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1.
Proc Natl Acad Sci U S A ; 117(42): 26218-26225, 2020 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-33020275

RESUMO

Understanding the activation mechanism of the µ-opioid receptor (µ-OR) and its selective coupling to the inhibitory G protein (Gi) is vital for pharmaceutical research aimed at finding treatments for the opioid overdose crisis. Many attempts have been made to understand the mechanism of the µ-OR activation, following the elucidation of new crystal structures such as the antagonist- and agonist-bound µ-OR. However, the focus has not been placed on the underlying energetics and specificity of the activation process. An energy-based picture would not only help to explain this coupling but also help to explore why other possible options are not common. For example, one would like to understand why µ-OR is more selective to Gi than a stimulatory G protein (Gs). Our study used homology modeling and a coarse-grained model to generate all of the possible "end states" of the thermodynamic cycle of the activation of µ-OR. The end points were further used to generate reasonable intermediate structures of the receptor and the Gi to calculate two-dimensional free energy landscapes. The results of the landscape calculations helped to propose a plausible sequence of conformational changes in the µ-OR and Gi system and for exploring the path that leads to its activation. Furthermore, in silico alanine scanning calculations of the last 21 residues of the C terminals of Gi and Gs were performed to shed light on the selective binding of Gi to µ-OR. Overall, the present work appears to demonstrate the potential of multiscale modeling in exploring the action of G protein-coupled receptors.


Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Receptores Opioides mu/química , Receptores Opioides mu/metabolismo , Humanos , Modelos Moleculares , Conformação Proteica , Transdução de Sinais , Termodinâmica
2.
Proc Natl Acad Sci U S A ; 117(37): 23096-23105, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32868434

RESUMO

The ß2-adrenergic receptor (ß2AR) is a prototypical G protein-coupled receptor (GPCR) that preferentially couples to the stimulatory G protein Gs and stimulates cAMP formation. Functional studies have shown that the ß2AR also couples to inhibitory G protein Gi, activation of which inhibits cAMP formation [R. P. Xiao, Sci. STKE 2001, re15 (2001)]. A crystal structure of the ß2AR-Gs complex revealed the interaction interface of ß2AR-Gs and structural changes upon complex formation [S. G. Rasmussen et al., Nature 477, 549-555 (2011)], yet, the dynamic process of the ß2AR signaling through Gs and its preferential coupling to Gs over Gi is still not fully understood. Here, we utilize solution nuclear magnetic resonance (NMR) spectroscopy and supporting molecular dynamics (MD) simulations to monitor the conformational changes in the G protein coupling interface of the ß2AR in response to the full agonist BI-167107 and Gs and Gi1 These results show that BI-167107 stabilizes conformational changes in four transmembrane segments (TM4, TM5, TM6, and TM7) prior to coupling to a G protein, and that the agonist-bound receptor conformation is different from the G protein coupled state. While most of the conformational changes observed in the ß2AR are qualitatively the same for Gs and Gi1, we detected distinct differences between the ß2AR-Gs and the ß2AR-Gi1 complex in intracellular loop 2 (ICL2). Interactions with ICL2 are essential for activation of Gs These differences between the ß2AR-Gs and ß2AR-Gi1 complexes in ICL2 may be key determinants for G protein coupling selectivity.


Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Benzoxazinas/farmacologia , Sítios de Ligação/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Humanos , Espectroscopia de Ressonância Magnética/métodos , Simulação de Dinâmica Molecular , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
3.
Nature ; 585(7823): 135-140, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32610344

RESUMO

Chemokines and their receptors mediate cell migration, which influences multiple fundamental biological processes and disease conditions such as inflammation and cancer1. Although ample effort has been invested into the structural investigation of the chemokine receptors and receptor-chemokine recognition2-4, less is known about endogenous chemokine-induced receptor activation and G-protein coupling. Here we present the cryo-electron microscopy structures of interleukin-8 (IL-8, also known as CXCL8)-activated human CXC chemokine receptor 2 (CXCR2) in complex with Gi protein, along with a crystal structure of CXCR2 bound to a designed allosteric antagonist. Our results reveal a unique shallow mode of binding between CXCL8 and CXCR2, and also show the interactions between CXCR2 and Gi protein. Further structural analysis of the inactive and active states of CXCR2 reveals a distinct activation process and the competitive small-molecule antagonism of chemokine receptors. In addition, our results provide insights into how a G-protein-coupled receptor is activated by an endogenous protein molecule, which will assist in the rational development of therapeutics that target the chemokine system for better pharmacological profiles.


