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Molecular Structure and Physiological Functions of GABA_B Receptors

Pharmazentrum, Department of Clinical-Biological Sciences, Institute of Physiology, University of Basel, and Novartis Institutes for Biomedical Research, Novartis Pharma AG, Basel, Switzerland

ABSTRACT

I. INTRODUCTION

II. NATIVE GABAB RECEPTORS

A. Coupling to G Proteins

B. Coupling to Ca2+ Channels

C. Coupling to K+ Channels

D. Coupling to Adenylyl Cyclase

E. Pharmacology of Native GABAB Receptors

III. CLONED GABAB RECEPTORS

A. Molecular Structure

1. Expression cloning of GABAB receptors

2. GABAB receptor heteromerization

3. Invertebrate GABAB receptors

4. GABAB-receptor isoforms

A) PREDOMINANT SUBUNIT VARIANTS: GABAB(1A) AND GABAB(1B).

B) MINOR SUBUNIT VARIANTS.

5. The GABA binding site

6. The Ca2+ binding site

7. Molecular determinants of G protein coupling

A) MOLECULAR DETERMINANTS IN THE G PROTEIN.

B) MOLECULAR DETERMINANTS IN GABAB RECEPTOR SUBUNITS.

8. Intra- and intermolecular events controlling receptor function

9. Interacting proteins

10. Phosphorylation and desensitization studies

B. Molecular Studies on Native GABAB Receptors

1. GABAB-deficient mice

2. GHB activity at GABAB receptors

3. Cellular and subcellular distribution

A) MRNA DISTRIBUTION.

B) RECEPTOR IMMUNOHISTOCHEMISTRY AND AUTORADIOGRAPHY.

C) GABAB RECEPTORS IN PERIPHERAL TISSUES.

IV. IMPLICATION IN DISEASE

A. Addiction

B. Epilepsy

C. Gastrointestinal Disease

D. Nociception

E. Genetic Linkage Studies

V. NOVEL GABAB COMPOUNDS

A. Allosteric Modulators

B. Subtype-Selective Ligands

VI. SUMMARY AND OUTLOOK

ACKNOWLEDGMENTS

REFERENCES

	ABSTRACT

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	I. INTRODUCTION

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GABA is the major inhibitory neurotransmitter in the central nervous system (CNS) and as such plays a key role in modulating neuronal activity. GABA mediates its action via distinct receptor systems, the ionotropic GABA_A and metabotropic GABA_B receptors. Unlike GABA_A receptors that form ion channels, GABA_B receptors address second messenger systems through the binding and activation of guanine nucleotide-binding proteins (G proteins; for other recent reviews on GABA_B receptors, see Refs. 45, 55). Dysfunction of GABA-mediated synaptic transmission in the CNS is believed to underlie various nervous system disorders. For example, hypoactivity of the GABA system was linked to epilepsy, spasticity, anxiety, stress, sleep disorders, depression, addiction, and pain. On the contrary, hyperactivity of the GABAergic system was associated with schizophrenia (20). GABA research, because of its medical relevance, has always attracted a great deal of attention in academia and industry. Over the years pharmaceutical companies successfully exploited the GABA system and introduced a number of drugs to the market. However, despite considerable drug-discovery efforts, baclofen (-chlorophenyl-GABA, Lioresal) currently remains the only available GABA_B medication. Baclofen, a lipophilic derivative of GABA, was synthesized in 1962 in an attempt to enhance the blood-brain barrier penetrability of the endogenous neurotransmitter. Baclofen was introduced to the market in 1972 and is used to treat spasticity and skeletal muscle rigidity in patients with spinal cord injury, multiple sclerosis, amyotrophic lateral sclerosis, and cerebral palsy (44). GABA_B agonists showed promising therapeutic effects in a whole range of other indications, but their side effects, including sedation, tolerance, and muscle relaxation, prevented further development (see sect. IV). Many researchers in the field assumed that a dissociation of the therapeutic effects from the side effects was achievable with more selective GABA_B drugs. This assumption was based on a large body of literature suggesting the existence of pharmacologically distinct GABA_B receptor subtypes in the brain (see sect. IIE). A more selective interference with the GABA_B system appeared feasible but crucially depended on the cloning of the predicted receptor subtypes. Therefore, it was mostly commercial interests that were driving efforts to isolate a GABA_B receptor cDNA. GABA_B receptors were not cloned until 1997 and thus remained the last of the major neurotransmitter receptors to be characterized at the molecular level (169). At first glance this is quite surprising, considering that GABA_B receptors were identified as early as in 1980 (46). In retrospect, it is clear that many cloning attempts failed because of the unexpected properties of GABA_B receptors. We therefore review some of the strategies that were applied when trying to isolate GABA_B receptors and discuss why these approaches failed (see sect. IIIA1). Identification of the first GABA_B cDNAs renewed commercial interests in these receptors for a number of reasons. Most importantly, the cloning generated the necessary tools to isolate the expected additional members of the GABA_B receptor family. Moreover, high-throughput compound screening, using molecularly defined receptors and functional assay systems, suddenly became practicable. GABA_B receptors were now also amenable to gene-targeting technology. This was supposed to help validate the most promising drug targets and at the same time provide a means to determine the specificity of GABA_B compounds in vivo. Throughout this review emphasis is given to developments in the field that followed the initial cloning of GABA_B receptors. However, where molecular findings extend or challenge earlier work, special attention is given to older literature.

