G protein-coupled receptors (GPCRs) have key roles in cell-cell communication. Recent data suggest that these receptors can form large complexes, a possibility expected to expand the complexity of this regulatory system. Among the brain GPCRs, the heterodimeric GABA(B) receptor is one of the most abundant, being distributed in most brain regions, on either pre- or post-synaptic elements. Here, using specific antibodies labelled with time-resolved FRET compatible fluorophores, we provide evidence that the heterodimeric GABA(B) receptor can form higher-ordered oligomers in the brain, as suggested by the close proximity of the GABA(B1) subunits. Destabilizing the oligomers using a competitor or a GABA(B1) mutant revealed different G protein coupling efficiencies depending on the oligomeric state of the receptor. By examining, in heterologous system, the G protein coupling properties of such GABA(B) receptor oligomers composed of a wild-type and a non-functional mutant heterodimer, we provide evidence for a negative functional cooperativity between the GABA(B) heterodimers. 相似文献
GABAB receptors assemble from principle and auxiliary subunits. The principle subunits GABAB1 and GABAB2 form functional heteromeric GABAB(1,2) receptors that associate with homotetramers of auxiliary KCTD8, -12, -12b, or -16 (named after their K+ channel tetramerization domain) subunits. These auxiliary subunits constitute receptor subtypes with distinct functional properties. KCTD12 and -12b generate desensitizing receptor responses while KCTD8 and -16 generate largely non-desensitizing receptor responses. The structural elements of the KCTDs underlying these differences in desensitization are unknown. KCTDs are modular proteins comprising a T1 tetramerization domain, which binds to GABAB2, and a H1 homology domain. KCTD8 and -16 contain an additional C-terminal H2 homology domain that is not sequence-related to the H1 domains. No functions are known for the H1 and H2 domains. Here we addressed which domains and sequence motifs in KCTD proteins regulate desensitization of the receptor response. We found that the H1 domains in KCTD12 and -12b mediate desensitization through a particular sequence motif, T/NFLEQ, which is not present in the H1 domains of KCTD8 and -16. In addition, the H2 domains in KCTD8 and -16 inhibit desensitization when expressed C-terminal to the H1 domains but not when expressed as a separate protein in trans. Intriguingly, the inhibitory effect of the H2 domain is sequence-independent, suggesting that the H2 domain sterically hinders desensitization by the H1 domain. Evolutionary analysis supports that KCTD12 and -12b evolved desensitizing properties by liberating their H1 domains from antagonistic H2 domains and acquisition of the T/NFLEQ motif. 相似文献
Pretreatment with 100 M GABA of synaptosomes purified from rat brain results in an increased uptake of the labelled neurotransmitter in subsequent incubations. The effect is blocked by a GABAB receptor antagonist, 2-hydroxy-saclofen. The effect is mimicked by baclofen and the baclofen effect is blocked by saclofen too. Lower GABA concentrations (up to 50 M) do not result in an increase of subsequent GABA uptake. Treatment of synaptosomes with 8-Br-cAMP results in a decreased GABA uptake. Since the uptake incubations were run with saturating concentrations of labelled GABA, the data indicates that GABAB receptor activation in brain synaptosomes up-regulates their GABA uptake capacity by an increase in Vmax. This mechanism appears of physiological relevance under conditions of sustained GABA release and substantial increase of its extracellular concentration. 相似文献
G protein-coupled receptors are involved in the modulation of complex neuronal networks in the brain. To investigate the impact of a cell-specific G(i/o) protein-mediated signaling pathway on brain function, we created a new optogenetic mouse model in which the G(i/o) protein-coupled receptor vertebrate rhodopsin can be cell-specifically expressed with the aid of Cre recombinase. Here we use this mouse model to study the functional impact of G(i/o) modulation in cerebellar Purkinje cells (PCs). We show that in vivo light activation of vertebrate rhodopsin specifically expressed in PCs reduces simple spike firing that is comparable with the reduction in firing observed for the activation of cerebellar G(i/o)-coupled GABA(B) receptors. Notably, the light exposure of the cerebellar vermis in freely moving mice changes the motor behavior. Thus, our studies directly demonstrate that spike modulation via G(i/o)-mediated signaling in cerebellar PCs affects motor coordination and show a new promising approach for studying the physiological function of G protein-coupled receptor-mediated signaling in a cell type-specific manner. 相似文献