Assuntos
Modelos Moleculares , Receptores de Interleucina-8B/química , Receptores de Interleucina-8B/metabolismo , Transdução de Sinais , Regulação Alostérica , Sítio Alostérico , Quimiocinas/classificação , Quimiocinas/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Humanos , Interleucina-8/metabolismo , Ligação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato
4.
Biochem Pharmacol ; 180: 114143, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32653592

RESUMO

A novel mechanism of action was described for the protease-activated receptor 4 (PAR4)-derived pepducin (P4Pal10), when it was shown to exhibit inhibitory efficacy towards G protein coupling to multiple Gαq-coupled receptors (Carr, R., 3rd et al., Mol. Pharmacol. 2016(89) 94). We could confirm that P4Pal10, similar to an earlier-characterized Gαq inhibitor (YM-254890), inhibited platelet aggregation induced by agonists for the Gαq-coupled receptors PAR1 and PAR4. Next, we applied P4Pal10 as a tool compound and investigated its modulatory effect on several Gαq- and Gαi-coupled GPCRs expressed by human neutrophils. P4Pal10 had, however, no inhibitory effects on signaling downstream of the Gαq-coupled receptors for ATP (P2Y2R) and PAF (PAFR). Instead, P4Pal10 inhibited signaling downstream the Gαi-coupled FPR2. The inhibition was not due to a direct effect on Gαi as the closely related FPR1 was unaffected. In addition, we found that the pepducin activated allosterically modulated short chain fatty acid receptor (FFAR2), a Gαi/Gαq coupled GPCR that is functionally expressed in neutrophils. Taken together, we show that pepducins are unique tool-compounds for mechanistic studies of GPCR signaling and modulation in neutrophils. The data presented add also lipopeptides into the known ligand recognition lists for the two pattern recognition receptors FPR2 and FFAR2, receptors that primarily sense formylated peptides and short free fatty acids, respectively, inflammatory mediators of microbial origin.


Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Neutrófilos/metabolismo , Oligopeptídeos/farmacologia , Receptores de Superfície Celular/metabolismo , Receptores de Formil Peptídeo/metabolismo , Receptores de Lipoxinas/metabolismo , Receptores de Trombina/metabolismo , Relação Dose-Resposta a Droga , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Humanos , Neutrófilos/efeitos dos fármacos , Ligação Proteica/fisiologia , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
5.
Nature ; 584(7819): 125-129, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32528175

RESUMO

The D2 dopamine receptor (DRD2) is a therapeutic target for Parkinson's disease1 and antipsychotic drugs2. DRD2 is activated by the endogenous neurotransmitter dopamine and synthetic agonist drugs such as bromocriptine3, leading to stimulation of Gi and inhibition of adenylyl cyclase. Here we used cryo-electron microscopy to elucidate the structure of an agonist-bound activated DRD2-Gi complex reconstituted into a phospholipid membrane. The extracellular ligand-binding site of DRD2 is remodelled in response to agonist binding, with conformational changes in extracellular loop 2, transmembrane domain 5 (TM5), TM6 and TM7, propagating to opening of the intracellular Gi-binding site. The DRD2-Gi structure represents, to our knowledge, the first experimental model of a G-protein-coupled receptor-G-protein complex embedded in a phospholipid bilayer, which serves as a benchmark to validate the interactions seen in previous detergent-bound structures. The structure also reveals interactions that are unique to the membrane-embedded complex, including helix 8 burial in the inner leaflet, ordered lysine and arginine side chains in the membrane interfacial regions, and lipid anchoring of the G protein in the membrane. Our model of the activated DRD2 will help to inform the design of subtype-selective DRD2 ligands for multiple human central nervous system disorders.