As a consequence of intense research efforts that followed isolation of the first GABA_B cDNAs, several laboratories reported the cloning of a second GABA_B receptor cDNA, termed GABA_B(2) (see sect. IIIA2). Several groups published a most important discovery related to GABA_B(2). As shown for the first time for a G protein-coupled receptor (GPCR), the GABA_B receptor was not a single protein but instead consisted of two distinct subunits, neither of which was functional on its own. This finding was of interest to a large scientific community, and very quick progress was made in dissecting the roles of the individual subunits in receptor activation, assembly, and signaling (see sect. IIIA). Much of this review is devoted to this topic, as it fundamentally changed our view on the structure and functioning of GPCRs. The search for GABA_B receptor subtypes did not lead to the expected identification of additional GABA_B cDNAs, although a number of GABA_B-related cDNAs were identified in the process. The apparent lack of molecular heterogeneity was a surprise to many in the field who expected a variety of pharmacologically distinct GABA_B subunits, as predicted from work on native receptors. Efforts therefore turned toward identifying GABA_B isoforms (see sect. IIIA4), receptor-associated proteins (see sect. IIIA9), receptor modifications (see sect. IIIA10), and endogenous factors (see sect. IIIA6) that possibly were responsible for generating pharmacological differences. Once more, some unexpected findings were made. Several groups reported that transcription factors, such as CREB2/ATF-4, are able to directly interact with GABA_B receptors. At the same time, with the puzzling lack of pharmacologically distinct receptor subtypes, it became important to understand to which known GABA_B functions the cloned receptor subunits contribute in vivo. To address this question, many laboratories studied the expression of cloned GABA_B subunits in vivo (see sect. IIIB3) or disabled GABA_B genes in mice (see sect. IIIB1), which greatly clarified the role of individual subunits in GABA_B receptor physiology. The overt phenotypes of GABA_B knockout mice also pointed at the neuronal systems that crucially depend on GABA_B-receptor activity. A long-standing question in the field concerns the relationship between GABA_B receptors and the receptors for -hydroxybutyrate (GHB), a metabolite of GABA and emerging drug of abuse. It is a matter of much debate whether specific GHB receptors exist and whether they are related to GABA_B receptors. This question was addressed using molecular tools (see sect. IIIB2). Following receptor cloning, several studies tried to establish a link between polymorphisms in GABA_B genes and congenital human diseases (see sect. IVE). Taking recent developments in the field into account, we also touch on the most promising indications for GABA_B drugs (see sect. IV). Last but not least, the cloned receptors were used to establish high-throughput compound screens based on functional assays, which yielded the first allosteric compounds acting at GABA_B receptors (see sect. VA).

	II. NATIVE GABAB RECEPTORS

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A. Coupling to G Proteins

Bowery et al. (46) were the first to discover that GABA reduces norepinephrine release by activating a bicuculline- and isoguvacine-insensitive receptor, which they named the GABA_B receptor. Evidence for a coupling of GABA_B receptors to G proteins came from the sensitivity of agonist affinity to GTP analogs (10, 144). Studies using N-ethylmaleimide (NEM), islet activated protein (IAP), pertussis toxin, or antisense knock-down provided evidence that GABA_B receptors predominantly couple to G_i- and G_o-type G proteins (9, 59, 122, 223, 228). It is now well established that presynaptic GABA_B receptors repress Ca²⁺ influx by inhibiting Ca²⁺ channels in a membrane-delimited manner via the G subunits (see sect. IIB). Postsynaptic GABA_B receptors trigger the opening of K⁺ channels, again through the G subunits (see sect. IIC). This results in a hyperpolarization of the postsynaptic neuron that underlies the late phase of inhibitory postsynaptic potentials (IPSPs) (201). Besides modulating ion channels through G, GABA_B receptors activate and inhibit adenylyl cyclase via the G_i/G_o and G subunits (see sect. IID). Recent work by Hirono et al. (145) shows that GABA_B activity enhances mGlu1 responses at excitatory synapses in the cerebellum. This facilitation is suggested to require Ca²⁺ release from internal stores through a mechanism that involves G_i/G_o-linked phospholipase C (PLC) activation and a cooperative upregulation of GPCR signaling by the G subunits. This is reminiscent of synergistic interactions seen with GABA_B and -adrenergic receptors (42). Importantly, G protein-independent GABA_B effects on neurotransmitter release were also suggested (132).

B. Coupling to Ca²⁺ Channels

Presynaptic GABA_B receptors are subdivided into those that control GABA release (autoreceptors) and those that inhibit all other neurotransmitter release (heteroreceptors). In most preparations, GABA_B receptors mediate their presynaptic effects through a voltage-dependent inhibition of high-voltage activated Ca²⁺ channels of the N type (Ca_v2.2) or P/Q type (Ca_v2.1) (4, 61, 223, 225, 236, 261, 265, 299, 320). Both types of Ca²⁺ channels are expressed in presynaptic terminals and were shown to trigger neurotransmitter release (349). A postsynaptic inhibition of Ca²⁺ channels by GABA_B receptors was also postulated (130). It was shown that GABA_B receptors couple to different types of Ca²⁺ channels depending on the input site (266). The inhibition of Ca²⁺ inward currents is voltage dependent and varies between 10 and 42% among studies (73, 78, 294). Since Ca²⁺ influx and transmitter release are correlated with a third to fourth power law (349), GABA_B agonists frequently inhibit more than 90% of neurotransmitter release with a less than 50% inhibition of Ca²⁺-channel activity. This inhibition is modulated by the action potential frequency, where strong depolarization relieves Ca²⁺ channels from their G-mediated inhibition (140, 152, 354). This particular property of presynaptic Ca²⁺ channels may differentially modulate action potential trains, depending on their frequency (53). GABA_B receptors are also described to either inhibit (4, 202, 208) or facilitate (309) L-type Ca²⁺ channels. The latter effect was shown to be indirect and to depend on protein kinase C (PKC) activity. Similarly, GABA_B receptors also inhibit or disinhibit T-type Ca²⁺ channels (83, 107, 219, 303, 304).

C. Coupling to K⁺ Channels

GABA_B receptors induce a slow inhibitory postsynaptic current (late IPSC) through activation of inwardly rectifying K⁺ channels (GIRK or Kir3) (201, 300). Accordingly, GABA_B-induced late IPSCs can be inhibited by the Kir3 channel blocker Ba²⁺ (159, 264, 325), and they usually exhibit a reversal potential similar to the K⁺ equilibrium potential (188, 220). The physiological effect of Kir3-channel activation is normally a K⁺ efflux, resulting in a hyperpolarization. The time course of the late IPSC, with a time to peak of 50–250 ms and decay times of 100 and 500 ms, clearly differs from that of the fast IPSC, which is GABA_A receptor mediated (188, 249). Baclofen-induced outward currents in hippocampal neurons are absent in Kir3.2 and GABA_B(1) knockout mice, corroborating the prominent role of Kir3 channels in mediating the effects of GABA_B receptors (201, 300). The rectification properties of synaptically evoked late IPSCs differed between studies. On the one hand, the stimulus-evoked and spontaneous late IPSCs in dopaminergic neurons are inwardly rectifying and similar to those activated by baclofen (134). On the other hand, baclofen also induces linear or even outwardly rectifying conductances, suggesting that channels other than Kir3 can contribute to the late IPSC. These other channels may include fast inactivating, voltage-gated K⁺ channels (289) and small-conductance Ca²⁺-activated K⁺ channels (SK channels) (116). Accordingly, the fast inactivating A-type K⁺-channel blocker 4-aminopyridine inhibits a baclofen-induced current in guinea pig hippocampal neurons (153, 243). Moreover, GABA activates Ca²⁺-sensitive K⁺ channels (36) and small-conductance K⁺ channels (36, 89) in rat hippocampal neurons. Possibly, GABA_B receptors enhance the activity of SK channels by inhibiting the production of cAMP after an action potential-induced Ca²⁺ influx (116). In addition to the well-documented coupling of GABA_B receptors to postsynaptic K⁺ channels, GABA_B receptors also appear to activate Ba²⁺-sensitive K⁺ channels at presynaptic sites (325). Likely these presynaptic K⁺ channels are of the Kir3 type, devoid of the Kir3.2 subunit and assembled from Kir3.1 and Kir3.4 subunits (201).

