The heptahelical domain of GABA(B2) is activated directly by CGP7930, a positive allosteric modulator of the GABA(B) receptor

The gamma-aminobutyric acid, type B (GABA(B)) receptor is well recognized as being composed of two subunits, GABA(B1) and GABA(B2). Both subunits share structural homology with other class-III G-protein-coupled receptors. They are composed of two main domains: a heptahelical domain (HD) typical of all G-protein-coupled receptors and a large extracellular domain (ECD). Although GABA(B1) binds GABA, GABA(B2) is required for GABA(B1) to reach the cell surface. However, it is still not demonstrated whether the association of these two subunits is always required for function in the brain. Indeed, GABA(B2) plays a major role in the coupling of the heteromer to G-proteins, such that it is possible that GABA(B2) can transmit a signal in the absence of GABA(B1). Today only ligands interacting with GABA(B1) ECD have been identified. Thus, the compounds acting exclusively on the GABA(B2) subunit will be helpful in analyzing the specific role of this subunit in the brain. Here, we explored the mechanism of action of CGP7930, a compound described as a positive allosteric regulator of the GABA(B) receptor. We showed that it activates the wild type GABA(B) receptor but with a low efficacy. The GABA(B2) HD is necessary for this effect, although one cannot exclude that CGP7930 could also bind to GABA(B1). Of interest, CGP7930 could activate GABA(B2) expressed alone and is the first described agonist of GABA(B2). Finally, we show that CGP7930 retains its agonist activity on a GABA(B2) subunit deleted of its ECD. This demonstrates that the HD of GABA(B2) behaves similar to a rhodopsin-like receptor, because it can reach the cell surface alone, can couple to G-protein, and be activated by agonists. These data open new strategies for studying the mechanism of activation of GABA(B) receptor and examine any possible role of homomeric GABA(B2) receptors.     

Identification of a preassembled TRH receptor-G(q/11) protein complex in HEK293 cells

Protein-protein interactions define specificity in signal transduction and these interactions are central to transmembrane signaling by G-protein-coupled receptors (GPCRs). It is not quite clear, however, whether GPCRs and the regulatory trimeric G-proteins behave as freely and independently diffusible molecules in the plasma membrane or whether they form some preassociated complexes. Here we used clear-native polyacrylamide gel electrophoresis (CN-PAGE) to investigate the presumed coupling between thyrotropin-releasing hormone (TRH) receptor and its cognate G(q/11) protein in HEK293 cells expressing high levels of these proteins. Under different solubilization conditions, the TRH receptor (TRH-R) was identified to form a putative pentameric complex composed of TRH-R homodimer and G(q/11) protein. The presumed association of TRH-R with G(q/11)α or Gβ proteins in plasma membranes was verified by RNAi experiments. After 10- or 30-min hormone treatment, TRH-R signaling complexes gradually dissociated with a concomitant release of receptor homodimers. These observations support the model in which GPCRs can be coupled to trimeric G-proteins in preassembled signaling complexes, which might be dynamically regulated upon receptor activation. The precoupling of receptors with their cognate G-proteins can contribute to faster G-protein activation and subsequent signal transfer into the cell interior.     

Allosteric interactions between GB1 and GB2 subunits are required for optimal GABA(B) receptor function

Recent studies on G-protein-coupled receptors revealed that they can dimerize. However, the role of each subunit in the activation process remains unclear. The gamma-amino-n-butyric acid type B (GABA(B)) receptor is comprised of two subunits: GB1 and GB2. Both consist of an extracellular domain (ECD) and a heptahelical domain composed of seven transmembrane alpha-helices, loops and the C-terminus (HD). Whereas GB1 ECD plays a critical role in ligand binding, GB2 is required not only to target GB1 subunit to the cell surface but also for receptor activation. Here, by analysing chimeric GB subunits, we show that only GB2 HD contains the determinants required for G-protein signalling. However, the HD of GB1 improves coupling efficacy. Conversely, although GB1 ECD is sufficient to bind GABA(B) ligands, the ECD of GB2 increases the agonist affinity on GB1, and is necessary for agonist activation of the receptor. These data indicate that multiple allosteric interactions between the two subunits are required for wild-type functioning of the GABA(B) receptor and highlight further the importance of the dimerization process in GPCR activation.     

