Phosphorylation-independent desensitization of GABA(B) receptor by GRK4

Agonist-promoted desensitization of the heterodimeric metabotropic GABA(B) receptor was investigated. Whereas no desensitization was observed in HEK293 cells heterologously expressing the receptor, GABA and the synthetic agonist baclofen induced a robust desensitization in cerebellar granule cells endogenously expressing the receptor. Taking advantage of this cell-specific desensitization phenotype, we identified GRK4 as the kinase involved in the neuronal desensitization. Transfection of small interference RNA directed against GRK4 significantly reduced GRK4 levels in cerebellar granule cells and strongly inhibited the agonist-promoted desensitization. Reciprocally, transfection of GRK4 in HEK293 cells restored agonist-promoted desensitization, confirming that this kinase is sufficient to support desensitization. Surprisingly, this desensitization occurred in the absence of ligand-induced receptor phosphorylation and could be promoted by GRK4 mutants deleted of their kinase domain. Taken together, these results suggest that GRK4 plays a central role in the agonist-promoted desensitization of GABA(B) receptor and that it does so through an atypical mechanism that challenges the generally accepted model linking the kinase activity of GRKs to their role in receptor desensitization.     

Phospho-dependent functional modulation of GABA(B) receptors by the metabolic sensor AMP-dependent protein kinase

GABA(B) receptors are heterodimeric G protein-coupled receptors composed of R1 and R2 subunits that mediate slow synaptic inhibition in the brain by activating inwardly rectifying K(+) channels (GIRKs) and inhibiting Ca(2+) channels. We demonstrate here that GABA(B) receptors are intimately associated with 5'AMP-dependent protein kinase (AMPK). AMPK acts as a metabolic sensor that is potently activated by increases in 5'AMP concentration that are caused by enhanced metabolic activity, anoxia, or ischemia. AMPK binds the R1 subunit and directly phosphorylates S783 in the R2 subunit to enhance GABA(B) receptor activation of GIRKs. Phosphorylation of S783 is evident in many brain regions, and is increased dramatically after ischemic injury. Finally, we also reveal that S783 plays a critical role in enhancing neuronal survival after ischemia. Together our results provide evidence of a neuroprotective mechanism, which, under conditions of metabolic stress or after ischemia, increases GABA(B) receptor function to reduce excitotoxicity and thereby promotes neuronal survival.     

A cleavable signal peptide enhances cell surface delivery and heterodimerization of Cerulean-tagged angiotensin II AT1 and bradykinin B2 receptor

Heterodimerization of the angiotensin II AT1 receptor with the receptor for the vasodepressor bradykinin, B2R, is known to sensitize the AT1-stimulated response of hypertensive individuals in vivo. To analyze features of that prototypic receptor heterodimer in vitro, we established a new method that uses fluorescence resonance energy transfer (FRET) and applies for the first time AT1-Cerulean as a FRET donor. The Cerulean variant of the green fluorescent protein as donor fluorophore was fused to the C-terminus of AT1, and the enhanced yellow fluorescent protein (EYFP) as acceptor fluorophore was fused to B2R. In contrast to AT1–EGFP, the AT1-Cerulean fusion protein was retained intracellularly. To facilitate cell surface delivery of AT1-Cerulean, a cleavable signal sequence was fused to the receptor’s amino terminus. The plasma membrane-localized AT1-Cerulean resembled the native AT1 receptor regarding ligand binding and receptor activation. A high FRET efficiency of 24.7% between membrane-localized AT1-Cerulean and B2R-EYFP was observed with intact, non-stimulated cells. Confocal FRET microscopy further revealed that the AT1/B2 receptor heterodimer was functionally coupled to receptor desensitization mechanisms because activation of the AT1-Cerulean/B2R-EYFP heterodimer with a single agonist triggered the co-internalization of AT1/B2R. Receptor co-internalization was sensitive to inhibition of G protein-coupled receptor kinases, GRKs, as evidenced by a GRK-specific peptide inhibitor. In agreement with efficient AT1/B2R heterodimerization, confocal FRET imaging of co-enriched receptor proteins immobilized on agarose beads also detected a high FRET efficiency of 24.0%. Taken together confocal FRET imaging revealed efficient heterodimerization of co-enriched and cellular AT1/B2R, and GRK-dependent co-internalization of the AT1/B2R heterodimer.     