D. Coupling to Adenylyl Cyclase

All of the known nine adenylyl cyclase isoforms are expressed in neuronal tissue. G_i and G_o proteins, the predominant transducers of GABA_B receptors, inhibit most of them (311). Many studies have reported that GABA_B receptors inhibit forskolin-stimulated cAMP formation, but others also observed a stimulation of cAMP production (45, 55). G_i and G_o proteins inhibit adenylyl cyclase types I, III, V, and VI, while G stimulates adenylyl cyclase types II, IV, and VII. This stimulation depends on the presence of G_s, which results from the activation of GPCRs by, e.g., norepinephrine, isoprenaline, histamine, or vasoactive intestinal polypeptide (311, 321). Therefore, the stimulatory action of GABA_B receptors on cAMP levels is a consequence of G protein cross-talk and depends on the expression of adenylyl cyclase isoforms together with GABA_B and G_s-coupled GPCRs. Both the inhibition and enhancement of cAMP levels by GABA_B receptor activation were confirmed in vivo using microdialysis (133). Many ion channels are targets of the cAMP-dependent kinase (protein kinase A or PKA). Accordingly, a GABA_B receptor-mediated modulation of K⁺ channels via cAMP was reported (116). Significantly, the activity of GABA_B receptors on adenylyl cyclase is expected to modulate neuronal function on a longer time scale (see sect. IIIA9).

GABA_B receptors were repeatedly implicated in synaptic plasticity (87, 229, 246, 247, 334). Until recently, it was unclear whether GABA_B receptors can influence plasticity processes through the cAMP pathway. Recent experiments now demonstrate that G protein-mediated signaling through GABA_B receptors retards the recruitment of synaptic vesicles during sustained activity and after short-term depression (290). This retardation occurs through a lowering of cAMP, which blocks the stimulatory effect of the increased Ca²⁺ concentration on vesicle recruitment. In this signaling pathway, cAMP and Ca²⁺/calmodulin cooperate to enhance vesicle priming.

E. Pharmacology of Native GABA_B Receptors

Bowery et al. (45) recently published a comprehensive and detailed review on currently available GABA_B ligands (45). Here, we only focus on one particular aspect of GABA_B receptor pharmacology: the discrepancies in the potency of agonists and antagonists in different biochemical and physiological paradigms. Numerous biochemical studies indicate pharmacological differences between auto- and heteroreceptors and even within auto- and heteroreceptors (15, 39–41, 248, 323). However, the proposal of presynaptic receptor subtypes based on neurotransmitter release experiments has been open to dispute (336). Electrophysiological and release experiments suggest distinctions between pre- and postsynaptic GABA_B receptors as well (65, 76, 85, 88, 96, 115, 262, 268, 325, 352). Accordingly, published half-maximal effective concentrations for baclofen in pharmacological studies vary considerably and range between 100 nM and 100 µM (Table 1).

	III. CLONED GABAB RECEPTORS

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A. Molecular Structure

1. Expression cloning of GABA_B receptors

GABA_B receptors were cloned in 1997 (169), close to 20 years after their discovery by Bowery et al. (46). In retrospect, we understand the reasons that prevented an earlier isolation of GABA_B cDNAs. To begin with, a purification of GABA_B receptor proteins proved difficult. There were no radioligands that bound irreversibly or with high affinity to the receptor. Moreover, receptor function was inevitably lost in the presence of solubilizing concentrations of detergents, most likely because of the dissociation of GABA_B(1) and GABA_B(2) subunits. This made it impossible to trace the protein during the purification steps using functional assay systems, such as, e.g., GTP[³⁵S] binding. Nevertheless, the purification of a putative 80-kDa GABA_B-receptor protein was reported (233). The molecular mass of the 80-kDa GABA_B protein does not match the molecular mass of cloned GABA_B subunits, and a relationship with the latter is unlikely. No amino acid sequence of the 80-kDa protein was disclosed, and hence, its molecular structure remains enigmatic. As a consequence of the problems associated with purifying a GABA_B-receptor protein, no antibodies for cloned GABA_B subunits were available prior to cloning.

In pregenome times, expression cloning was the most successful approach for isolation of a neurotransmitter receptor. In essence, expression cloning circumvents the requirement for protein purification and instead uses a specific biological activity as the basis of cDNA identification. An inherent advantage of such a screening approach is that the isolated cDNA clones are usually full-length and immediately available for functional studies. Xenopus oocytes are the expression system of choice when using electrophysiological screening techniques. The method relies on the ability of oocytes to translate protein from injected cRNA (RNA transcribed from cDNA). Expressed receptors in the oocyte membrane can then be detected using two-electrode voltage-clamp or similar recording techniques. This procedure was used to isolate a number of G protein-coupled neurotransmitter receptors, including mGlu receptors (218). Several laboratories, including ours, explored strategies using Xenopus oocytes when attempting to clone a GABA_B cDNA. Because GABA_B receptors do not couple to PLC, it was impossible to apply expression-cloning strategies based on increases in intracellular [Ca²⁺] and subsequent activation of Ca²⁺-activated Cl^– channels. Others and we therefore supplied Xenopus oocytes with effector Kir3 channels (201) or promiscuous G proteins that permitted the detection of weak functional GABA_B responses (306). We eventually abandoned expression cloning in Xenopus oocytes because the electrophysiological GABA_B responses were unreliable and were lost in the process of splitting the brain cDNA pool into smaller pools. This loss of functional responses is readily explained by the fact that both GABA_B(1) and GABA_B(2) subunits are required to assemble a functional receptor. The concomitant serial isolation of the two distinct cDNA clones is virtually impossible because the functional response is missing whenever the two cDNAs segregate into different pools in the course of narrowing down the active cDNAs.