Clathrin-independent endocytosis of GABA(A) receptors in HEK 293 cells.

The endocytosis of GABA(A) receptors was investigated in HEK 293 cells expressing receptor alpha1beta2- and alpha1beta2gamma2-subunit combinations. For assessment of internalized receptors by radioimmunoassay or immunofluorescence, a triple c-myc epitope was introduced into the amino terminus of the beta2 subunit. An assay based on biotin inaccessibility was used for alpha1 subunits. GABA(A) alpha1beta2- and alpha1beta2gamma2-subunit receptors were internalized with a t(1/2) of 5.5 min at 37 degrees C. With both subunit combinations, phorbol 12-myristate 3-acetate enhanced internalization by nearly 100%. Treatment of the cells with hypertonic sucrose prevented both the basal and phorbol ester-induced endocytosis of GABA(A) receptors. GF 109203X, an inhibitor of protein kinase C, blocked the stimulation by phorbol ester but had no detectable effect on basal receptor endocytosis. Coexpression with a dominant-negative mutant of dynamin (K44A) led to a 100% enhancement of GABA(A) receptor internalization, while the endocytosis of beta(2)-adrenergic receptors was completely prevented. The results indicate that the endocytosis of GABA(A) alpha1beta2-subunit receptors in HEK cells is constitutive, positively modulated by activation of protein kinase C, and occurs by a mechanism that requires neither the participation of a GABA(A) receptor gamma2 subunit nor a clathrin-mediated pathway.     

VIP activates G(s) and G(i3) in rat alveolar macrophages and G(s) in HEK293 cells transfected with the human VPAC(1) receptor

We have characterized vasoactive intestinal peptide (VIP) receptor/G-protein coupling in rat alveolar macrophage (AM) membranes and find that pertussis toxin treatment and antisera against G(alphai3) and G(alphas) reduce high-affinity (125)I-VIP binding, indicating that both G(alphas) and G(alphai3) couple to the VIP-receptor. The predominant VIP-receptor subtype in AM is VPAC(1) and we examined the G-protein interactions of the human VPAC(1) that had been transfected into HEK293 cells. VPAC(1) has a molecular mass of 56 kDa; GTP analogs reduced (125)I-VIP binding to this protein demonstrating that high-affinity binding of VIP to the receptor requires coupling to G-protein. Functional VIP/VPAC(1)/G-protein complexes were captured by covalent cross-linking and analyzed by Western blotting. The transfected human VPAC(1) receptor in HEK293 was found to be coupled to G(alphas) but not G(alphai) or G(alphaq). Furthermore, pertussis toxin treatment had no effect on VPAC(1)/G-protein coupling in these cells. These observations suggest that the G-proteins activated by VPAC(1) may be dependent upon species and cell type.     

Selective regulation of endogenous G protein-coupled receptors by arrestins in HEK293 cells