Inhibitory GABAB receptors (GABABRs) can down-regulate most excitatory synapses in the CNS by reducing postsynaptic excitability. Functional GABABRs are heterodimers of GABAB1 and GABAB2 subunits and here we show that the trafficking and surface expression of GABABRs is differentially regulated by synaptic or pathophysiological activation of NMDA receptors (NMDARs). Activation of synaptic NMDARs using a chemLTP protocol increases GABABR recycling and surface expression. In contrast, excitotoxic global activation of synaptic and extrasynaptic NMDARs by bath application of NMDA causes the loss of surface GABABRs. Intriguingly, exposing neurons to extreme metabolic stress using oxygen/glucose deprivation (OGD) increases GABAB1 but decreases GABAB2 surface expression. The increase in surface GABAB1 involves enhanced recycling and is blocked by the NMDAR antagonist AP5. The decrease in surface GABAB2 is also blocked by AP5 and by inhibiting degradation pathways. These results indicate that NMDAR activity is critical in GABABR trafficking and function and that the individual subunits can be separately controlled to regulate neuronal responsiveness and survival. 相似文献
Thrombin is a potent modulator of endothelial function and, through stimulation of NF-κB, induces endothelial expression of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). These cell surface adhesion molecules recruit inflammatory cells to the vessel wall and thereby participate in the development of atherosclerosis, which is increasingly recognized as an inflammatory condition. The principal receptor for thrombin on endothelial cells is protease-activated receptor-1 (PAR-1), a member of the G protein-coupled receptor superfamily. Although it is known that PAR-1 signaling to NF-κB depends on initial PKC activation, the subsequent steps leading to stimulation of the canonical NF-κB machinery have remained unclear. Here, we demonstrate that a complex of proteins containing CARMA3, Bcl10, and MALT1 links PAR-1 activation to stimulation of the IκB kinase complex. IκB kinase in turn phosphorylates IκB, leading to its degradation and the release of active NF-κB. Further, we find that although this CARMA3·Bcl10·MALT1 signalosome shares features with a CARMA1-containing signalosome found in lymphocytes, there are significant differences in how the signalosomes communicate with their cognate receptors. Specifically, whereas the CARMA1-containing lymphocyte complex relies on 3-phosphoinositide-dependent protein kinase 1 for assembly and activation, the CARMA3-containing endothelial signalosome functions completely independent of 3-phosphoinositide-dependent protein kinase 1 and instead relies on β-arrestin 2 for assembly. Finally, we show that thrombin-dependent adhesion of monocytes to endothelial cells requires an intact endothelial CARMA3·Bcl10·MALT1 signalosome, underscoring the importance of the signalosome in mediating one of the most significant pro-atherogenic effects of thrombin. 相似文献
Gamma‐aminobutyric acid (GABA) is a non‐protein amino acid commonly present in all organisms. Because cellular levels of GABA in plants are mainly regulated by synthesis (glutamate decarboxylase, GAD) and catabolism (GABA‐transaminase, GABA‐T), we attempted seed‐specific manipulation of the GABA shunt to achieve stable GABA accumulation in rice. A truncated GAD2 sequence, one of five GAD genes, controlled by the glutelin (GluB‐1) or rice embryo globulin promoters (REG) and GABA‐T‐based trigger sequences in RNA interference (RNAi) cassettes controlled by one of these promoters as well, was introduced into rice (cv. Koshihikari) to establish stable transgenic lines under herbicide selection using pyriminobac. T1 and T2 generations of rice lines displayed high GABA concentrations (2–100 mg/100 g grain). In analyses of two selected lines from the T3 generation, there was a strong correlation between GABA level and the expression of truncated GAD2, whereas the inhibitory effect of GABA‐T expression was relatively weak. In these two lines both with two T‐DNA copies, their starch, amylose, and protein levels were slightly lower than non‐transformed cv. Koshihikari. Free amino acid analysis of mature kernels of these lines demonstrated elevated levels of GABA (75–350 mg/100 g polished rice) and also high levels of several amino acids, such as Ala, Ser, and Val. Because these lines of seeds could sustain their GABA content after harvest (up to 6 months), the strategy in this study could lead to the accumulation GABA and for these to be sustained in the edible parts. 相似文献