Coupling of GABAB receptor GABAB2 subunit to G proteins: evidence from Xenopus oocyte and baby hamster kidney cell expression system

Coupling of functional GABA_B receptors (GABA_BR) to G proteins was investigated with an expression system of baby hamster kidney (BHK) cells and Xenopus oocytes. Fluorescence resonance energy transfer (FRET) analysis of BHK cells coexpressing GABA_B1a receptor (GB_1aR) fused to Cerulean, a brighter variant of cyan fluorescent protein, and GABA_B2 receptor (GB₂R) fused to Venus, a brighter variant of yellow fluorescent protein, revealed that GB_1aR-Cerulean and GB₂R-Venus form a heterodimer. The GABA_BR agonists baclofen and 3-aminopropylphosphonic acid (3-APPA) elicited inward-rectifying K⁺ currents in a concentration-dependent manner in oocytes expressing GB_1aR and GB₂R, or GB_1aR-Cerulean and GB₂R-Venus, together with G protein-activated inward-rectifying K⁺ channels (GIRKs), but not in oocytes expressing GB_1aR alone or GB₂R alone together with GIRKs. Oocytes coexpressing GB_1aR + G_i2-fused GB₂R (GB₂R-G_i2) caused faster K⁺ currents in response to baclofen. Furthermore, oocytes coexpressing GB_1aR + GB₂R fused to G_qi5 (a chimeric G_q protein that activates PLC pathways) caused PLC-mediated Ca²⁺-activated Cl^– currents in response to baclofen. In contrast, these responses to baclofen were not observed in oocytes coexpressing GB_1aR-G_i2 or GB_1aR-G_qi5 together with GB₂R. BHK cells and Xenopus oocytes coexpressing GB_1aR-Cerulean + a triplet tandem of GB₂R-Venus-G_qi5 caused FRET and Ca²⁺-activated Cl^– currents, respectively, with a similar potency in BHK cells coexpressing GB_1aR-Cerulean + GB₂R-Venus and in oocytes coexpressing GB_1aR + GB₂R-G_qi5. Our results indicate that functional GABA_BR forms a heterodimer composed of GB₁R and GB₂R and that the signal transducing G proteins are directly coupled to GB₂R but not to GB₁R. fluorescence resonance energy transfer     

A trafficking checkpoint controls GABA(B) receptor heterodimerization

Surface expression of GABA(B) receptors requires heterodimerization of GB1 and GB2 subunits, but little is known about mechanisms that ensure efficient heterodimer assembly. We found that expression of the GB1 subunit on the cell surface is prevented through a C-terminal retention motif RXR(R); this sequence is reminiscent of the ER retention/retrieval motif RKR identified in subunits of the ATP-sensitive K+ channel. Interaction of GB1 and GB2 through their C-terminal coiled-coil alpha helices masks the retention signal in GB1, allowing the plasma membrane expression of the assembled complexes. Because individual GABA(B) receptor subunits and improperly assembled receptor complexes are not functional even if expressed on the cell surface, we conclude that a trafficking checkpoint ensures efficient assembly of functional GABA(B) receptors.     

The G protein-coupled receptor kinase (GRK) interactome: role of GRKs in GPCR regulation and signaling

G protein-coupled receptor kinases (GRKs) and arrestins are key participants in the canonical pathways leading to phosphorylation-dependent GPCR desensitization, endocytosis, intracellular trafficking and resensitization as well as in the modulation of important intracellular signaling cascades by GPCR. Novel studies have revealed a phosphorylation-independent desensitization mechanism operating through their RGS-homology (RH) domain and the recent determination of the crystal structures of GRK2 and GRK6 has uncovered interesting details on the structure-function relationships of these kinases. Emerging evidence indicates that the activity of GRKs is tightly modulated by mechanisms including phosphorylation by different kinases and interaction with several cellular proteins such as calmodulin, caveolin or RKIP. In addition, GRKs are involved in multiple interactions with non-receptor proteins (PI3K, Akt, GIT or MEK) that point to novel GRK cellular roles. In this article, our purpose is to describe the ever increasing map of functional interactions for GRK proteins as a basis to better understand its contribution to cellular processes.     