We reasoned that for the isolation of a GABA_B cDNA, a screening approach using a radioligand binding assay rather than an electrophysiological read-out would be more promising. Such a screening does not depend on a functional coupling of the receptor and solely relies on the exposure of a high-affinity binding site. With the development of high-affinity GABA_B radioligand antagonists, such as ¹²⁵I-CGP64213 (K_d = 1.2 ± 0.2 nM), expression cloning using a binding assay became feasible and allowed the isolation of GABA_B(1a) and GABA_B(1b) cDNAs (169). The two encoded proteins derive from the same gene and differ in their extracellular NH₂-terminal domains (see sect. IIIA4A). The molecular structure of the cloned GABA_B proteins revealed all the hallmarks of a GPCR. Specifically, the sequence of GABA_B(1) subunits exhibited seven transmembrane domains and similarity with Family 3 (also named Family C) GPCRs. It is now known that Family 3 GPCRs comprise metabotropic glutamate (mGlu), the Ca²⁺-sensing (CaS), vomeronasal, and taste receptors (Fig. 1).

View larger version (16K): [in this window] [in a new window]   FIG. 1. Phylogenetic tree of human Family 3 GPCRs. The predicted seven transmembrane domains for receptors were aligned using Clustal W (version 1.82) (324), using default parameters (gap open penalty, 10; gap extension penalty, 1.0; and protein weight matrix blosum). The receptors are as follows: GABAB(1) (GenBank accession number NW001470); GABAB(2) (AF056085); GABAB-related receptor, GABABL (XM093467); Ca2+-sensing receptor, CaSR (S83176); metabotropic glutamate receptors, mGluR1 (NM_000838), mGluR2 (NM_000839), mGluR3 (NM_000840), mGluR4 (NM_000841), mGluR5 (NM_000842), mGluR6 (NM_000843), mGluR7 (NM_000844), mGluR8 (NM_00845); taste receptors, T1R1 (NM_138697), TIR2 (AH011576), TIR3 (BK000152); and a family of orphan receptors, RAIG1 (AF095448), GPRC5B (AF202640), GPRC5C (AF207989), GPRC5D (NM_018654). Not indicated are the members of a large family of putative pheromone receptors that were identified in rodents.

While GABA_B(1) subunits showed many of the expected features of native GABA_B receptors in terms of structure and distribution, they surprisingly did not efficiently couple to their effector systems (214). Moss and colleagues (80) were the first to demonstrate that GABA_B(1) proteins are retained in the endoplasmatic reticulum (ER) when expressed in heterologous cells. This is taken to explain the 100- to 150-fold lower affinity for agonists that is observed with recombinant GABA_B(1) subunits compared with native GABA_B receptors (169). Presumably, the failure of GABA_B(1) to traffic to the cell surface in the absence of GABA_B(2) prevents the interaction with the G protein in the plasma membrane, which is necessary to stabilize the high-affinity conformation of the binding site (see sect. IIIA8). The search for a missing factor, which traffics GABA_B(1) subunits to the cell surface and renders them functional, therefore became an important objective. The search ended with the remarkable publication of three consecutive papers in Nature by groups from GlaxoWellcome, Novartis, and Synaptic Pharmaceuticals, as well as three subsequent papers from BASF-LYNX Bioscience, Merck, and the Laboratory of Molecular Neurobiology at Boston University (163, 170, 185, 216, 239, 343). All six papers describe the identification of the GABA_B(2) subunit, which must be coexpressed with GABA_B(1a) or GABA_B(1b) subunits to form a functional receptor. This finding represented the first compelling evidence for heteromerization among the GPCRs. Recombinant heteromeric GABA_B(1,2) receptors couple to all prominent effector systems of native GABA_B receptors, that is, adenylyl cyclase, Kir3-type K⁺ channels, and P/Q- and N-type Ca²⁺ channels (98, 100, 214). When the GABA_B(2) subunit is coexpressed with GABA_B(1), agonist potency more closely approximates that of native receptors (214). Kaupmann et al. (170) still observed a 10-fold lower affinity of recombinant GABA_B(1,2) receptors as opposed to brain receptors, which may be explained by limiting amounts of the G protein in the heterologous cells (170).

The reason for the intracellular retention of GABA_B(1) subunits is the presence of an ER-retention signal, the four-amino acid motif RSRR, in its cytoplasmic tail (210, 250). ER-retention signals of the RXR type were also observed in other multisubunit proteins, such as, for example, the K_ATP channels (355) or N-methyl-D-aspartate (NMDA) receptors (302). It was recently proposed that the sequence context of the RSRR motif in GABA_B(1) is crucial for ER retention, and the motif was extended to include the sequence QLQXRQQLRSRR (125). The ER-retention signal in GABA_B(1) is masked from its ER-anchoring mechanism through the interaction with the COOH terminus of GABA_B(2), thus allowing for delivery of the GABA_B(1,2) complex to the cell surface. This ER-retention mechanism is suggested to prevent incorrectly folded GABA_B receptors from reaching the cell surface and to represent a quality control mechanism. Some laboratories identified GABA_B(2) in the yeast two-hybrid system when using GABA_B(1) COOH-terminal sequences as bait, directly demonstrating that the COOH-terminal domains of GABA_B(1) and GABA_B(2) interact with each other (185, 343). Subsequent deletion analysis mapped the interaction to -helical coiled-coil domains of 32–35 amino acids in length. Coiled-coil domains are dimerization motifs that are found in numerous structural, trafficking, and regulatory proteins, such as, e.g., in leucine-zipper transcription factors. Mixing equimolar amounts of recombinant GABA_B(1) and GABA_B(2) peptides corresponding to the predicted coiled-coil domains indeed produced parallel coiled-coil heteromers under physiological conditions (166). In contrast, individual GABA_B(1) or GABA_B(2) peptides folded into relatively unstable homodimers or remained largely unstructured, suggesting that homodimeric receptors are not efficiently formed through coiled-coil interaction. Mutational analysis confirmed that the COOH-terminal coiled-coil interaction of GABA_B receptors is essential for surface trafficking of the heteromer (57, 210, 250). Surprisingly, however, this interaction is not an absolute requirement for assembly of the receptor complex. It was demonstrated that COOH-terminally truncated GABA_B(1) and GABA_B(2) subunits can form fully functional receptors when expressed in heterologous mammalian cells (57, 250). This indicates that the transmembrane domains and/or extracellular domains (ECDs) encode surfaces that are sufficient for heteromerization, which is in agreement with findings with the COOH-terminally truncated GABA_B(1e) splice variant (301). Furthermore, this agrees with data that suggest that other Family 3 GPCRs, e.g., the mGlu1, mGlu5, and CaS receptors, homodimerize in their ECDs (278, 286, 328).

