共查询到20条相似文献,搜索用时 31 毫秒
1.
Mei‐ling A Joiner Marie‐France Lisé Eunice Y Yuen Angel Y F Kam Mingxu Zhang Duane D Hall Zulfiqar A Malik Hai Qian Yucui Chen Jason D Ulrich Alain C Burette Richard J Weinberg Ping‐Yee Law Zhen Yan Johannes W Hell 《The EMBO journal》2010,29(2):482-495
Central noradrenergic signalling mediates arousal and facilitates learning through unknown molecular mechanisms. Here, we show that the β2‐adrenergic receptor (β2AR), the trimeric Gs protein, adenylyl cyclase, and PKA form a signalling complex with the AMPA‐type glutamate receptor subunit GluR1, which is linked to the β2AR through stargazin and PSD‐95 and their homologues. Only GluR1 associated with the β2AR is phosphorylated by PKA on β2AR stimulation. Peptides that interfere with the β2AR–GluR1 association prevent this phosphorylation of GluR1. This phosphorylation increases GluR1 surface expression at postsynaptic sites and amplitudes of EPSCs and mEPSCs in prefrontal cortex slices. Assembly of all proteins involved in the classic β2AR–cAMP cascade into a supramolecular signalling complex and thus allows highly localized and selective regulation of one of its major target proteins. 相似文献
2.
It is generally assumed that antagonists of Gs‐coupled receptors do not activate cAMP signalling, because they do not stimulate cAMP production via Gs‐protein/adenylyl cyclase activation. Here, we report a new signalling pathway whereby antagonists of β1‐adrenergic receptors (β1ARs) increase cAMP levels locally without stimulating cAMP production directly. Binding of antagonists causes dissociation of a preformed complex between β1ARs and Type‐4 cyclic nucleotide phosphodiesterases (PDE4s). This reduces the local concentration of cAMP‐hydrolytic activity, thereby increasing submembrane cAMP and PKA activity. Our study identifies receptor/PDE4 complex dissociation as a novel mechanism of antagonist action that contributes to the pharmacological properties of β1AR antagonists and might be shared by other receptor subtypes. 相似文献
3.
Makoto Arakawa Naveena Yanamala Jasbir Upadhyaya Andrew Halayko Judith Klein‐Seetharaman Prashen Chelikani 《Protein science : a publication of the Protein Society》2010,19(1):85-93
G‐protein coupled receptors (GPCRs) are transmembrane signaling molecules, with a majority of them performing important physiological roles. β2‐Adrenergic receptor (β2‐AR) is a well‐studied GPCRs that mediates natural responses to the hormones adrenaline and noradrenaline. Analysis of the ligand‐binding region of β2‐AR using the recently solved high‐resolution crystal structures revealed a number of highly conserved amino acids that might be involved in ligand binding. However, detailed structure‐function studies on some of these residues have not been performed, and their role in ligand binding remains to be elucidated. In this study, we have investigated the structural and functional role of a highly conserved residue valine 114, in hamster β2‐AR by site‐directed mutagenesis. We replaced V114 in hamster β2‐AR with a number of amino acid residues carrying different functional groups. In addition to the complementary substitutions V114I and V114L, the V114C and V114E mutants also showed significant ligand binding and agonist dependent G‐protein activation. However, the V114G, V114T, V114S, and V114W mutants failed to bind ligand in a specific manner. Molecular modeling studies were conducted to interpret these results in structural terms. We propose that the replacement of V114 influences not only the interaction of the ethanolamine side‐chains but also the aryl‐ring of the ligands tested. Results from this study show that the size and orientation of the hydrophobic residue at position V114 in β2‐AR affect binding of both agonists and antagonists, but it does not influence the receptor expression or folding. 相似文献
4.
Generation of cAMP through Gs-coupled G protein-coupled receptor (GPCR) [e.g. β2-adrenoceptor (β2AR), adenosine A2B receptor (A2BR)] activation, induces arterial smooth muscle relaxation, counteracting the actions of vasoconstrictors. Gs-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 A2BR 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 A2BR-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 A2BR 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 A2BR-AC signalling and attenuated A2BR 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. 相似文献
5.
