hyperpolarization. The former mechanism appears to be mediated by the inhibition of an inward calcium conductance whereas the latter is * Corresponding author. Tel.: 141 1 635 59 11; fax: 141 1 635 57 08. E-mail address: [email protected] (H. Möhler) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 0 1 8 -9
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produced by an increase in potassium conductance. In both cases the GABAB-receptor is coupled via G-proteins to its conductance mechanism. GABAB-receptor variants Structurally, GABAB-receptors represent a novel principle of receptor assembly and signal transduction. Most, if not all, GABAB-receptors are heterodimers formed from two closely related seven transmembrane proteins that interact at the C-terminus in a stochiometry of 1:1, the GABABR1- and GABABR2-subunits. In addition, the GABABR1-receptor occurs in the two splice variants termed GABABR1a and GABABR1b (130 und 100 kDa, respectively). The heterodimer displays an agonist affinity that is comparable to that of native receptors. Furthermore, the heterodimer shows robust coupling to effector systems such as inhibition of forscolin-stimulated adenylate cyclase activity, coupling to a potassium channel (Kir 3.1) and coupling to voltage-dependent calcium channels. These results were largely achieved in studying GABAB-receptors in heterologous expression systems (for review [3]). However, the subunit architecture and the regional and subcellular distribution of GABAB-receptor subtypes in situ remained largely unknown. The GABAB-receptors variants where therefore analyzed with regard to their subunit composition as well as their regional and subcellular distribution in situ [4]. The analysis was based on the use of antisera recognizing selectively the receptor components GABABR1a (GBR1a), GABABR1b (GBR1b) and GABABR2 (GBR2). Following their solubilization from brain tissue, GBR1a and GBR1b where both found by immunoprecipitation to occur as heterodimers each associated with GBR2. Furthermore, monomers of GBR1a, GBR1b or GBR2 were not detectable, suggesting that practically all GABABreceptors are heterodimers in situ. Finally, there was no evidence for an association of GBR1a with GBR1b indicating that these two constituents represent two different receptor population. Thus, these two receptor subtypes appear to be the prevalent structure of GABAB-receptors [4]. Regulation of GABAB-receptor expression A distinctive localization of the GABAB-receptor isoforms has not yet been accomplished. The cellular and subcellular localization of the GBR1a and GBR1b isoforms was therefore investigated in rat brain using specific antibodies in combination with confocal laser scanning microscopy and electromicroscopy. The staining pattern of antibodies recognizing GBR1a,b and GBR1b was compared and the presence of the GBR1a isoform was deduced by subtractive analysis [5]. In newborn rats GBR1a immunoreactivity was found to be intense and uniformly distributed across the brain. In contrast, GABABR1b immunoreactivity was largely absent from the brain at birth with the exception of the habenula. It was only during the third postnatal week that GBR1b increased rapidly to become the predominant GBR1b isoform in cerebral cortex, thalamus and cerebellum in adult brain and in the spinal cord dorsal horn (Fig. 1). Thus, the two GABAB-receptors appear to serve distinct functional roles in developing and adult brain. A very intense GABABR1b staining was detected in the medial habenula and interpeduncular nucleus with distinct axonal labelling present in the fasciulus retroflexus which connects these two nuclei, suggesting an anterograde axonal transport of the receptor protein.
H. Möhler et al. / Life Sciences 68 (2001) 2297–2300
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Fig. 1. Comparitive regional distribution of GBR1b and GBR2 in adult rat brain, as seen by immunoperoxidase staining in parasagital sections using subunit-specific antisera. Note that while GBR2 is present is all regions stained for GBR1b (e.g., cerebral cortex, thalamus, cerebellum), it is in addition found in other regions (hippocampal formation, tectum, olfactory bulb), presumably associated with GBR1a. In the striatum, where no GBR2 mRNA has been reported, GBR2 staining is nevertheless prominent, suggesting its presence on afferent terminals. Scale bar, 5mm.