Challenging the general assumption that GABA_B receptors necessarily need to heteromerize for function are infrequent responses seen with receptor subunits expressed in isolation. For example, GABA_B(2) was found to couple to adenylyl cyclase in the absence of GABA_B(1) (185, 216), which is at odds with the proposal that only GABA_B(1) subunits are able to bind ligands (179). Similarly, when expressed alone in heterologous cells, GABA_B(1) yields infrequent electrophysiological and small biochemical responses (169, 171). This suggests that GABA_B(1) could be functional either alone or in combination with an unknown protein. It is difficult to reconcile functional GABA_B(1) responses with the efficient ER retention observed with this subunit. Even when wild-type GABA_B(1) is artificially targeted to the cell surface by masking the ER-retention signal with the COOH-terminal domain of GABA_B(2), functional responses are not observed (250). It may be speculated that the weak and infrequent coupling of homomeric GABA_B(1) or GABA_B(2) to cAMP and Kir3 channels depends on presently unknown endogenous factors. On the other hand, it remains unclear whether occasional endogenous expression of the partner subunit in heterologous cells could be responsible for the rare responses that were seen when GABA_B(2) or GABA_B(1) was transfected alone. In this context it is interesting to compare GABA_B receptors with NMDA receptors that are similarly assembled with subunits that contain the RXR-type ER-retention signal (302). For unknown reasons, and similar to GABA_B subunits, the NMDA receptor subunit NR1 occasionally generates functional responses in HEK293 cells or Xenopus oocytes in the absence of the masking subunit (222).

Despite several observations suggesting that GPCRs could form dimers or higher-order oligomers (7), the conventional perception was that GPCRs exist at the cell surface as monomers that couple to G proteins. For the most part it is the characterization of GABA_B receptors that changed our view. It is now generally agreed that GPCRs can form homo- and heterodimers. In the last couple of years, it became more and more evident that heteromerization between GPCRs is not that rare a phenomenon. There are now several examples where even distantly related GPCRs form heteromeric complexes (1, 117, 164, 284). This raises fascinating combinatorial possibilities and may generate a level of regulatory and pharmacological diversity that we did not anticipate. Because GPCRs are major pharmacological targets, this recognition will have important implications for the development and screening of new drugs.

3. Invertebrate GABA_B receptors

Development and organization of the nervous system are substantially different between vertebrates and invertebrates. Neurotransmitters evolved the ability to activate a range of ion channels and second messenger systems, but their relative importance as sensory, inter- or motorneuron signaling molecules varies widely between phyla. Although the evidence is still sketchy, it currently appears that most mammalian neurotransmitter receptors have counterparts in invertebrates (337). Invertebrate GABA_B receptors were described in echinoderms (90, 91), mollusks (11, 287), and arthropods (101, 226, 254, 276, 335). Invertebrate GABA_B functions were mostly studied at the neuromuscular junction of echinoderms, where GABA elicits inhibitory and excitatory responses (90). GABA_B responses were also observed in invertebrate CNS neurons (150, 287). Like their mammalian orthologs, invertebrate GABA_B receptors activate G proteins and regulate K⁺ and Ca²⁺ currents (226, 287).

The only cloned invertebrate GABA_B receptors are those of Drosophila melanogaster (224). Two of the three subunits that were described, d-GABA_B(1) and d-GABA_B(2), show clear sequence identity to the mammalian subunits. A third subunit, d-GABA_B(3), seems to be specific for insects and is of unknown function. Surprisingly, Drosophila GABA_B receptors do not contain the sushi domains that are found in the mammalian GABA_B(1a) subunit (see sect. IIIA4A), although sushi domains exist in the Drosophila genome. For example, sushi repeats are found in hikaru genki, a Drosophila protein that is secreted from presynaptic terminals during the period of synapse development (148). Analysis of the expression pattern in the embryonic nervous system revealed that d-GABA_B(1) and d-GABA_B(2) are expressed in overlapping regions. Upon coexpression in Xenopus oocytes or mammalian cell lines, d-GABA_B(1) and d-GABA_B(2) form a functional heteromeric d-GABA_B(1,2) receptor that couples to G_i/G_o-type G proteins. Therefore, heteromerization is not only a prerequisite for mammalian but also for invertebrate GABA_B function. d-GABA_B(1,2) receptors exhibit a unique pharmacology. GABA and 3-aminopropylphosphonous acid (3-APPA) are, as expected, d-GABA_B(1,2) agonists. However, baclofen has no longer agonistic properties and instead acts as an antagonist. Furthermore, neither saclofen nor CGP35348antagonizes d-GABA_B(1,2) receptors, in contrast to their activity at mammalian receptors. d-GABA_B(1,2) receptors closely reproduce the pharmacology that was described for insect GABA_B receptors (12, 150, 295) and therefore do not suggest the existence of additional GABA_B subunits.

Physiological or pharmacological approaches have not found any evidence for GABA_B receptors in helminths (337). GABA-induced muscle relaxation in the nematode Caenorhabditis only depends on the unc-49 gene products, which form GABA_A-like ionotropic receptors (14). However, the Caenorhabditis genome database reveals the presence of orthologs for both GABA_B(1) and GABA_B(2) (179). Most residues of the GABA binding-pocket in GABA_B(1) are conserved from Caenorhabditis to human (179). This is in contrast to the complete lack of evolutionary constraint placed on the binding pocket of GABA_B(2), which suggests that this subunit does not constitute a binding site for an endogenous ligand (see sect. IIIA5).

4. GABA_B-receptor isoforms

When it became apparent that probably all GABA_B receptors in the vertebrate brain are the sole products of the GABA_B(1) and GABA_B(2) genes, much attention focused on subunit isoforms. Many in the field wondered whether isoforms encoded pharmacological differences and accounted for the heterogeneity observed with native GABA_B receptors. Rapidly numerous GABA_B isoforms were identified (recently reviewed in Ref. 29). A close inspection of GABA_B gene structures indicates that not all of these splice variants are real and that some do not occur across different species. A summary of confirmed GABA_B isoforms is shown in Figure 2. While in most laboratories mixing and matching of isoforms did not produce GABA_B receptors with distinct functional and pharmacological properties, others reported differences that are, however, highly controversial (see sect. IIIA4A). GABA_B isoforms may afford the means for a differential subcellular targeting and/or coupling to distinct intracellular signaling pathways. To some extent a coupling to different effector systems could mimic a differential pharmacology and explain some of the differences that were observed with native GABA_B receptors.