Melissa R. Dent Paramjit S. Tappia Naranjan S. Dhalla 《Journal of cellular physiology》2011,226(1):181-186
This study was undertaken to determine alterations in the β‐adrenoceptor (β‐AR) signaling system in male and female rats at 4 weeks after the induction of arteriovenous (AV) fistula or shunt. AV shunt produced a greater degree of cardiac hypertrophy and larger increase in cardiac output in male than in female animals. Increases in plasma levels of norepinephrine and epinephrine (EPI) due to AV shunt were also higher in male than females. While no difference in the β1‐AR affinity was seen in males and females, AV shunt induced increase in β1‐AR density in female rats was higher than that in males. Furthermore, no changes in basal adenylyl cyclase (AC) V/VI mRNA levels were seen; however, the increase in EPI‐stimulated AC activities was greater in AV shunt females than in males. AV shunt decreased myocardial β1‐AR mRNA level in male rats and increased β2‐AR mRNA level in female hearts; an increase in Gi‐protein mRNA was detected only in male hearts. Although GRK2 gene expression was increased in both sexes, an increase in GRK3 mRNA was seen only in AV shunt female rats. β‐arrestin1 mRNA was elevated in females whereas β‐arrestin 2 gene expression was increased in both male and female AV shunt rats. While these data demonstrate gender associated differences in various components of the β‐AR system in cardiac hypertrophy due to AV shunt, only higher levels of plasma catecholamines may account for the greater increase in cardiac output and higher degree of cardiac hypertrophy in males. J. Cell. Physiol. 226: 181–186, 2010. © 2010 Wiley‐Liss, Inc. 相似文献
6.
Yulia Koryakina Stacie M. Jones Lawrence E. Cornett Kathryn Seely Lisa Brents Paul L. Prather Alexander Kofman Richard C. Kurten 《Cell biology international》2012,36(12):1171-1183
The β2‐AR (β2‐adrenergic receptor) is an important target for respiratory and CVD (cardiovascular disease) medications. Clinical studies suggest that N‐terminal polymorphisms of β2‐AR may act as disease modifiers. We hypothesized that polymorphisms at amino acids 16 and 27 result in differential trafficking and down‐regulation of β2‐AR variants following β‐agonist exposure. The functional consequences of the four possible combinations of these polymorphisms in the human β2‐AR (designated β2‐AR‐RE, β2‐AR‐GE, β2‐AR‐RQ and β2‐AR‐GQ) were studied using site‐directed mutagenesis and recombinant expression in HEK‐293 cells (human embryonic kidney cells). Ligand‐binding assays demonstrated that after 24 h exposure to 1 μM isoprenaline, isoforms with Arg16 (β2‐AR‐RE and β2‐AR‐RQ) underwent increased down‐regulation compared with isoforms with Gly16 (β2‐AR‐GE and β2‐AR‐GQ). Consistent with these differences in down‐regulation between isoforms, prolonged isoprenaline treatment resulted in diminished cAMP response to subsequent isoprenaline challenge in β2‐AR‐RE relative to β2‐AR‐GE. Confocal microscopy revealed that the receptor isoforms had similar co‐localization with the early endosomal marker EEA1 following isoprenaline treatment, suggesting that they had similar patterns of internalization. None of the isoforms exhibited significant co‐localization with the recycling endosome marker Rab11 in response to isoprenaline treatment. Furthermore, we found that prolonged isoprenaline treatment led to a higher degree of co‐localization of β2‐AR‐RE with the lysosomal marker LAMP1 (lysosome‐associated membrane protein 1) compared with that of β2‐AR‐GE. Taken together, these results indicate that a mechanism responsible for differential responses of these receptor isoforms to the β‐agonist involves differences in the efficiency with which agonist‐activated receptors are trafficked to the lysosomes for degradation, or differences in degradation in the lysosomes. 相似文献
7.