Assuntos
Microscopia Crioeletrônica , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/ultraestrutura , Lipídeos de Membrana/metabolismo , Membranas Artificiais , Receptores de Dopamina D2/química , Receptores de Dopamina D2/ultraestrutura , Bromocriptina/química , Bromocriptina/metabolismo , Dopamina/química , Dopamina/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Humanos , Lipídeos de Membrana/química , Modelos Moleculares , Conformação Proteica , Receptores de Dopamina D2/agonistas , Receptores de Dopamina D2/metabolismo , Transdução de Sinais
6.
Nat Commun ; 11(1): 2995, 2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32532984

RESUMO

Adipocyte dysfunction links obesity to insulin resistance and type 2 diabetes. Adipocyte function is regulated by receptor-mediated activation of heterotrimeric G proteins. Little is known about the potential in vivo metabolic roles of Gi-type G proteins expressed by adipocytes, primarily due to the lack of suitable animal models. To address this question, we generated mice lacking functional Gi proteins selectively in adipocytes. Here we report that these mutant mice displayed significantly impaired glucose tolerance and reduced insulin sensitivity when maintained on an obesogenic diet. In contrast, using a chemogenetic strategy, we demonstrated that activation of Gi signaling selectively in adipocytes greatly improved glucose homeostasis and insulin signaling. We also elucidated the cellular mechanisms underlying the observed metabolic phenotypes. Our data support the concept that adipocyte Gi signaling is essential for maintaining euglycemia. Drug-mediated activation of adipocyte Gi signaling may prove beneficial for restoring proper glucose homeostasis in type 2 diabetes.


Assuntos
Adipócitos/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Resistência à Insulina/genética , Transdução de Sinais/genética , Adipócitos/citologia , Animais , Glicemia/metabolismo , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Perfilação da Expressão Gênica/métodos , Intolerância à Glucose/genética , Homeostase/genética , Insulina/sangue , Insulina/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Obesidade/sangue , Obesidade/genética , Obesidade/metabolismo
7.
Am J Physiol Cell Physiol ; 319(1): C183-C193, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32432925

RESUMO

The vasa vasorum (VV), the microvascular network around large vessels, has been recognized as an important contributor to the pathological vascular remodeling in cardiovascular diseases. In bovine and rat models of hypoxic pulmonary hypertension (PH), we have previously shown that chronic hypoxia profoundly increased pulmonary artery (PA) VV permeability, associated with infiltration of inflammatory and progenitor cells in the arterial wall, perivascular inflammation, and structural vascular remodeling. Extracellular adenosine was shown to exhibit a barrier-protective effect on VV endothelial cells (VVEC) via cAMP-independent mechanisms, which involved adenosine A1 receptor-mediated activation of Gi-phosphoinositide 3-kinase-Akt pathway and actin cytoskeleton remodeling. Using VVEC isolated from the adventitia of calf PA, in this study we investigated in more detail the mechanisms linking Gi activation to downstream barrier protection pathways. Using a small-interference RNA (siRNA) technique and transendothelial electrical resistance assay, we found that the adaptor protein, engulfment and cell motility 1 (ELMO1), the tyrosine phosphatase Src homology region 2 domain-containing phosphatase-2, and atypical Gi- and Rac1-mediated protein kinase A activation are implicated in VVEC barrier enhancement. In contrast, the actin-interacting GTP-binding protein, girdin, and the p21-activated kinase 1 downstream target, LIM kinase, are not involved in this response. In addition, adenosine-dependent cytoskeletal rearrangement involves activation of cofilin and inactivation of ezrin-radixin-moesin regulatory cytoskeletal proteins, consistent with a barrier-protective mechanism. Collectively, our data indicate that targeting adenosine receptors and downstream barrier-protective pathways in VVEC may have a potential translational significance in developing pharmacological approach for the VV barrier protection in PH.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenosina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células Endoteliais/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Vasa Vasorum/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Adenosina/farmacologia , Animais , Bovinos , Células Endoteliais/efeitos dos fármacos , Líquido Extracelular/efeitos dos fármacos , Líquido Extracelular/metabolismo , Masculino , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Vasa Vasorum/efeitos dos fármacos
8.
J Genet ; 992020.
Artigo em Inglês | MEDLINE | ID: mdl-32366735