Arrestins play an important role in regulating desensitization and trafficking of G protein-coupled receptors (GPCRs). However, limited insight into the specificity of arrestin-mediated regulation of GPCRs is currently available. Recently, we used an antisense strategy to reduce arrestin levels in HEK293 cells and characterize the role of arrestins on endogenous G(s)-coupled receptors (Mundell, S. J., Loudon, R. B., and Benovic, J. L. (1999) Biochemistry 38, 8723-8732). Here, we characterized GPCRs coupled to either G(q) (M(1) muscarinic acetylcholine receptor (M(1)AchR) and P2y(1) and P2y(2) purinergic receptors) or G(i) (somatostatin and AT1 angiotensin receptors) in wild type and arrestin antisense HEK293 cells. The agonist-specific desensitization of the M(1)Ach and somatostatin receptors was significantly attenuated in antisense-expressing cells, whereas desensitization of P2y(1) and P2y(2) purinergic and AT1 angiotensin receptors was unaffected by reduced arrestin levels. To further examine arrestin/GPCR specificity, we studied the effects of endogenous GPCR activation on the redistribution of arrestin-2 epitope tagged with the green fluorescent protein (arrestin-2-GFP). These studies revealed a receptor-specific movement of arrestin-2-GFP that mirrored the arrestin-receptor specificity observed in the antisense cells. Thus, agonist-induced activation of endogenous beta(2)-adrenergic, prostaglandin E(2), M(1)Ach, and somatostatin receptors induced arrestin-2-GFP redistribution to early endosomes, whereas P2y(1) and P2y(2) purinergic and AT1 angiotensin receptor activation did not. Thus, endogenous arrestins mediate the regulation of selective G(q)- and G(i)-coupled receptors in HEK293 cells.     

Heterotrimeric G-protein signaling in Arabidopsis: Puzzling G-protein-coupled receptor

Seven transmembrane G-protein-coupled receptors (GPCRs) are commonly used by eukaryotes to sense extracellular signals to switch on cellular responses through the activation of cognate heterotrimeric G-proteins. In Arabidopsis thaliana, GCR2 has been proposed as a GPCR for the plant hormone abscisic acid. On the other hand, biochemical analysis demonstrates that the sole Arabidopsis heterotrimeric G-protein α subunit, GPA1, is in the activated state (GTP-bound) by default, suggesting that the heterotrimeric G-proteins may act without any GPCRs.Key words: heterotrimeric G-proteins, GCR2, GPA1, G-protein-coupled receptor (GPCR), AtRGS1     

Developmental regulation of GABA(B) receptor function in rat spinal cord

GABA(B) receptors are heterodimers coupled to G-proteins. The present study was undertaken to investigate activation of GABA(B) receptors in cerebral cortex and spinal cord using [35S]GTPgammaS binding assays, a direct measure of G-protein activity. The results revealed that the GABA(B) agonist baclofen stimulates GTPgammaS binding in cerebral cortex, with an ED50 of 50microM. This response is blocked by the GABA(B) receptor antagonist CGP 55845A (100nM). In contrast, baclofen-stimulated GTPgammaS binding was not observed in adult spinal cord tissue under similar incubation conditions, or after varying magnesium, calcium, GDP, [35S]GTPgammaS, or membrane concentrations in the assay medium. Stimulation of adult rat spinal cord muscarinic receptors did result in a concentration-related increase in [35S]GTPgammaS binding. Baclofen-stimulated GTPgammaS binding in adult spinal cord did not appear after peripheral inflammation, despite significant increases in GABA(B) subunit mRNA levels. As opposed to adult, appreciable GTPgammaS binding was observed in membranes prepared from spinal cords of rats within the first 14 days of postnatal development, suggesting that GABA(B) receptor function in the rat spinal cord is developmentally regulated. The results indicate that GABA(B) receptors may not be coupled to G-proteins in the adult rat spinal cord, or couple in a way that differs from that in newborns or adult cerebral cortex.     

Desensitization of GABA(B) receptor signaling by formation of protein complexes of GABA(B2) subunit with GRK4 or GRK5