Most strikingly, the GBR1b isoform in addition to its synaptic localization was also found extrasynaptically. The presence of the GBR1a isoform was evident in the adult brain in the striatum, hippocampus, dentate gyrus and other areas indicating that GBR1a and GBR1b immunoreactivities correspond to distinct subtypes of GABAB-receptors with largely different distribution patterns in the CNS (Fig. 1). These results suggest distinct functional roles for the two isoforms of receptors in adult brain [5]. Receptor heterogeneity It is evident that the extensive pharmacological heterogeneity attributed to GABAB-receptors [6, 7, 8, 9] cannot be accounted for solely by the differential distribution of the GBR1a and GBR1b isoforms. For example, Calabresi et al. [10] postulated various distinct receptor subtypes in the neostriatum, in which GB1a appears to be virtually the only isoform expressed. Post-translational modifications of GABAB-isoforms e.g. protein phosphorylation and coupling to different effector systems are likely to add to the functional and pharmacological receptor heterogeneity. In the future, gene knock-out experiments, performed in a region or
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cell-specific manner, will help to identify the role of GABAB-receptors in complex CNS functions and pathologies. Recognizing the regional and cellular relevance of GABAB-receptors will be essential in identifying the receptor isoforms as novel drug targets. A new pharmacology of GABAB-receptors may arise from the progress in the recognition of receptor modifications and novel signal transduction cascades. References 1. Bowery, N.G. Pharmacology of mammalian GABAB-receptors. In: S.J. Enna and N.G. Bowery editors: The GABA receptors, Totowa N. J., Humana Press, 1997; pp. 209–236. 2. Möhler, H. and Fritschy, J.-M. GABAB-receptors make it to the top – as dimers. Trends Pharm. Sci. 1999; 20, 87–89. 3. Bettler, B., Kaupmann, K. and Bowery N. GABAB-receptors: drugs meet clones. Curr. Opinion Neurobiol. 1998; 8, 345–350. 4. Benke, D., Honer, M., Michel, C. Bettler, B. and Möhler, H. g-Aminobutyric acid type B receptor splice variant proteins GBR1a and GBR1b are both associated with GBR2 in situ and display differential regional and subcellular distribution. J. Biol. Chem. 1999; 274, 27323–27330. 5. Fritschy, J.-M., Meskenaite, V., Weinmann, O., Honer, M., Benke, D. and Möhler, H. GABAB-receptor splice variants GB1a and GB1b in rat brain: developmental regulation, cellular distribution and extrasynaptic localization. Europ. J. Neurosci. 1999; 11, 761–768. 6. Bonanno, G. and Raiteri, M. g-Aminobutryric acid (GABA) autoreceptors in rat cerebral cortex and spinal cord represent pharmacologically distinct subtypes of the GABAB-receptor. J. Pharmacol. Exp. Ther. 1993; 265, 765–768. 7. Lanza, M., Fassio, A., Gemignani, A., Bonanno, G. and Raiteri, M. CGP 52432: a novel potent and selective GABAB autoreceptor antagonist in rat cerebral cortex. Eur. J. Pharmacol. 1993; 237, 191–195. 8. Cunningham, M.D. and Enna S. Evidence for pharmacologically distinct GABAB receptors associated with cAMP production in rat brain. Brain Res. 1996; 720, 220–224. 9. Deisz, R.A., Billard, J.M. and Zieglgansberger, W Presynaptic and postsynaptic GABAB receptors of neocortical neurons of the rat in vitro: differences in pharmacology and ionic mechanisms. Synapse 1997;25, 62–72. 10. Calabresi, P., Mercuri, N.B., De Murtas, M. and Bernardi, G. Endogenous GABA mediates presynaptic inhibition of spontaneous and evoked excitatory synaptic potentials in the rat neostriatum. Neurosci. Lett.1990; 118, 99–102.