Nicole Bgin-Heick 《Journal of cellular biochemistry》1995,58(4):464-473
Lipolysis and adenylyl cyclase (AC) activation in response to β-adrenergic agents are abnormally low in white epididymal adipose tissue (WAT) of the ob/ob mouse. The abundance of G-proteins (Gsα and Giα) linked to AC is also abnormally low. By contrast, β-adrenergic receptor (β-AR) levels were previously found to be normal in WAT and elevated in liver. The relative importance of various forms of the β-AR in mouse WAT was reassessed in view of the discovery of the β3-AR. The results show that (1) the β3-AR is mainly responsible for AC activation in lean-mouse WAT; (2) the β3-AR is only partly responsible for AC activation in obese mouse WAT; and (3) GTP modulates β3—-but not β1—-or β2-AR activation of AC in a biphasic manner. Therefore, the β3-AR appears responsible for the well-known bimodal effect of GTP on β-adrenergic receptor-mediated AC activity in WAT. 相似文献
8.
Phosphorylation of Cav1.2 on S1928 uncouples the L‐type Ca2+ channel from the β2 adrenergic receptor
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Tommaso Patriarchi Hai Qian Valentina Di Biase Zulfiquar A Malik Dhrubajyoti Chowdhury Jennifer L Price Erik A Hammes Olivia R Buonarati Ruth E Westenbroek William A Catterall Franz Hofmann Yang K Xiang Geoffrey G Murphy Chao‐Ye Chen Manuel F Navedo Johannes W Hell 《The EMBO journal》2016,35(12):1330-1345
Agonist‐triggered downregulation of β‐adrenergic receptors (ARs) constitutes vital negative feedback to prevent cellular overexcitation. Here, we report a novel downregulation of β2AR signaling highly specific for Cav1.2. We find that β2‐AR binding to Cav1.2 residues 1923–1942 is required for β‐adrenergic regulation of Cav1.2. Despite the prominence of PKA‐mediated phosphorylation of Cav1.2 S1928 within the newly identified β2AR binding site, its physiological function has so far escaped identification. We show that phosphorylation of S1928 displaces the β2AR from Cav1.2 upon β‐adrenergic stimulation rendering Cav1.2 refractory for several minutes from further β‐adrenergic stimulation. This effect is lost in S1928A knock‐in mice. Although AMPARs are clustered at postsynaptic sites like Cav1.2, β2AR association with and regulation of AMPARs do not show such dissociation. Accordingly, displacement of the β2AR from Cav1.2 is a uniquely specific desensitization mechanism of Cav1.2 regulation by highly localized β2AR/cAMP/PKA/S1928 signaling. The physiological implications of this mechanism are underscored by our finding that LTP induced by prolonged theta tetanus (PTT‐LTP) depends on Cav1.2 and its regulation by channel‐associated β2AR. 相似文献
9.
Viktoriya Syrovatkina Xin‐Yun Huang 《Protein science : a publication of the Protein Society》2019,28(2):305-312
Heterotrimeric G‐proteins are cellular signal transducers. They mainly relay signals from G‐protein‐coupled receptors (GPCRs). GPCRs function as guanine nucleotide‐exchange factors to active these G‐proteins. Based on the sequence and functional similarities, these G‐proteins are grouped into four subfamilies: Gs, Gi, Gq, and G12/13. The G12/13 subfamily consists of two members: G12 and G13. G12/13‐mediated signaling pathways play pivotal roles in a variety of physiological processes, while aberrant regulation of this pathway has been identified in various human diseases. Here we summarize the signaling mechanisms and physiological functions of Gα13 in blood vessel formation and bone homeostasis. We further discuss the expanding roles of Gα13 in cancers, serving as oncogenes as well as tumor suppressors. 相似文献
10.