View larger version (22K): [in this window] [in a new window]   FIG. 2. Protein structure of vertebrate GABAB(1) isoforms. The sushi domains (SD), seven transmembrane domains (7TM), coiled-coil domains (CC), and the ER-retention signals (RSRR) are indicated. Unfortunately, unrelated variants identified in humans and rats both were named GABAB(1c). Thus the variants described in the rat are better referred to as GABAB(1c-a) or GABAB(1c-b), depending on whether their amino-terminal domain is related to GABAB(1a) or GABAB(1b), respectively. The 31-amino acid insertion between the second extracellular loop (ECL2) and the fifth transmembrane domain in GABAB(1c-a), GABAB(1c-b), and GABAB(1f) corresponds to a 93-bp exon located between exons 19 and 20, which is not conserved in human and mouse. Dark gray segments represent the unique carboxy-terminal tails of GABAB(1d), GABAB(1e), and GABAB(1g). In GABAB(1f), skipping of exon 5 results in an in-frame deletion of 7 amino acids (7aa). References for all published variants are shown in parentheses.

View larger version (33K): [in this window] [in a new window]   FIG. 3. Evolutionary conservation of the GABAB receptor antagonist binding site. Photoaffinity labeling of GABAB receptors from different species. Brain membranes from the species indicated were labeled with 125I-CGP71872. In the case of Drosophila melanogaster and Haemonchus concortus, whole animals were analyzed. cDNA cloning revealed that Drosophila GABAB receptors exhibit a unique pharmacology and do not interact with a number of known agonists and antagonists.

View larger version (46K): [in this window] [in a new window]   FIG. 4. Differential expression of GABAB(1a) and GABAB(1b) transcripts. A: schematic representation of the 5'-end of the GABAB(1) gene. The GABAB(1b) isoform uses an alternative transcription initiation site within the GABAB(1a) intron upstream of exon 6. Thus the first 147 amino acids of the mature GABAB(1a) isoform are replaced in GABAB(1b) with a sequence of 18 amino acids. The amino-terminal extracellular domains of GABAB(1a) and GABAB(1b) primarily differ by the presence of a pair of sushi repeats encoded by GABAB(1a) exons 3 and 4. B: spatial distribution of GABAB(1) mRNA in the cerebellum using variant-specific riboprobes. GABAB(1a) transcripts are predominantly observed in the granule cell layer (GL), whereas GABAB(1b) mRNA is present in Purkinje cells (P). C: photoaffinity cross-linking of GABAB receptor proteins. GABAB(1a) and GABAB(1b) are differentially expressed throughout the central nervous system. D: developmental expression of GABAB receptors. GABAB(1b) protein is generally expressed at higher levels in the adult brain compared with fetal brain, whereas GABAB(1a) protein is more abundant during development. Numbers refer to postnatal days.

A number of laboratories compared the pharmacology of GABA_B(1a) and GABA_B(1b) and did not detect significant differences (49, 121, 170, 206). However, there are isolated reports that claim that GABA_B(1a) and GABA_B(1b) can be separated by pharmacological or functional means (26, 192, 237). Especially, the proposal that the anticonvulsant gabapentin is an agonist at GABA_B(1a,2), but not at GABA_B(1b,2), attracted a great deal of attention (26, 27, 237). Not only was gabapentin suggested to be subunit specific, but it was also shown to selectively activate postsynaptic GABA_B receptors. This remains highly controversial, as a number of other laboratories were unable to reproduce these findings, using similar and additional experimental approaches (161, 190, 255). In these latter studies, no clear effect of gabapentin on GABA_B receptors was seen in recombinant systems, in brain slice preparations, or in vivo, even when using high concentrations of the drug.

B) MINOR SUBUNIT VARIANTS.  Several additional GABA_B(1) isoforms were reported. GABA_B(1c) and GABA_B(1d) were identified in rat cDNA libraries (156, 260). GABA_B(1c) is characterized by an in-frame insertion of 31 amino acids between the second extracellular loop and the fifth transmembrane domain. This extra sequence is not conserved in humans, casting doubt on its significance. Nonetheless, the rat GABA_B(1c) protein forms a functional receptor when expressed with GABA_B(2) in heterologous cells (260). An isoform that differs at the NH₂ terminus from the GABA_B(1a) isoform was identified in humans (56, 217). Unfortunately, this variant was also named GABA_B(1c). Human GABA_B(1c) is similar to GABA_B(1a) yet lacks one sushi repeat because the splice machinery skips exon 4. The human GABA_B(1c) mRNA expression pattern parallels that of GABA_B(1a). GABA_B(1d) results from the failure to splice out the last intron of the GABA_B(1) gene and has a divergent COOH terminus that deletes half the coiled-coiled domain, including the ER-retention motif. It is impossible to generate GABA_B(1d) in human and mouse due to poor sequence conservation, again rising doubts about the physiological relevance of this splice event. GABA_B(1e) encodes the extracellular ligand-binding domain of the GABA_B(1a) subunit (301). GABA_B(1e) is generated by skipping of exon 15 and is detected both in rats and humans. While the GABA_B(1e) transcript is a minor component in the CNS, it is very prominent in peripheral tissues. GABA_B(1e) is secreted into the culture medium when expressed in transfected mammalian cells. Additionally, it also forms stable heteromeric complexes with GABA_B(2) at the plasma membrane, providing additional evidence that the coiled-coil interaction is not the only dimerization interface between GABA_B(1) and GABA_B(2) (57, 125, 250). Should the GABA_B(1e) protein occur in vivo, then it could affect GABA_B receptor function in a dominant-negative manner. GABA_B(1f) was identified as a rat transcript. It contains the in-frame deletion of exon 5 resulting in a 21-bp deletion in the ECD, as well as a COOH-terminal insertion corresponding to intron 22 (156, 339). GABA_B(1g), an additional truncated GABA_B(1a) isoform, was identified in rat tissue (340). GABA_B(1g) is characterized by an insertion of 124 bp between exon 4 and 5, which generates a frameshift. GABA_B(1g) encodes a COOH-terminally truncated polypeptide of 239 amino acid residues of unknown function. The distribution of GABA_B(c-g) isoform mRNA was exclusively studied using Northern blot, PCR, or in situ hybridization. For none of the GABA_B(c-g) splice variants the existence of a protein in vivo was demonstrated yet [in contrast to the GABA_B(1a) and GABA_B(1b) variants].