The G protein-coupled receptor kinase (GRK) interactome: Role of GRKs in GPCR regulation and signaling

G protein-coupled receptor kinases (GRKs) and arrestins are key participants in the canonical pathways leading to phosphorylation-dependent GPCR desensitization, endocytosis, intracellular trafficking and resensitization as well as in the modulation of important intracellular signaling cascades by GPCR. Novel studies have revealed a phosphorylation-independent desensitization mechanism operating through their RGS-homology (RH) domain and the recent determination of the crystal structures of GRK2 and GRK6 has uncovered interesting details on the structure-function relationships of these kinases. Emerging evidence indicates that the activity of GRKs is tightly modulated by mechanisms including phosphorylation by different kinases and interaction with several cellular proteins such as calmodulin, caveolin or RKIP. In addition, GRKs are involved in multiple interactions with non-receptor proteins (PI3K, Akt, GIT or MEK) that point to novel GRK cellular roles. In this article, our purpose is to describe the ever increasing map of functional interactions for GRK proteins as a basis to better understand its contribution to cellular processes.     

A single subunit (GB2) is required for G-protein activation by the heterodimeric GABA(B) receptor.

Although G-protein-coupled receptors (GPCRs) have been shown to assemble into functional homo or heteromers, the role of each protomer in G-protein activation is not known. Among the GPCRs, the gamma-aminobutyric acid (GABA) type B receptor (GABA(B)R) is the only one known so far that needs two subunits, GB1 and GB2, to function. The GB1 subunit contains the GABA binding site but is unable to activate G-proteins alone. In contrast the GB2 subunit, which does not bind GABA, has an heptahelical domain able to activate G-proteins when assembled into homodimers (Galvez, T., Duthey, B., Kniazeff, J., Blahos, J., Rovelli, G., Bettler, B., Prézeau, L., and Pin, J.-P. (2001) EMBO J. 20, 2152-2159). In the present study, we have examined the role of each subunit within the GB1-GB2 heteromer, in G-protein coupling. To that end, point mutations in the highly conserved third intracellular loop known to prevent G-protein activation of the related Ca-sensing or metabotropic glutamate receptors were introduced into GB1 and GB2. One mutation, L686P introduced in GB2 prevents the formation of a functional receptor, even though the heteromer reaches the cell surface, and even though the mutated subunit still associates with GB1 and increases GABA affinity on GB1. This was observed either in HEK293 cells where the activation of the G-protein was assessed by measurement of inositol phosphate accumulation, or in cultured neurons where the inhibition of the Ca(2+) channel current was measured. In contrast, the same mutation when introduced into GB1 does not modify the G-protein coupling properties of the heteromeric GABA(B) receptor either in HEK293 cells or in neurons. Accordingly, whereas in all GPCRs the same protein is responsible for both agonist binding and G-protein activation, these two functions are assumed by two distinct subunits in the GABA(B) heteromer: one subunit, GB1, binds the agonists whereas the other, GB2, activates the G-protein. This illustrates the importance of a single subunit for G-protein activation within a dimeric receptor.     

G Protein-coupled Receptor Kinases of the GRK4 Protein Subfamily Phosphorylate Inactive G Protein-coupled Receptors (GPCRs)

G protein-coupled receptor (GPCR) kinases (GRKs) play a key role in homologous desensitization of GPCRs. It is widely assumed that most GRKs selectively phosphorylate only active GPCRs. Here, we show that although this seems to be the case for the GRK2/3 subfamily, GRK5/6 effectively phosphorylate inactive forms of several GPCRs, including β2-adrenergic and M2 muscarinic receptors, which are commonly used as representative models for GPCRs. Agonist-independent GPCR phosphorylation cannot be explained by constitutive activity of the receptor or membrane association of the GRK, suggesting that it is an inherent ability of GRK5/6. Importantly, phosphorylation of the inactive β₂-adrenergic receptor enhanced its interactions with arrestins. Arrestin-3 was able to discriminate between phosphorylation of the same receptor by GRK2 and GRK5, demonstrating preference for the latter. Arrestin recruitment to inactive phosphorylated GPCRs suggests that not only agonist activation but also the complement of GRKs in the cell regulate formation of the arrestin-receptor complex and thereby G protein-independent signaling.     

The G-protein-coupled receptor kinase GRK4 mediates homologous desensitization of metabotropic glutamate receptor 1.