《Journal of receptor and signal transduction research》2013,33(1):75-85
AbstractThe β2-adrenergic receptor (β2AR) couples to Gs, activating adenylyl cyclase (AC) and increasing cAMP. Such signaling undergoes desensitization with continued agonist exposure. β2AR also couple to Gi after receptor phosphorylation by the cAMP dependent protein kinase A, but the efficiency of such coupling is not known. Given the PKA dependence of β2AR-Gi coupling, we explored whether this may be a mechanism of agonist-promoted desensitization. HEK293 cells were transfected to express β2AR or β2AR and Giα2, and then treated with vehicle or the agonist isoproterenol to evoke agonist-promoted β2AR desensitization. Membrane AC activities showed that Giα2 overexpression decreased basal levels, but the fold-stimulation of the AC over basal by agonist was not altered. However, with treatment of the cells with isoproterenol prior to membrane preparation, a marked decrease in agonist-stimulated AC was observed with the cells overexpressing Giα2. in the absence of such overexpression, β2AR desensitization was 23 ± 7%, while with 5-fold Giα2 overexpression desensitization was 58 ± 5% (p<0.01, n=4). the effect of Gi on desensitization was receptor-specific, in that forskolin responses were not altered by Giα2 overexpression. Thus, acquired β2AR coupling to Gi is an important mechanism of agonist-promoted desensitization, and pathologic conditions that increase Gi levels contribute to β2AR dysfunction. 相似文献
11.
Matthew T. Duvernay Chunmin Dong Xiaoping Zhang Mélanie Robitaille Terence E. Hébert Guangyu Wu 《Traffic (Copenhagen, Denmark)》2009,10(5):552-566
The intrinsic structural determinants for export trafficking of G protein‐coupled receptors (GPCRs) have been mainly identified in the termini of the receptors. In this report, we determined the role of the first intracellular loop (ICL1) in the transport from the endoplasmic reticulum (ER) to the cell surface of GPCRs. The α2B‐adrenergic receptor (AR) mutant lacking the ICL1 is unable to traffic to the cell surface and to initiate signaling measured as ERK1/2 activation. Mutagenesis studies identify a single Leu48 residue in the ICL1 modulates α2B‐AR export from the ER. The ER export function of the Leu48 residue can be substituted by Phe, but not Ile, Val, Tyr and Trp, and is unlikely involved in correct folding or dimerization of α2B‐AR in the ER. Importantly, the isolated Leu residue is remarkably conserved in the center of the ICL1s among the family A GPCRs and is also required for the export to the cell surface of β2‐AR, α1B‐AR and angiotensin II type 1 receptor. These data indicate a crucial role for a single Leu residue within the ICL1 in ER export of GPCRs. 相似文献
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Céline M'Kadmi Jean-Philippe Leyris Lauriane Onfroy Céline Galés Aude Saulière Didier Gagne Marjorie Damian Sophie Mary Mathieu Maingot Séverine Denoyelle Pascal Verdié Jean-Alain Fehrentz Jean Martinez Jean-Louis Banères Jacky Marie 《The Journal of biological chemistry》2015,290(45):27021-27039
The G protein-coupled receptor GHS-R1a mediates ghrelin-induced growth hormone secretion, food intake, and reward-seeking behaviors. GHS-R1a signals through Gq, Gi/o, G13, and arrestin. Biasing GHS-R1a signaling with specific ligands may lead to the development of more selective drugs to treat obesity or addiction with minimal side effects. To delineate ligand selectivity at GHS-R1a signaling, we analyzed in detail the efficacy of a panel of synthetic ligands activating the different pathways associated with GHS-R1a in HEK293T cells. Besides β-arrestin2 recruitment and ERK1/2 phosphorylation, we monitored activation of a large panel of G protein subtypes using a bioluminescence resonance energy transfer-based assay with G protein-activation biosensors. We first found that unlike full agonists, Gq partial agonists were unable to trigger β-arrestin2 recruitment and ERK1/2 phosphorylation. Using G protein-activation biosensors, we then demonstrated that ghrelin promoted activation of Gq, Gi1, Gi2, Gi3, Goa, Gob, and G13 but not Gs and G12. Besides, we identified some GHS-R1a ligands that preferentially activated Gq and antagonized ghrelin-mediated Gi/Go activation. Finally, we unambiguously demonstrated that in addition to Gq, GHS-R1a also promoted constitutive activation of G13. Importantly, we identified some ligands that were selective inverse agonists toward Gq but not of G13. This demonstrates that bias at GHS-R1a signaling can occur not only with regard to agonism but also to inverse agonism. Our data, combined with other in vivo studies, may facilitate the design of drugs selectively targeting individual signaling pathways to treat only the therapeutically relevant function. 相似文献
14.