RESUMO

Goat is the most preferred domesticated animal in Indian subcontinent. However, the climatic change-induced heat stresscauses a formidable challenge for maintaining optimum productivity. G protein subunit alpha i3 (GNAI3) is one of the genes that may have significant role in heat tolerance mechanism in goats. The caprine GNAI3 gene was searched for homology analysis and its three dimensional protein structure was predicted followed by its validation through in silico approach. Nucleotide sequence-based phylogenetic tree analysis showed that the caprine GNAI3 gene has close evolutionary relationship with that of Ovis aries. Homology modelling of caprine GNAI3 protein was done in MODELLER 9.18 (P1), PHYRE2 (P2), GENO3D (P3) and SWISS MODEL (P4). The modelled structures were further validated after observing the Ramachandran and hydrophobicity plots. In the best of three dimensional protein structure (P4 as produced by SWISS MODEL), 330 (98.8%), three (0.9%) and one (0.3%) amino acid residues were found in favoured region, allowed region and outlier region, respectively. Degree of hydrophobicity of the generated protein structures revealed the presence of alternate hydrophobic and hydrophilic regions. The ligand receptor interaction site of the predicted 3D model was traced out using Discovery Studio 3.5. STRING database revealed protein interactions with Plcb1, Plcb2, Plcb3 and other proteins of G family such as Gnb1, Gnb2, Gnb3,Gnb4, Gng2, Gng4 and Gpsm1. KEGG pathway maps revealed interaction with eNOS, iNOS, VEGF and MAPK, which are reported to be transcribed in response to heat stress. Thus, caprine GNAI3 can be used as a possible biomarker for studying heattolerance mechanism in goats.


Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Cabras/genética , Termotolerância/genética , Sequência de Aminoácidos , Animais , Biologia Computacional , Simulação por Computador , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Cabras/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Modelos Moleculares , Filogenia , Conformação Proteica , Mapeamento de Interação de Proteínas , Homologia de Sequência de Aminoácidos
9.
Science ; 367(6484): 1346-1352, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32193322

RESUMO

Class B G protein-coupled receptors, an important class of therapeutic targets, signal mainly through the Gs class of heterotrimeric G proteins, although they do display some promiscuity in G protein binding. Using cryo-electron microscopy, we determined the structures of the human glucagon receptor (GCGR) bound to glucagon and distinct classes of heterotrimeric G proteins, Gs or Gi1 These two structures adopt a similar open binding cavity to accommodate Gs and Gi1 The Gs binding selectivity of GCGR is explained by a larger interaction interface, but there are specific interactions that affect Gi more than Gs binding. Conformational differences in the receptor intracellular loops were found to be key selectivity determinants. These distinctions in transducer engagement were supported by mutagenesis and functional studies.


Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Glucagon/química , Receptores de Glucagon/química , Sítios de Ligação , Microscopia Crioeletrônica , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/ultraestrutura , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/ultraestrutura , Glucagon/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Conformação Proteica em alfa-Hélice , Receptores de Glucagon/metabolismo , Receptores de Glucagon/ultraestrutura , Transdução de Sinais
10.
Cell ; 180(4): 645-654.e13, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-32004460

RESUMO

Drugs selectively targeting CB2 hold promise for treating neurodegenerative disorders, inflammation, and pain while avoiding psychotropic side effects mediated by CB1. The mechanisms underlying CB2 activation and signaling are poorly understood but critical for drug design. Here we report the cryo-EM structure of the human CB2-Gi signaling complex bound to the agonist WIN 55,212-2. The 3D structure reveals the binding mode of WIN 55,212-2 and structural determinants for distinguishing CB2 agonists from antagonists, which are supported by a pair of rationally designed agonist and antagonist. Further structural analyses with computational docking results uncover the differences between CB2 and CB1 in receptor activation, ligand recognition, and Gi coupling. These findings are expected to facilitate rational structure-based discovery of drugs targeting the cannabinoid system.


Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Receptor CB2 de Canabinoide/química , Transdução de Sinais , Animais , Sítios de Ligação , Células CHO , Agonistas de Receptores de Canabinoides/síntese química , Agonistas de Receptores de Canabinoides/farmacologia , Antagonistas de Receptores de Canabinoides/síntese química , Antagonistas de Receptores de Canabinoides/farmacologia , Cricetinae , Cricetulus , Microscopia Crioeletrônica , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Receptor CB2 de Canabinoide/agonistas , Receptor CB2 de Canabinoide/antagonistas & inibidores , Receptor CB2 de Canabinoide/metabolismo , Células Sf9 , Spodoptera
11.
Proc Natl Acad Sci U S A ; 117(10): 5502-5509, 2020 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-32098843

RESUMO

The habenula, an ancient small brain area in the epithalamus, densely expresses nicotinic acetylcholine receptors and is critical for nicotine intake and aversion. As such, identification of strategies to manipulate habenular activity may yield approaches to treat nicotine addiction. Here we show that GPR151, an orphan G-protein-coupled receptor (GPCR) highly enriched in the habenula of humans and rodents, is expressed at presynaptic membranes and synaptic vesicles and associates with synaptic components controlling vesicle release and ion transport. Deletion of Gpr151 inhibits evoked neurotransmission but enhances spontaneous miniature synaptic currents and eliminates short-term plasticity induced by nicotine. We find that GPR151 couples to the G-alpha inhibitory protein Gαo1 to reduce cyclic adenosine monophosphate (cAMP) levels in mice and in GPR151-expressing cell lines that are amenable to ligand screens. Gpr151- knockout (KO) mice show diminished behavioral responses to nicotine and self-administer greater quantities of the drug, phenotypes rescued by viral reexpression of Gpr151 in the habenula. These data identify GPR151 as a critical modulator of habenular function that controls nicotine addiction vulnerability.


Assuntos
Habenula/fisiologia , Plasticidade Neuronal/fisiologia , Nicotina/metabolismo , Agonistas Nicotínicos/metabolismo , Receptores Acoplados a Proteínas-G/fisiologia , Transtornos Relacionados ao Uso de Substâncias/metabolismo , Animais , Células CHO , Cricetulus , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Habenula/metabolismo , Humanos , Camundongos Knockout , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/genética , Nicotina/administração & dosagem , Agonistas Nicotínicos/administração & dosagem , Receptores Acoplados a Proteínas-G/genética , Transmissão Sináptica/genética , Transmissão Sináptica/fisiologia
12.
Cell ; 180(4): 655-665.e18, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-32004463

RESUMO

Human endocannabinoid systems modulate multiple physiological processes mainly through the activation of cannabinoid receptors CB1 and CB2. Their high sequence similarity, low agonist selectivity, and lack of activation and G protein-coupling knowledge have hindered the development of therapeutic applications. Importantly, missing structural information has significantly held back the development of promising CB2-selective agonist drugs for treating inflammatory and neuropathic pain without the psychoactivity of CB1. Here, we report the cryoelectron microscopy structures of synthetic cannabinoid-bound CB2 and CB1 in complex with Gi, as well as agonist-bound CB2 crystal structure. Of important scientific and therapeutic benefit, our results reveal a diverse activation and signaling mechanism, the structural basis of CB2-selective agonists design, and the unexpected interaction of cholesterol with CB1, suggestive of its endogenous allosteric modulating role.