With regard to GABA_B(2), all initial papers report a single transcript (163, 170, 185, 216, 239, 343). Subsequently, two additional transcripts, GABA_B(2b) and GABA_B(2c), were identified in human tissue (75). Analysis of human GABA_B(2) genomic sequences reveals that the intron-exon boundaries required to generate these transcripts do not match known consensus sequences for splice junctions (217). GABA_B(2b) and GABA_B(2c) transcripts are likely to represent artifacts arising during cDNA synthesis and/or PCR amplification. Therefore, at the present time, there are no confirmed GABA_B(2) splice variants.

5. The GABA binding site

All GABA_B agonists and competitive antagonists bind to the ECD of the GABA_B(1) subunit, as shown for other Family 3 GPCRs as well (207). The ECD of GABA_B(1) can be expressed as a soluble protein. The truncated protein mostly retains the binding properties of wild-type receptors, indicating that it folds independently from the transmembrane domains. Structural analysis of the GABA_B(1) ECD reveals a weak sequence homology with bacterial periplasmic binding proteins, such as the leucine-binding protein (LBP) (169). GABA_B(1a) and GABA_B(1b), which differ in their ECD (see sect. IIIA4A), share the entire bacterial homology domain (110). In all GABA_B subunits the LBP-like domain is linked to the first transmembrane domain via a short sequence that lacks the cysteine-rich region conserved between the other members of Family 3 GPCRs. In the mGlu receptors, this cysteine-rich region appears to be necessary for the LBP-like domain to bind glutamate (244).

The X-ray structure of periplasmic-binding proteins reveals a binding pocket that is made up by two globular lobes (lobes I and II) separated by a hinge region. The two lobes close upon ligand binding, similar to a Venus flytrap when touched by an insect (275). Homology models of the GABA_B(1) ligand-binding domain, based on the X-ray structures of the bacterial proteins, have guided mutational analysis of the GABA binding site (110, 111). Key for both agonist and antagonist binding are S246, S269, D471, and E465 in lobe I, as well as Y366 in lobe II (Fig. 5). GABA and baclofen are thought to bind via their carboxylic group to the hydroxyl groups of S246 and Y366. E465 is then believed to bind to the NH₂-terminal end of GABA. D471, which was originally proposed to undergo an ionic interaction with GABA, now appears more important for correct folding of lobe I. Mutation of S247 and Q312 increases the affinity of agonists while decreasing the affinity of antagonists. This supports a model where the LBP-like domain exists in two conformational states, an open and a closed state, where binding of ligands favors the closed state (110). According to the three-dimensional model, a direct interaction with the second lobe is only possible in the closed form of the LBP-like domain, as shown for other receptors (8, 245). Some mutations differentially affect the binding of agonists. For example, the potency of GABA is decreased 30-fold by the S269A mutation, whereas the potency of baclofen remains unaltered (112). As is discussed in section IIIA6, this correlates with distinct effects of Ca²⁺ on GABA and baclofen binding (112). Interestingly, mutation of Y366 in lobe II not only decreases the affinity of GABA and baclofen, but also converts baclofen into an antagonist (111). The recently obtained crystal structure of the dimeric ECD of mGlu1 in the presence and absence of glutamate has essentially validated the homology models for GABA_B subunits described above (186).

View larger version (36K): [in this window] [in a new window]   FIG. 5. Three-dimensional model of the Venus flytrap module (VFTM) of GABAB(1). A model of GABA docked into the ligand-binding site of GABAB(1). The putative interactions between GABA and the amino acid residues of GABAB(1) are shown with dotted lines (see sect. IIIA5 for details). [Adapted from Kniazeff et al. (179); courtesy of Dr. J.-P. Pin.]

6. The Ca²⁺ binding site

GABA_B receptors share sequence similarity with mGlu and CaS receptors that are sensitive to Ca²⁺. In the CaS receptor the primary determinant for Ca²⁺ recognition is in the ECD (129). The effect of Ca²⁺ on mGlu receptors is heavily disputed. Some reports show that Ca²⁺ can directly activate mGlu1, mGlu3, and mGlu5 receptors (183), whereas others claim that Ca²⁺ is an allosteric modulator rather than an agonist (234, 296). Because of the effects of Ca²⁺ on Family 3 GPCRs, two groups investigated the possible regulation of GABA_B receptors by Ca²⁺ (112, 345). This led to the discovery of a Ca²⁺ binding site in the GABA_B(1) subunit as the first allosteric site of GABA_B receptors. Accordingly, it was shown that Ca²⁺ potentiates GABA-stimulated GTP[³⁵S] binding in membranes expressing native or recombinant GABA_B receptors. The effect of Ca²⁺ depends on the agonist, with baclofen being less sensitive than GABA or 3-APPA.

The residues that confer to GABA_B receptors the ability to sense Ca²⁺ were identified (112). The S269A mutation (see sect. IIIA5) in the LBP-like domain of GABA_B(1) renders the otherwise functional GABA_B(1,2) receptor Ca²⁺ insensitive. S269 localizes to the GABA-binding pocket, next to S246 that interacts with agonists (111). S269 in GABA_B(1) aligns with S170 in the CaS receptor, a residue that is involved in Ca²⁺ activation of the CaS receptor (48). Allosteric regulation of GABA_B(1) by Ca²⁺ is supposed to stabilize the activated closed conformational state (112). Possibly, Ca²⁺ compensates for the lack of the -amino group in GABA, and the contact of Ca²⁺ with S269 optimizes positioning of the carboxylic group of GABA for contacting S246. Alternatively Ca²⁺ does not directly interact with S269 but affects the positioning of its hydroxyl group. This may allow the formation of an additional hydrogen bond with the carboxylic group of GABA. The EC₅₀ value for Ca²⁺ modulation of GABA binding at GABA_B receptors is with 37 µM rather low. Under normal physiological conditions, with [Ca²⁺] in the blood and cerebrospinal fluid in the millimolar range, the Ca²⁺ site of GABA_B receptors is saturated. Allosteric regulation by Ca²⁺ may, however, become significant under pathological conditions, when extracellular [Ca²⁺] is low. This could be the case following ischemia (191) or epileptic seizures (138).