Beilei Lei Daniel P. Morris Michael P. Smith Debra A. Schwinn 《Cellular signalling》2009,21(10):1532-1539
We have reported that the α1A-adrenergic receptor (α1AAR) in rat-1 fibroblasts is a lipid raft protein. Here we examined whether disrupting lipid rafts by methyl-β-cyclodextrin (MCD) sequestration of cholesterol affects α1AAR signaling. Unexpectedly, MCD increased α1AAR-dependent basal inositol phosphate formation and p38 mitogen-activated protein kinase activation in a cholesterol-dependent manner. It also initiated internalization of surface α1AAR, which was partially blocked by receptor inhibition. Binding assays revealed MCD-mediated increases in receptor agonist affinity as well as reciprocal decreases in inverse agonist affinity, a behavior that is usually interpreted as a shift toward the active receptor conformation. In untreated cells a fraction of the receptor was found to be present in preassociated receptor/G protein complexes, which rapidly dissociate upon receptor stimulation. Consistent with MCD-induced signaling, raft disruption resulted in an increase in receptor/G protein complexes. These results strongly suggest that lipid rafts constrain basal α1AAR activity; however, preassembled receptor/G protein complexes could still provide a mechanism for accelerating α1AAR signaling following stimulation. 相似文献
15.
Juan José Fung Xavier Deupi Leonardo Pardo Xiao Jie Yao Gisselle A Velez‐Ruiz Brian T DeVree Roger K Sunahara Brian K Kobilka 《The EMBO journal》2009,28(21):3315-3328
The β2‐adrenoceptor (β2AR) was one of the first Family A G protein‐coupled receptors (GPCRs) shown to form oligomers in cellular membranes, yet we still know little about the number and arrangement of protomers in oligomers, the influence of ligands on the organization or stability of oligomers, or the requirement for other proteins to promote oligomerization. We used fluorescence resonance energy transfer (FRET) to characterize the oligomerization of purified β2AR site‐specifically labelled at three different positions with fluorophores and reconstituted into a model lipid bilayer. Our results suggest that the β2AR is predominantly tetrameric following reconstitution into phospholipid vesicles. Agonists and antagonists have little effect on the relative orientation of protomers in oligomeric complexes. In contrast, binding of inverse agonists leads to significant increases in FRET efficiencies for most labelling pairs, suggesting that this class of ligand promotes tighter packing of protomers and/or the formation of more complex oligomers by reducing conformational fluctuations in individual protomers. The results provide new structural insights into β2AR oligomerization and suggest a possible mechanism for the functional effects of inverse agonists. 相似文献
16.