G-protein-coupled receptor kinases (GRKs) are involved in the regulation of many G-protein-coupled receptors. As opposed to the other GRKs, such as rhodopsin kinase (GRK1) or beta-adrenergic receptor kinase (beta ARK, GRK2), no receptor substrate for GRK4 has been so far identified. Here we show that GRK4 is expressed in cerebellar Purkinje cells, where it regulates mGlu(1) metabotropic glutamate receptors, as indicated by the following: 1) When coexpressed in heterologous cells (HEK293), mGlu(1) receptor signaling was desensitized by GRK4 in an agonist-dependent manner (homologous desensitization). 2) In transfected HEK293 and in cultured Purkinje cells, the exposure to glutamate agonists induced internalization of the receptor and redistribution of GRK4. There was a substantial colocalization of the receptor and kinase both under basal condition and after internalization. 3) Kinase activity was necessary for desensitizing mGlu(1a) receptor and agonist-dependent phosphorylation of this receptor was also documented. 4) Antisense treatment of cultured Purkinje cells, which significantly reduced the levels of GRK4 expression, induced a marked modification of the mGlu(1)-mediated functional response, consistent with an impaired receptor desensitization. The critical role for GRK4 in regulating mGlu(1) receptors implicates a major involvement of this kinase in the physiology of Purkinje cell and in motor learning.     

The structure of G protein-coupled receptor kinase (GRK)-6 defines a second lineage of GRKs

We describe the 2.6-A crystal structure of human G protein-coupled receptor kinase (GRK)-6, a key regulator of dopaminergic signaling and lymphocyte chemotaxis. GRK6 is a member of the GRK4 subfamily of GRKs, which is represented in most, if not all, metazoans. Comparison of GRK6 with GRK2 confirms that the catalytic core of all GRKs consists of intimately associated kinase and regulator of G protein signaling (RGS) homology domains. Despite being in complex with an ATP analog, the kinase domain of GRK6 remains in an open, presumably inactive conformation, suggesting that G protein-coupled receptors activate GRKs by inducing kinase domain closure. The structure reveals a putative phospholipid-binding site near the N terminus of GRK6 and structural elements within the kinase substrate channel that likely influence G protein-coupled receptor access and specificity. The crystalline GRK6 RGS homology domain forms an extensive dimer interface using conserved hydrophobic residues distinct from those in GRK2 that bind Galpha(q), although dimerization does not appear to occur in solution and is not required for receptor phosphorylation.     

Interaction between the conserved region in the C-terminal domain of GRK2 and rhodopsin is necessary for GRK2 to catalyze receptor phosphorylation

The C-terminal domain of G protein-coupled receptor kinases (GRKs) consists of a conserved region and a variable region, and the variable region has been shown to direct the membrane translocation of cytosolic enzymes. The present work has revealed that the C-terminal domain may also be involved in kinase-receptor interaction that is primarily mediated by the conserved region. Truncation of the C-terminal domain or deletion of the conserved region in this domain of GRK2 resulted in a complete loss of its ability to phosphorylate rhodopsin and in an obvious decrease in its sensitivity to receptor-mediated phosphorylation of a peptide substrate. On the contrary, deletion of the betagamma subunit binding region in the C-terminal domain of GRK2 did not significantly alter the ability of the enzyme to phosphorylate rhodopsin. In addition, the recombinant proteins that represent the C-terminal domain and the conserved region of GRK2 could inhibit GRK2-mediated phosphorylation of rhodopsin and receptor-mediated activation of GRK2 but not GRK2-mediated phosphorylation of the peptide substrate. Furthermore, the conserved region as well as the C-terminal domain could directly bind rhodopsin in vitro. These results indicate that the C-terminal domain, or more precisely, the conserved region of this domain, is important for enzyme-receptor interaction and that this interaction is required for GRK2 to catalyze receptor phosphorylation.     

Utilizing a structure-based docking approach to develop potent G protein-coupled receptor kinase (GRK) 2 and 5 inhibitors

G protein-coupled receptor (GPCR) kinases (GRKs) regulate the desensitization and internalization of GPCRs. Two of these, GRK2 and GRK5, are upregulated in heart failure and are promising targets for heart failure treatment. Although there have been several reports of potent and selective inhibitors of GRK2 there are few for GRK5. Herein, we describe a ligand docking approach utilizing the crystal structures of the GRK2–Gβγ·GSK180736A and GRK5·CCG215022 complexes to search for amide substituents predicted to confer GRK2 and/or GRK5 potency and selectivity. From this campaign, we successfully generated two new potent GRK5 inhibitors, although neither exhibited selectivity over GRK2.     