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. 相似文献
The D4 dopamine receptor belongs to the D2‐like family of dopamine receptors, and its exact regional distribution in the central nervous system is still a matter of considerable debate. The availability of a selective radioligand for the D4 receptor with suitable properties for positron emission tomography (PET) would help resolve issues of D4 receptor localization in the brain, and the presumed diurnal change of expressed protein in the eye and pineal gland. We report here on in vitro and in vivo characteristics of the high‐affinity D4 receptor‐selective ligand N‐{2‐[4‐(3‐cyanopyridin‐2‐yl)piperazin‐1‐yl]ethyl}‐3‐[11C]methoxybenzamide ([11C] 2 ) in rat. The results provide new insights on the in vitro properties that a brain PET dopamine D4 radioligand should possess in order to have improved in vivo utility in rodents. 相似文献
This study reports the isolation and characterization of a new type of transparent zebrafish Danio rerio mutant called pinky (pk), which has been visually isolated from a spontaneous mutation in a D. rerio colony. The pk larvae possess complex mutations affecting pigmentation because of missing pigment cells or a dramatic reduction in the chromatophore number. The pk displays a totally colourless phenotype and adult body transplant with no other obvious external morphological abnormalities, except for a red retina. The molecular analysis results in several candidate genes, hps1, ap3m2 and rabggta, implicated in the Hermansky–Pudlak syndrome (HPS) genes associated with HPS in pk. To demonstrate its applications of deep‐tissue imaging, this study examines green fluorescent protein alone or with other fluorescent proteins to investigate their capability for using multilabelling purposes in live adult pk. In this study, pk is particularly valuable for tissue cell labelling and internal organogenesis studies because of its optical clarity in the adult body. 相似文献
Domestic pigs possess a well‐developed sense of smell. However, the morphology of the porcine olfactory epithelium (OE) is poorly understood. Recently, several strains of transgenic cloned pigs that are presumed to ubiquitously express green fluorescent protein (GFP) have been created. Thus, the purpose of this study was to elucidate the features of porcine OE using the tissues of GFP transgenic cloned pigs. Based on observations of Hematoxylin and Eosin staining and measurements of thickness, porcine OE tissue portions were classified into three categories (thick, standard, and thin). Cryosections revealed that the prominent GFP signals were expressed in olfactory sensory neurons (OSN), Bowman's glands, and olfactory nerve. A few GFP‐expressing sustentacular cells were seen; however, the intensity of GFP fluorescence was slight. In the thick portion, numerous GFP‐expressing polygonal OSN that did not possess dendrites were found. In the standard portions, GFP‐expressing cells had longitudinal dendrites. A few GFP‐expressing cells were found in the thin portion. In the thick and standard portions, most of the prominent GFP‐expressing cells were positive for olfactory marker protein. Moreover, double immunofluorescence staining with boiled GFP and Sox2 antibody revealed that GFP expression patterns in OSN are synchronized with Sox2 immunoreactive patterns. 相似文献
Insect-resistant plants have been developed throughexpression of insecticidal proteins from Bacillusthuringiensis (Bt) in the early 1980s [1,2]. However, forcontrol of insect pests, it is necessary to increase theexpression of Bt protein overall or in specific plant tissues.To increase the expression level, synthetic Bt genes havebeen developed and used to produce transgenic plants[2–5]. A number of approaches have been taken to increasethe expression level of foreign proteins in transgeni… 相似文献