Richard Carr III Yang Du Julie Quoyer Reynold A. Panettieri Jr. Jay M. Janz Michel Bouvier Brian K. Kobilka Jeffrey L. Benovic 《The Journal of biological chemistry》2014,289(52):35668-35684
The β2-adrenergic receptor (β2AR) is a prototypical G protein-coupled receptor that mediates many hormonal responses, including cardiovascular and pulmonary function. β-Agonists used to combat hypercontractility in airway smooth muscle stimulate β2AR-dependent cAMP production that ultimately promotes airway relaxation. Chronic stimulation of the β2AR by long acting β-agonists used in the treatment of asthma can promote attenuated responsiveness to agonists and an increased frequency of fatal asthmatic attacks. β2AR desensitization to β-agonists is primarily mediated by G protein-coupled receptor kinases and β-arrestins that attenuate receptor-Gs coupling and promote β2AR internalization and degradation. A biased agonist that can selectively stimulate Gs signaling without promoting receptor interaction with G protein-coupled receptor kinases and β-arrestins should serve as an advantageous asthma therapeutic. To identify such molecules, we screened ∼50 lipidated peptides derived from the intracellular loops of the β2AR, known as pepducins. This screen revealed two classes of Gs-biased pepducins, receptor-independent and receptor-dependent, as well as several β-arrestin-biased pepducins. The receptor-independent Gs-biased pepducins operate by directly stimulating G protein activation. In contrast, receptor-dependent Gs-biased pepducins appear to stabilize a Gs-biased conformation of the β2AR that couples to Gs but does not undergo G protein-coupled receptor kinase-mediated phosphorylation or β-arrestin-mediated internalization. Functional studies in primary human airway smooth muscle cells demonstrate that Gs-biased pepducins are not subject to conventional desensitization and thus may be good candidates for the development of next generation asthma therapeutics. Our study reports the first Gs-biased activator of the β2AR and provides valuable tools for the study of β2AR function. 相似文献
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Xiaohui Cang Linlin Yang Jing Yang Cheng Luo Mingyue Zheng Kunqian Yu Huaiyu Yang Hualiang Jiang 《Proteins》2014,82(5):760-770
Two 8‐µs all‐atom molecular dynamics simulations have been performed on the two highly homologous G protein‐coupled receptor (GPCR) subtypes, β1‐ and β2‐adrenergic receptors, which were embedded in a lipid bilayer with randomly dispersed cholesterol molecules. During the simulations, cholesterol molecules accumulate to different surface regions of the two receptors, suggesting the subtype specificity of cholesterol–β‐adrenergic receptor interaction and providing some clues to the physiological difference of the two subtypes. Meanwhile, comparison between the two receptors in interacting with cholesterols shed some new light on general determinants of cholesterol binding to GPCRs. Our results indicate that although the concave surface, charged residues and aromatic residues are important, neither of these stabilizing factors is indispensable for a cholesterol interaction site. Different combinations of these factors lead to the diversified binding modes of cholesterol binding to the receptors. Our long‐time simulations, for the first time, revealed the pathway of a cholesterol molecule entering the consensus cholesterol motif (CCM) site, and the binding process of cholesterol to CCM is accompanied by a side chain flipping of the conserved Trp4.50. Moreover, the simulation results suggest that the I‐/V‐/L‐rich region on the extracellular parts of helix 6 might be an alternatively conserved cholesterol‐binding site for the class‐A GPCRs. Proteins 2014; 82:760–770. © 2013 Wiley Periodicals, Inc. 相似文献
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20.
Prokineticin 1 (pk1) and prokineticin 2 (pk2) interact with two structurally related G-protein coupled receptors, prokineticin receptor 1 (PKR1) and prokineticin receptor 2 (PKR2). Cellular signalling studies show that the activated receptors can evoke Ca2+-mobilization, pertussis toxin-sensitive ERK phosphorylation, and intracellular cAMP accumulation, which suggests the partecipation of several G protein subtypes, such as Gq/11, Gi/o and Gs. However, direct interactions with these transduction proteins have not been studied yet. Here we measured by bioluminescence resonance energy transfer (BRET) the association of PKR1 and PKR2 with different heterotrimeric Gα proteins in response to pk1 and pk2 activation. Using host-cell lines carrying gene deletions of Gαq/11 or Gαs, and pertussis toxin treatment to abolish the receptor interactions with Gαi/o, we determined that both receptors could couple with comparable efficiency to Gq/11 and Gi/o, but far less efficiently to Gs or other pertussis toxin-insensitive G proteins. We also used BRET methodology to assess the association of prokineticin receptors with β-arrestin isoforms. Fluorescent versions of the isoforms were transfected both in HEK293 cells and in double KO β-arrestin 1/2 mouse fibroblasts, to study receptor interaction with the reconstituted individual β-arrestins without background expression of the endogenous genes. Both receptors formed stable BRET-emitting complexes with β-arrestin 2 but not with β-arrestin 1, indicating strong selectivity for the former. In all the studied transducer interactions and in both receptors, pk2 was more potent than pk1 in promoting receptor binding to transduction proteins. 相似文献