We investigated the role of G protein coupled-receptor kinases (GRKs) in the desensitization of GABA(B) receptor-mediated signaling using Xenopus oocytes and baby hamster kidney (BHK) cells. Baclofen elicited inward K(+) currents in oocytes coexpressing heterodimeric GABA(B) receptor, GABA(B1a) subunit (GB(1a)R) and GABA(B2) subunit (GB(2)R), together with G protein-activated inwardly rectifying K(+) channels (GIRKs), in a concentration-dependent manner. Repetitive application of baclofen to oocytes coexpressing GABA(B)R and GIRKs did not change peak K(+) currents in the first and second responses, but the latter responses were significantly attenuated by coexpression of either GRK4 or GRK5 with attenuation efficacy of GRK4 > GRK5. Coexpression of other GRKs including GRK2, GRK3, and GRK6 had no effect on GABA(B) receptor-mediated desensitization processes. In BHK cells coexpressing GRK4 fused to Venus (brighter variant of yellow fluorescent protein, GRK4-Venus) with GB(1a)R and GB(2)R, GRK4-Venus was expressed in the cytosol but was translocated to the plasma membranes by GABA(B)R activation. In BHK cells coexpressing GRK4 fused to Cerulean (brighter variant of cyan fluorescent protein, GRK4-Cerulean) with GB(1a)R and GB(2)R-Venus, fluorescence resonance energy transfer (FRET) analysis demonstrated that GRK4-Cerulean formed a protein complex with GB(2)R-Venus. Immunoprecipitation and Western blot analysis confirmed GB(2)R-GRK4 complex formation. GRK5 also formed a complex with GB(2)R on the plasma membranes as determined by FRET and Western blotting but not GRK2, GRK3, and GRK6. Our results indicate that GRK4 and GRK5 desensitize GABA(B) receptor-mediated responses by forming protein complexes with GB(2)R subunit of GABA(B)R at the plasma membranes.     

On the mechanism of a mutated and abnormally functioning gamma-aminobutyric acid (A) receptor linked to epilepsy

A recent report indicates that a lysine-to-methionine mutation (K289M) in the gamma2 subunit of a human gamma-aminobutyric acid neurotransmitter receptor, the GABA(A) receptor, is linked to generalized epilepsy with febrile seizures [Baulac et al. (2001) Nat. Genet. 28, 46-48]. This mutation caused a decreased current response to GABA [Baulac et al. (2001) Nat. Genet. 28, 46-48]. Here we determine changes that occur in the mechanism of opening and closing of transmembrane channels formed by the GABA(A) receptor as a result of this mutation. The K289M mutation was introduced into the gamma2L subunit of the rat GABA(A) receptor, and the mutated subunit was coexpressed with the alpha1 and beta2 subunits in HEK293 cells. Transient kinetic techniques suitable for investigating reactions on cell surfaces with a microsecond-to-millisecond time resolution [Hess, G. P., and Grewer, C. (1998) Methods Enzymol. 291, 443-473] were used. They allow one to determine not only the channel-opening probability and rates of receptor desensitization but also the opening and closing rates of the mutated GABA(A) receptor channel. The channel-opening equilibrium constant of the mutated receptor was found to be 5-fold lower than that of the wild type. We calculated that this decrease in the channel-opening equilibrium accounts for the dysfunction of the mutated receptor. We discuss how a knowledge of the mechanism of the mutated receptor indicates an approach for alleviating this dysfunction.     

The dynamics of formation and action of the ternary complex revealed in living cells using a G-protein-gated K+ channel as a biosensor

Traditionally the consequences of activation of G-protein-coupled receptors (GPCRs) by an agonist are studied using biochemical assays. In this study we use live cells and take advantage of a G-protein-gated inwardly rectifying potassium channel (Kir3.1+3.2A) that is activated by the direct binding of Gbetagamma subunit to the channel complex to report, in real-time, using the patch clamp technique the activity of the "ternary complex" of agonist/receptor/G-protein. This analysis is further facilitated by the use of pertussis toxin-resistant fluorescent and non-fluorescent Galpha(i/o) subunits and a series of HEK293 cell lines stably expressing both channel and receptors (including the adenosine A(1) receptor, the adrenergic alpha(2A) receptor, the dopamine D(2S) receptor, the M4 muscarinic receptor, and the dimeric GABA-B(1b/2) receptor). We systematically analyzed the contribution of the various inputs to the observed kinetic response of channel activation. Our studies indicate that the combination of agonist, GPCR, and G-protein isoform uniquely specify the behavior of these channels and thus support the importance of the whole ternary complex at a kinetic level.     