Synthesis of the nanomolar photoaffinity GABA(B) receptor ligand CGP 71872 reveals diversity in the tissue distribution of GABA(B) receptor forms

A radioiodinated probe, [125I]-CGP 71872, containing an azido group that can be photoactivated, was synthesized and used to characterize GABA(B) receptors. Photoaffinity labeling experiments using crude membranes prepared from rat brain revealed two predominant ligand binding species at approximately 130 and approximately 100 kDa believed to represent the long (GABA(B)R1a) and short (GABA(B)R1b) forms of the receptor. Indeed, these ligand binding proteins were immunoprecipitated using a GABA(B) receptor-specific antibody confirming the receptor specificity of the photoaffinity probe. Most convincingly, [125I]-CGP 71872 binding was competitively inhibited in a dose-dependent manner by cold CGP 71872, GABA, saclofen, (-)-baclofen, (+)-baclofen and (L)-glutamic acid with a rank order and stereospecificity characteristic of the GABA(B) receptor. Photoaffinity labeling experiments revealed that the recombinant GABA(B)R2 receptor does not bind [125I]-CGP 71872, providing surprising and direct evidence that CGP 71872 is a GABA(B)R1 selective antagonist. Photoaffinity labeling experiments using rat tissues showed that both GABA(B)R1a and GABA(B)R1b are co-expressed in the brain, spinal cord, stomach and testis, but only the short GABA(B)R1b receptor form was detected in kidney and liver whereas the long GABA(B)R1a form was selectively expressed in the adrenal gland, pituitary, spleen and prostate. We report herein the synthesis and biochemical characterization of the nanomolar affinity [125I]-CGP 71872 and CGP 71872 GABA(B)R1 ligands, and differential tissue expression of the long GABA(B)R1a and short GABA(B)R1b receptor forms in rat and dog.     

A predicted amphipathic helix mediates plasma membrane localization of GRK5

G protein-coupled receptor kinases (GRKs) specifically phosphorylate agonist-occupied G protein-coupled receptors at the inner surface of the plasma membrane (PM), leading to receptor desensitization. GRKs utilize a variety of mechanisms to bind tightly, and sometimes reversibly, to cellular membranes. Previous studies demonstrated the presence of a membrane binding domain in the C terminus of GRK5. Here we define a mechanism by which this short C-terminal stretch of amino acids of GRK5 mediates PM localization. Secondary structure predictions suggest that a region contained within amino acids 546-565 of GRK5 forms an amphipathic helix, with the key features of the predicted helix being a hydrophobic patch of amino acids on one face of the helix, hydrophilic amino acids on the opposite face, and a number of basic amino acids surrounding the hydrophobic patch. We show that amino acids 546-565 of GRK5 are sufficient to target the cytoplasmic green fluorescent protein (GFP) to the PM, and the hydrophobic amino acids are necessary for PM targeting of GFP-546-565. Moreover, full-length GRK5-GFP is localized to the PM, but mutation of the hydrophobic patch or the surrounding basic amino acids prevents PM localization of GRK5-GFP. Last, we show that mutation of the hydrophobic residues severely diminishes phospholipid-dependent autophosphorylation of GRK5 and phosphorylation of membrane-bound rhodopsin by GRK5. The findings in this report thus suggest the presence of a membrane binding motif in GRK5 and define the importance of a group of hydrophobic amino acids within this motif in mediating its PM localization.     

Selective regulation of Galpha(q/11) by an RGS domain in the G protein-coupled receptor kinase, GRK2

G protein-coupled receptor kinases (GRKs) are well characterized regulators of G protein-coupled receptors, whereas regulators of G protein signaling (RGS) proteins directly control the activity of G protein alpha subunits. Interestingly, a recent report (Siderovski, D. P., Hessel, A., Chung, S., Mak, T. W., and Tyers, M. (1996) Curr. Biol. 6, 211-212) identified a region within the N terminus of GRKs that contained homology to RGS domains. Given that RGS domains demonstrate AlF(4)(-)-dependent binding to G protein alpha subunits, we tested the ability of G proteins from a crude bovine brain extract to bind to GRK affinity columns in the absence or presence of AlF(4)(-). This revealed the specific ability of bovine brain Galpha(q/11) to bind to both GRK2 and GRK3 in an AlF(4)(-)-dependent manner. In contrast, Galpha(s), Galpha(i), and Galpha(12/13) did not bind to GRK2 or GRK3 despite their presence in the extract. Additional studies revealed that bovine brain Galpha(q/11) could also bind to an N-terminal construct of GRK2, while no binding of Galpha(q/11), Galpha(s), Galpha(i), or Galpha(12/13) to comparable constructs of GRK5 or GRK6 was observed. Experiments using purified Galpha(q) revealed significant binding of both Galpha(q) GDP/AlF(4)(-) and Galpha(q)(GTPgammaS), but not Galpha(q)(GDP), to GRK2. Activation-dependent binding was also observed in both COS-1 and HEK293 cells as GRK2 significantly co-immunoprecipitated constitutively active Galpha(q)(R183C) but not wild type Galpha(q). In vitro analysis revealed that GRK2 possesses weak GAP activity toward Galpha(q) that is dependent on the presence of a G protein-coupled receptor. However, GRK2 effectively inhibited Galpha(q)-mediated activation of phospholipase C-beta both in vitro and in cells, possibly through sequestration of activated Galpha(q). These data suggest that a subfamily of the GRKs may be bifunctional regulators of G protein-coupled receptor signaling operating directly on both receptors and G proteins.     