GABAB-receptor isoforms Molecular architecture and distribution H. Möhler*, D. Benke, J.-M. Fritschy Institute of Pharmacology, Swiss Federal Institute of Technology (ETH) and University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland
Abstract The slow component of GABAergic inhibition in the brain is mediated by the metabotropic GABABreceptors. Most if not all GABAB-receptors are heterodimers of GABABR1 (GBR1) and GABABR2 (GBR2) proteins. Distinctive receptor isoforms are based on the presence of two GBR1 splice variants termed GBR1a and GBR1b. Both were found to be associated with GBR2 suggesting that the isoforms GBR1a/GBR2 and GBR1b/GBR2 represent the vast majority of GABAB-receptors in the brain. The two isoforms differed strikingly in their pattern of expression on the regional, cellular and subcellular level. These results point to distinct funcional roles of the two receptor isoforms. © 2001 Elsevier Science Inc. All rights reserved. Keywords: GABAB-Receptor subtype; GABAB-Receptor expression; Immunohistochemistry
Functional role of GABAB-receptors in neurotransmission Inhibitory neurotransmission is mainly mediated by g-aminobutiric acid (GABA) that displays a fast and a slow component. Whereas the fast inhibitory response results from the activation of the postsynaptically localized GABAA-receptors by triggering the opening of an integral chloride channel the slow GABA action is mediated by the metabotropic GABABreceptors. GABAB-receptors are present in most regions of the mammalian brain on presynaptic terminals as well as on postsynaptic neurons, where they interact via G-proteins with a variety of effector systems [1, 2]. Activation of presynaptic GABAB-receptors located on GABA-containing nerve terminals (autoreceptors) or terminals of various other neurons (heteroreceptors) suppresses the release of neurotransmitters, whereas the stimulation of postsynaptic receptors produces a prolonged neuronal hyperpolarization. The former mechanism appears to be mediated by the inhibition of an inward calcium conductance whereas the latter is * Corresponding author. Tel.: 141 1 635 59 11; fax: 141 1 635 57 08. E-mail address: [email protected] (H. Möhler) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 0 1 8 -9
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H. Möhler et al. / Life Sciences 68 (2001) 2297–2300
produced by an increase in potassium conductance. In both cases the GABAB-receptor is coupled via G-proteins to its conductance mechanism. GABAB-receptor variants Structurally, GABAB-receptors represent a novel principle of receptor assembly and signal transduction. Most, if not all, GABAB-receptors are heterodimers formed from two closely related seven transmembrane proteins that interact at the C-terminus in a stochiometry of 1:1, the GABABR1- and GABABR2-subunits. In addition, the GABABR1-receptor occurs in the two splice variants termed GABABR1a and GABABR1b (130 und 100 kDa, respectively). The heterodimer displays an agonist affinity that is comparable to that of native receptors. Furthermore, the heterodimer shows robust coupling to effector systems such as inhibition of forscolin-stimulated adenylate cyclase activity, coupling to a potassium channel (Kir 3.1) and coupling to voltage-dependent calcium channels. These results were largely achieved in studying GABAB-receptors in heterologous expression systems (for review [3]). However, the subunit architecture and the regional and subcellular distribution of GABAB-receptor subtypes in situ remained largely unknown. The GABAB-receptors variants where therefore analyzed with regard to their subunit composition as well as their regional and subcellular distribution in situ [4]. The analysis was based on the use of antisera recognizing selectively the receptor components GABABR1a (GBR1a), GABABR1b (GBR1b) and GABABR2 (GBR2). Following their solubilization from brain tissue, GBR1a and GBR1b where both found by immunoprecipitation to occur as heterodimers each associated with GBR2. Furthermore, monomers of GBR1a, GBR1b or GBR2 were not detectable, suggesting that practically all GABABreceptors are heterodimers in situ. Finally, there was no evidence for an association of GBR1a with GBR1b indicating that these two constituents represent two different receptor population. Thus, these two receptor subtypes appear to be the prevalent structure of GABAB-receptors [4]. Regulation of GABAB-receptor expression A distinctive localization of the GABAB-receptor isoforms has not yet been accomplished. The cellular and subcellular localization of the GBR1a and GBR1b isoforms was therefore investigated in rat brain using specific antibodies in combination with confocal laser scanning microscopy and electromicroscopy. The staining pattern of antibodies recognizing GBR1a,b and GBR1b was compared and the presence of the GBR1a isoform was deduced by subtractive analysis [5]. In newborn rats GBR1a immunoreactivity was found to be intense and uniformly distributed across the brain. In contrast, GABABR1b immunoreactivity was largely absent from the brain at birth with the exception of the habenula. It was only during the third postnatal week that GBR1b increased rapidly to become the predominant GBR1b isoform in cerebral cortex, thalamus and cerebellum in adult brain and in the spinal cord dorsal horn (Fig. 1). Thus, the two GABAB-receptors appear to serve distinct functional roles in developing and adult brain. A very intense GABABR1b staining was detected in the medial habenula and interpeduncular nucleus with distinct axonal labelling present in the fasciulus retroflexus which connects these two nuclei, suggesting an anterograde axonal transport of the receptor protein.