Functional reconstitution of prostaglandin E receptor from bovine adrenal medulla with guanine nucleotide binding proteins

Prostaglandin E2 (PGE2) was found to bind specifically to a 100,000 x g pellet prepared from bovine adrenal medulla. The PGE receptor was associated with a GTP-binding protein (G-protein) and could be covalently cross-linked with this G-protein by dithiobis(succinimidyl propionate) in the 100,000 x g pellet (Negishi, M., Ito, S., Tanaka, T., Yokohama, H., Hayashi, H., Katada, T., Ui, M., and Hayaishi, O. (1987) J. Biol. Chem. 262, 12077-12084). In order to characterize the G-protein associated with the PGE receptor and reconstitute these proteins in phospholipid vesicles, we purified the G-protein to apparent homogeneity from the 100,000 x g pellet. The G-protein served as a substrate of pertussis toxin but differed in its alpha subunit from two known pertussis toxin substrate G-proteins (Gi and Go) purified from bovine brain. The molecular weight of the alpha subunit was 40,000, which is between those of Gi and Go. The purified protein was also distinguished immunologically from Gi and Go and was referred to as Gam. PGE receptor was solubilized by 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid and freed from G-proteins by wheat germ agglutinin column chromatography. Reconstitution of the PGE receptor with pure Gam, Gi, or Go in phospholipid vesicles resulted in a remarkable restoration of [3H]PGE2 binding activity in a GTP-dependent manner. The efficiency of these three G-proteins in this capacity was roughly equal. When pertussis toxin- or N-ethylmaleimide-treated G-proteins, instead of the native ones, were reconstituted into vesicles, the restoration of binding activity was no longer observed. The displacement of [3H]PGE2 binding was specific for PGE1 and PGE2. Furthermore, addition of PGE2 stimulated the GTPase activity of the G-proteins in reconstituted vesicles. These results indicate that the PGE receptor can couple functionally with Gam, Gi, or Go in phospholipid vesicles and suggest that Gam may be involved in signal transduction of the PGE receptor in bovine adrenal medulla.     

Activation and deactivation rates of recombinant GABA(A) receptor channels are dependent on alpha-subunit isoform.

The role of subunit composition in determining intrinsic maximum activation and deactivation kinetics of GABA(A) receptor channels is unknown. We used rapid ligand application (100-micros solution exchange) to examine the effects of alpha-subunit composition on GABA-evoked activation and deactivation rates. HEK 293 cells were transfected with human cDNAs encoding alpha1beta1gamma2- or alpha2beta1gamma2-subunits. Channel kinetics were similar across different transfections of the same subunits and reproducible across several GABA applications in the same patch. Current rise to peak was at least twice as fast for alpha2beta1gamma2 receptors than for alpha1beta1gamma2 receptors (reflected in 10-90% rise times of 0.5 versus 1.0 ms, respectively), and deactivation was six to seven times slower (long time constants of 208 ms versus 31 ms) after saturating GABA applications. Thus alpha-subunit composition determined activation and deactivation kinetics of GABA(A) receptor channels and is therefore likely to influence the kinetics and efficacy of inhibitory postsynaptic currents.     

The second intracellular loop of metabotropic glutamate receptors recognizes C termini of G-protein alpha-subunits