7. Molecular determinants of G protein coupling

A) MOLECULAR DETERMINANTS IN THE G PROTEIN.  The cloning of GABA_B receptors allowed characterizing the G protein interaction in heterologous systems. Franek et al. (103) used chimeric G proteins to identify the specific regions of the G_i/G_o subunits that are important for their interaction with GABA_B receptors. HEK293 cells expressing recombinant GABA_B(1) or GABA_B(2) alone or in combination do not activate PLC through G_q-type G proteins. However, heteromeric GABA_B(1,2) receptors do activate PLC via chimeric G_qi and G_qo proteins, in which the five COOH-terminal residues of G_i or G_o replace those of G_q. Therefore, like other GPCRs, GABA_B receptors recognize the very COOH terminus of G subunits (35). The amino acid residue at position –4 in the COOH terminus of G proteins was shown to be most important for the coupling to GABA_B receptors.

B) MOLECULAR DETERMINANTS IN GABA_B RECEPTOR SUBUNITS.  Questions were raised as to what domains of the heteromeric GABA_B(1,2) complex are involved in the interaction with G proteins. Two reports showed that neither the COOH-terminal intracellular domain of GABA_B(1) nor that of GABA_B(2) is needed for coupling to chimeric G proteins in a heterologous system (57, 250). Both groups expressed receptor constructs together with chimeric G_qi or G_qo proteins in HEK293 cells and measured the increase of the intracellular [Ca²⁺] following PLC activation. These findings were confirmed by others who found that the COOH termini of GABA_B(1) and GABA_B(2) influence G protein coupling but that they are not an absolute requirement for function (125). In contrast, it was reported that the deletion of the GABA_B(2) COOH terminus impairs the ability of recombinant receptors to activate Kir3-type K⁺ channels in Xenopus oocytes (211). The reason for this discrepancy is unclear but may relate to the fact that PLC is activated by G, whereas Kir3 channels are activated by G. There is now good evidence that the heptahelical region of the GABA_B(2) subunit directs the coupling to G proteins. With the use of chimeric GABA_B(1) and GABA_B(2) subunits with swapped ECDs, it was shown that only the heptahelical region of GABA_B(2) is absolutely necessary for G protein signaling (109, 212). However, the heptahelical region of GABA_B(1) significantly improves coupling efficacy. These studies further showed that both the GABA_B(1) and GABA_B(2) ECDs are required for function. It was later found that all GABA_B(2) intracellular loops are important for receptor coupling to Kir3 channels, whereas those of GABA_B(1) could be replaced with those of GABA_B(2) without affecting function (97, 211, 280). Particular attention was set on addressing the role of the second intracellular (i2) loop since there is evidence that this region is critical for G protein coupling in Family 3 GPCRs (119). Exchanging the i2 loops between GABA_B(1) and GABA_B(2) did not result in the formation of functional receptors. Hence, the i2 loop of GABA_B(2) needs to be correctly positioned with respect to the other intracellular domains. Sequence comparison between the i2 and i3 loops of GABA_B and the related mGlu receptors highlighted clear differences between GABA_B(1) and GABA_B(2). Mutational analysis confirmed the functional importance of conserved residues (K586, M587, and K590) in the i2 loop of GABA_B(2) and indicates that the GABA_B(1) i2 loop lacks the requirements for interaction with G proteins (97, 280). Mutation of K686, a basic residue in the i3 loop of GABA_B(2) that plays a critical role in G protein coupling of mGlu1 and CaS receptors (66, 102), suppresses functional coupling to G proteins in HEK293 and cerebellar granule cells, corroborating a similar role for this residue (97).

The question arises as to with how many G proteins a dimeric GPCR can interact. It was shown that the cytoplasmic surface of monomeric rhodopsin is too small to anchor both the G and G subunits (195). Only a rhodopsin homodimer provides sufficient interface to anchor both G and G. Although not generally accepted yet, the data on rhodopsin imply that all GPCRs need to homo- or heterodimerize for function. We therefore expect that the GABA_B(1,2) heterodimer binds one G protein only; one GABA_B subunit probably interacts with G, while the other subunit interacts with G.

8. Intra- and intermolecular events controlling receptor function

As described above, a large body of work has aimed at defining the structural requirements for ligand binding, subunit interaction, and G protein coupling (see sect. III, A5–A7). The interdependence of heteromerization, surface trafficking, and effector coupling makes it difficult to assign a defined molecular function to structural elements. Nevertheless, a number of laboratories reached similar conclusions regarding the sequence of intra- and intermolecular events that take place when activating a GABA_B receptor (109, 125, 253, 280). A scheme that accommodates most of the available data predicts that the ECD of GABA_B(1) is the only determinant for GABA binding, while the ECD of GABA_B(2) is necessary for receptor activation and for increasing agonist affinity. The hepathelical region and the cytoplasmic tail of GABA_B(2) is the prime determinant of G protein coupling, but GABA_B(1) is clearly necessary to optimize the coupling efficiency. A model was proposed where a conformational change within the dimeric ECDs of GABA_B(1) and GABA_B(2) is responsible for the stabilization of an active dimeric form of the transmembrane domains (Fig. 6). Hence, there is an allosteric interaction between the ligand-binding domain and the effector transmembrane domains. It is assumed that the GABA_B heteromer differs from the homodimers formed by other Family 3 GPCRs with respect to the functional coupling between binding and effector domains. In the GABA_B receptor the conformations of the effector and binding domains are probably tightly coupled, that is, the two domains are either both in an active or inactive conformation (253). This model is reminiscent of a two-state model for receptor activation.

View larger version (28K): [in this window] [in a new window]   FIG. 6. Putative activation mechanism of the GABAB heterodimer. Left: schematic representation of the inactive receptor with both Venus flytrap modules (VFTMs) in the open state and in the resting orientation. Binding of GABA in the VFTM of GABAB(1) induces its closing (step 1). The closed VFTM of GABAB(1) induces a change in the relative orientation of the two VFTMs to reach the active orientation (step 2). The possible closure of the VFTM of GABAB(2) without bound ligand is not indicated. The small white circles represent the axis allowing the opening and closing of each VFTM. The large white circle represents the axis for the change in the relative orientation of the two VFTMs. (Courtesy of Dr. J.-P. Pin.)

The discovery that receptor activity modifying proteins (RAMPs) can change the pharmacology of a GPCR triggered an intense search for GABA_B receptor-associated proteins (221). Many people in the field wondered whether interacting proteins could account for the pharmacological differences that were observed with native GABA_B receptors (see sect. IIE). A number of candidate proteins were identified in yeast two-hybrid screens, using the COOH-terminal domains of GABA_B(1) or GABA_B(2) as baits (Fig. 7). There are no reports claiming pharmacological changes upon expression of these proteins together with GABA_B(1), GABA_B(2), or GABA_B(1,2). This aside, interacting proteins constitute potential targets for ligands that modulate GABA_B function in a more specific way.