Differential regulation of β2-adrenoceptor and adenosine A2B receptor signalling by GRK and arrestin proteins in arterial smooth muscle

Generation of cAMP through G_s-coupled G protein-coupled receptor (GPCR) [e.g. β₂-adrenoceptor (β₂AR), adenosine A_2B receptor (A_2BR)] activation, induces arterial smooth muscle relaxation, counteracting the actions of vasoconstrictors. G_s-coupled GPCR signalling is regulated by G protein-coupled receptor kinases (GRK) and arrestin proteins, and dysregulation of Gs/GPCR signalling is thought play a role in the development of hypertension, which may be a consequence of enhanced GRK2 and/or arrestin expression. However, despite numerous studies indicating that β₂AR and A_2BR can be substrates for GRK/arrestin proteins, currently little is known regarding GRK/arrestin regulation of these endogenous receptors in arterial smooth muscle. Here, endogenous GRK isoenzymes and arrestin proteins were selectively depleted using RNA-interference in rat arterial smooth muscle cells (RASM) and the consequences of this for β₂AR- and A_2BR-mediated adenylyl cyclase (AC) signalling were determined by assessing cAMP accumulation. GRK2 or GRK5 depletion enhanced and prolonged β₂AR/AC signalling, while combined deletion of GRK2/5 has an additive effect. Conversely, activation of AC by A_2BR was regulated by GRK5, but not GRK2. β₂AR desensitization was attenuated following combined GRK2/GRK5 knockdown, but not by depletion of individual GRKs, arrestins, or by inhibiting PKA. Arrestin3 (but not arrestin2) depletion enhanced A_2BR-AC signalling and attenuated A_2BR desensitization, while β₂AR-AC signalling was regulated by both arrestin isoforms. This study provides a first demonstration of how different complements of GRK and arrestin proteins contribute to the regulation of signalling and desensitization of these important receptors mediating vasodilator responses in arterial smooth muscle.     

The sushi domains of secreted GABA(B1) isoforms selectively impair GABA(B) heteroreceptor function

GABA(B) receptors are the G-protein-coupled receptors for gamma-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the brain. GABA(B) receptors are promising drug targets for a wide spectrum of psychiatric and neurological disorders. Receptor subtypes exhibit no pharmacological differences and are based on the subunit isoforms GABA(B1a) and GABA(B1b). GABA(B1a) differs from GABA(B1b) in its ectodomain by the presence of a pair of conserved protein binding motifs, the sushi domains (SDs). Previous work showed that selectively GABA(B1a) contributes to heteroreceptors at glutamatergic terminals, whereas both GABA(B1a) and GABA(B1b) contribute to autoreceptors at GABAergic terminals or to postsynaptic receptors. Here, we describe GABA(B1j), a secreted GABA(B1) isoform comprising the two SDs. We show that the two SDs, when expressed as a soluble protein, bind to neuronal membranes with low nanomolar affinity. Soluble SD protein, when added at nanomolar concentrations to dissociated hippocampal neurons or to acute hippocampal slices, impairs the inhibitory effect of GABA(B) heteroreceptors on evoked and spontaneous glutamate release. In contrast, soluble SD protein neither impairs the activity of GABA(B) autoreceptors nor impairs the activity of postsynaptic GABA(B) receptors. We propose that soluble SD protein scavenges an extracellular binding partner that retains GABA(B1a)-containing heteroreceptors in proximity of the presynaptic release machinery. Soluble GABA(B1) isoforms like GABA(B1j) may therefore act as dominant-negative inhibitors of heteroreceptors and control the level of GABA(B)-mediated inhibition at glutamatergic terminals. Of importance for drug discovery, our data also demonstrate that it is possible to selectively impair GABA(B) heteroreceptors by targeting their SDs.