Heptahelical receptor coupling selectivity to G-proteins is controlled by a large contact area that involves several portions of the receptor and each subunit of the G-protein. In the G-protein alpha subunit, the C-terminal 5 residues, the N terminus, and the alpha N-beta 1 and alpha 4-alpha 5 loops play important roles. On the receptor side, both the second and third (i2 and i3) intracellular loops as well as the C-terminal tail probably contact these different regions of the G-protein. It is now accepted that the C terminus of the alpha subunit binds in a cavity formed by the i2 and i3 loops. Among the various G-protein-coupled receptors (GPCRs), class III receptors that include metabotropic glutamate (mGlu) receptors greatly differ from the rhodopsin-like GPCRs, but the contact zone between these receptors and the G-protein is less understood. The C terminus of the alpha subunit has been shown to play a pivotal role in the selective recognition of class III GPCRs. Indeed, the mGlu2 and mGlu4 and -8 receptors can discriminate between alpha subunits that differ at the level of their C-terminal end only (such as Gqo and Gqz). Here, we examine the role of the i2 loop of mGluRs in the selective recognition of this region of the alpha subunit. To that aim, we analyzed the coupling properties of mGlu2 and mGlu4 or -8 receptors and chimeras containing the i2 loop of the converse receptor to G-protein alpha subunits that only differ by their C termini (Gqo,Gqz, and their point mutants). Our data demonstrate that the central portion of the i2 loop is responsible for the selective recognition of the C-terminal end of the alpha subunit, especially the residue on position -4. These data are consistent with the proposal that the C-terminal end of the G-protein alpha subunit interacts with residues in a cavity formed by the i2 and i3 loops in class III GPCRs, as reported for class I GPCRs.     

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.     

Eugenol Inhibits the GABAA Current in Trigeminal Ganglion Neurons

Eugenol has sedative, antioxidant, anti-inflammatory, and analgesic effects, but also serves as an irritant through the regulation of a different set of ion channels. Activation of gamma aminobutyric acid (GABA) receptors on sensory neurons leads to the stabilization of neuronal excitability but contributes to formalin-induced inflammatory pain. In this study, we examined the effect of eugenol on the GABA-induced current in rat trigeminal ganglia (TG) neurons and in human embryonic kidney (HEK) 293 cells expressing the GABAA receptor α1β2γ2 subtype using the whole-cell patch clamp technique. RT-PCR and Western blot analysis were used to confirm the expression of GABA_A receptor γ2 subunit mRNA and protein in the TG and hippocampus. Eugenol decreased the amplitude ratio of the GABA-induced current to 27.5 ± 3.2% (p < 0.05) in TG neurons, which recovered after a 3-min washout. In HEK 293 cells expressing the α1β2γ2 subtype, eugenol inhibited GABA-induced currents in a dose-dependent manner. Application of eugenol also decreased the GABA response in the presence of a G-protein blocker. Eugenol pretreatment with different concentrations of GABA resulted in similar inhibition of the GABA-induced current in a non-competitive manner. In conclusion, eugenol inhibits the GABA-induced current in TG neurons and HEK 293 cells expressing the GABA_A receptor in a reversible, dose-dependent, and non-competitive manner, but not via the G-protein pathway. We suggest that the GABA_A receptor could be a molecular target for eugenol in the modulation of nociceptive information.     

GISP binding to TSG101 increases GABA receptor stability by down-regulating ESCRT-mediated lysosomal degradation

The neuron-specific G protein-coupled receptor interacting scaffold protein (GISP) is a multidomain, brain-specific protein derived from the A-kinase anchoring protein-9 gene. We originally isolated GISP as an interacting partner for the GABA(B) receptor subunit GABA(B1). Here, we show that the protein tumour susceptibility gene 101 (TSG101), an integral component of the endosomal sorting machinery that targets membrane proteins for lysosomal degradation, also interacts with GISP. TSG101 co-immunoprecipitates with GISP from adult rat brain, and using GST pull-downs, we identified that the eighth coiled-coiled region of GISP is critical for TSG101 association. Intriguingly, although there is no direct interaction between GISP and the GABA(B2) subunit, their co-expression in HEK293 cells increases levels of GABA(B2). GISP also inhibits TSG101-dependent GABA(B2) down-regulation in human embryonic kidney 293 cells whereas over-expression of a mutant GISP lacking the TSG101 binding domain has no effect on GABA(B2) degradation. These data suggest that GISP can function as a negative regulator of TSG101-dependent lysosomal degradation of transmembrane proteins in neurons to promote receptor stability.