To test the Cre/loxP recombination system in zebrafish, a stable transgenic zebrafish line was developed by using a floxed (loxP flanked) gfp(green fluorescent protein) gene construct under the muscle-specific mylz2 promoter. Like our previous non-floxed gfp transgenic line under the same promoter, the new transgenic line expresses GFP reporter faithfully in fast skeletal muscles to the same intensity. To demonstrate the excision of floxed gfp transgene, in vitro synthesized Cre RNA was injected into embryos of floxed gfp transgenic zebrafish and we found a dramatic reduction of GFP expression. To confirm the excision, PCR was performed and a DNA fragment of correct size was amplified as predicted from the Cre/loxP mediated excision. Finally, we cloned the fragment and sequence information confirmed that the excision occurred at the precise site as predicted. Our experiments demonstrated that the Cre/loxP system can function efficiently and accurately in the zebrafish system. 相似文献
We have examined the development of gamma-aminobutyric acidB (GABAB) receptors in rat cerebrum using a binding assay that has achieved specific binding levels of approximately 50% with the GABAB ligand (-)-[3H]baclofen. As early as postnatal day 1, GABAB receptors are present and are linked to both calcium- and guanosine triphosphate-binding protein (G protein)-regulatory sites, as indicated by the stimulation of binding by calcium and the inhibition of binding by the guanine nucleotide guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). However, whereas the EC50 for the calcium effect was at a mature value in the neonate, the IC50 for the inhibition of binding by GTP gamma S was not, and declined more than two orders of magnitude by adulthood. Moreover, while many previous studies had shown that manipulation of G proteins by guanine nucleotides affects receptors affinity rather than density, our saturation analysis of binding suggests that calcium affected GABAB receptor density rather than affinity. The results therefore suggest that calcium and the manipulation of G proteins by GTP gamma S may affect the GABAB receptor by different mechanisms. 相似文献
Marlin-1 is a GABA(B) receptor and Jak tyrosine kinase-binding protein that also associates with RNA and microtubules. In humans and rodents, expression of Marlin-1 is predominantly restricted to the brain, but expression in lymphoid cells has also been reported. Here, we have studied the distribution of Marlin-1 in testis and spermatozoa. Our results indicate that Marlin-1 is highly expressed in testis. The protein is abundant in spermatogonia, spermatocytes, spermatozoa, and Sertoli cells. We also have studied the subcellular distribution in spermatozoa. Marlin-1 is present in the tail and to a lesser degree in the head of the sperm cell. Finally, we have explored two protein interactions. Our findings demonstrate that Marlin-1 associates with a microtubule fraction and with GABA(B) receptors in testis suggesting that the set of protein interactions of Marlin-1 are conserved in different tissues. 相似文献
Serotonin (5‐HT)2C receptors play a role in psychoaffective disorders and often contribute to the antidepressant and anxiolytic effects of psychotropic drugs. During stress, activation of these receptors exerts a negative feedback on 5‐HT release, probably by increasing the activity of GABAergic interneurons. However, to date, the GABA receptor types that mediate the 5‐HT2C receptor‐induced feedback inhibition are still unknown. To address this question, we assessed the inhibition of 5‐HT turnover by a 5‐HT2C receptor agonist (RO 60‐0175) at the hippocampal level and under conditions of stress, after pharmacological or genetic inactivation of either GABA‐A or GABA‐B receptors in mice. Neither the GABA‐B receptor antagonist phaclofen nor the specific genetic ablation of either GABA‐B1a or GABA‐B1b subunits altered the inhibitory effect of RO 60‐0175, although 5‐HT turnover was markedly decreased in GABA‐B1a knock‐out mice in both basal and stress conditions. In contrast, the 5‐HT2C receptor‐mediated inhibition of 5‐HT turnover was reduced by the GABA‐A receptor antagonist bicuculline. However, a significant effect of 5‐HT2C receptor activation persisted in mutant mice deficient in the α3 subunit of GABA‐A receptors. It can be inferred that non‐α3 subunit‐containing GABA‐A receptors, but not GABA‐B receptors, mediate the 5‐HT2C‐induced inhibition of stress‐induced increase in hippocampal 5‐HT turnover in mice.