H. Möhler et al. / Life Sciences 68 (2001) 2297–2300
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Fig. 1. Comparitive regional distribution of GBR1b and GBR2 in adult rat brain, as seen by immunoperoxidase staining in parasagital sections using subunit-specific antisera. Note that while GBR2 is present is all regions stained for GBR1b (e.g., cerebral cortex, thalamus, cerebellum), it is in addition found in other regions (hippocampal formation, tectum, olfactory bulb), presumably associated with GBR1a. In the striatum, where no GBR2 mRNA has been reported, GBR2 staining is nevertheless prominent, suggesting its presence on afferent terminals. Scale bar, 5mm.
Most strikingly, the GBR1b isoform in addition to its synaptic localization was also found extrasynaptically. The presence of the GBR1a isoform was evident in the adult brain in the striatum, hippocampus, dentate gyrus and other areas indicating that GBR1a and GBR1b immunoreactivities correspond to distinct subtypes of GABAB-receptors with largely different distribution patterns in the CNS (Fig. 1). These results suggest distinct functional roles for the two isoforms of receptors in adult brain [5]. Receptor heterogeneity It is evident that the extensive pharmacological heterogeneity attributed to GABAB-receptors [6, 7, 8, 9] cannot be accounted for solely by the differential distribution of the GBR1a and GBR1b isoforms. For example, Calabresi et al. [10] postulated various distinct receptor subtypes in the neostriatum, in which GB1a appears to be virtually the only isoform expressed. Post-translational modifications of GABAB-isoforms e.g. protein phosphorylation and coupling to different effector systems are likely to add to the functional and pharmacological receptor heterogeneity. In the future, gene knock-out experiments, performed in a region or
2300
H. Möhler et al. / Life Sciences 68 (2001) 2297–2300
cell-specific manner, will help to identify the role of GABAB-receptors in complex CNS functions and pathologies. Recognizing the regional and cellular relevance of GABAB-receptors will be essential in identifying the receptor isoforms as novel drug targets. A new pharmacology of GABAB-receptors may arise from the progress in the recognition of receptor modifications and novel signal transduction cascades. References 1. Bowery, N.G. Pharmacology of mammalian GABAB-receptors. In: S.J. Enna and N.G. Bowery editors: The GABA receptors, Totowa N. J., Humana Press, 1997; pp. 209–236. 2. Möhler, H. and Fritschy, J.-M. GABAB-receptors make it to the top – as dimers. Trends Pharm. Sci. 1999; 20, 87–89. 3. Bettler, B., Kaupmann, K. and Bowery N. GABAB-receptors: drugs meet clones. Curr. Opinion Neurobiol. 1998; 8, 345–350. 4. Benke, D., Honer, M., Michel, C. Bettler, B. and Möhler, H. g-Aminobutyric acid type B receptor splice variant proteins GBR1a and GBR1b are both associated with GBR2 in situ and display differential regional and subcellular distribution. J. Biol. Chem. 1999; 274, 27323–27330. 5. Fritschy, J.-M., Meskenaite, V., Weinmann, O., Honer, M., Benke, D. and Möhler, H. GABAB-receptor splice variants GB1a and GB1b in rat brain: developmental regulation, cellular distribution and extrasynaptic localization. Europ. J. Neurosci. 1999; 11, 761–768. 6. Bonanno, G. and Raiteri, M. g-Aminobutryric acid (GABA) autoreceptors in rat cerebral cortex and spinal cord represent pharmacologically distinct subtypes of the GABAB-receptor. J. Pharmacol. Exp. Ther. 1993; 265, 765–768. 7. Lanza, M., Fassio, A., Gemignani, A., Bonanno, G. and Raiteri, M. CGP 52432: a novel potent and selective GABAB autoreceptor antagonist in rat cerebral cortex. Eur. J. Pharmacol. 1993; 237, 191–195. 8. Cunningham, M.D. and Enna S. Evidence for pharmacologically distinct GABAB receptors associated with cAMP production in rat brain. Brain Res. 1996; 720, 220–224. 9. Deisz, R.A., Billard, J.M. and Zieglgansberger, W Presynaptic and postsynaptic GABAB receptors of neocortical neurons of the rat in vitro: differences in pharmacology and ionic mechanisms. Synapse 1997;25, 62–72. 10. Calabresi, P., Mercuri, N.B., De Murtas, M. and Bernardi, G. Endogenous GABA mediates presynaptic inhibition of spontaneous and evoked excitatory synaptic potentials in the rat neostriatum. Neurosci. Lett.1990; 118, 99–102.