Assuntos
Agonistas de Receptores de Canabinoides/farmacologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Receptor CB1 de Canabinoide/química , Receptor CB2 de Canabinoide/química , Transdução de Sinais , Regulação Alostérica , Sítio Alostérico , Animais , Células CHO , Agonistas de Receptores de Canabinoides/química , Canabinoides/química , Canabinoides/farmacologia , Linhagem Celular Tumoral , Colesterol/química , Colesterol/farmacologia , Cricetinae , Cricetulus , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Humanos , Simulação de Dinâmica Molecular , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Células Sf9 , Spodoptera
13.
Int J Mol Sci ; 21(2)2020 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-31936673

RESUMO

The dopamine D2 receptor belongs to rhodopsin-like G protein-coupled receptors (GPCRs) and it is an important molecular target for the treatment of many disorders, including schizophrenia and Parkinson's disease. Here, computational methods were used to construct the full models of the dopamine D2 receptor short (D2S) and long (D2L) isoforms (differing with 29 amino acids insertion in the third intracellular loop, ICL3) and to study their coupling with Gi1 and Gi2 proteins. It was found that the D2L isoform preferentially couples with the Gi2 protein and D2S isoform with the Gi1 protein, which is in accordance with experimental data. Our findings give mechanistic insight into the interplay between isoforms of dopamine D2 receptors and Gi proteins subtypes, which is important to understand signaling by these receptors and their mediation by pharmaceuticals, in particular psychotic and antipsychotic agents.


Assuntos
Simulação por Computador , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Receptores de Dopamina D2/metabolismo , Dopamina , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Análise de Componente Principal , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estrutura Secundária de Proteína , Receptores de Dopamina D2/química , Água
14.
Clin Immunol ; 212: 108346, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31954803

RESUMO

Previous studies showed that circulating autoantibodies against M2 muscarinic receptors (anti-M2R Ab) are associated with decreased cardiac parasympathetic modulation in patients with chronic Chagas disease (CD). Here we investigated whether the exposure of M2R to such antibodies could impair agonist-induced receptor activation, leading to the inhibition of associated signaling pathways. Preincubation of M2R-expressing HEK 293T cells with serum IgG fractions from chagasic patients with cardiovascular dysautonomia, followed by the addition of carbachol, resulted in the attenuation of agonist-induced Gi protein activation and arrestin-2 recruitment. These effects were not mimicked by the corresponding Fab fractions, suggesting that they occur through receptor crosslinking. IgG autoantibodies did not enhance M2R/arrestin interaction or promote M2R internalization, suggesting that their inhibitory effects are not likely a result of short-term receptor regulation. Rather, these immunoglobulins could function as negative allosteric modulators of acetylcholine-mediated responses, thereby contributing to the development of parasympathetic dysfunction in patients with CD.


Assuntos
Autoanticorpos/imunologia , Doenças do Sistema Nervoso Autônomo/imunologia , Doença de Chagas/imunologia , Receptor Muscarínico M2/imunologia , Adulto , Idoso , Regulação Alostérica , Autoanticorpos/metabolismo , Autoanticorpos/farmacologia , Doenças do Sistema Nervoso Autônomo/etiologia , Doenças do Sistema Nervoso Autônomo/metabolismo , Doenças do Sistema Nervoso Autônomo/fisiopatologia , Carbacol/farmacologia , Doença de Chagas/complicações , Doença de Chagas/metabolismo , Doença de Chagas/fisiopatologia , Agonistas Colinérgicos/farmacologia , Feminino , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Frequência Cardíaca , Humanos , Masculino , Pessoa de Meia-Idade , Receptor Muscarínico M2/efeitos dos fármacos , Receptor Muscarínico M2/metabolismo , beta-Arrestina 1/metabolismo
15.
Eur J Pharmacol ; 868: 172886, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-31866407

RESUMO

Lysophosphatidic acid (LPA), as a bioactive lipid, plays a variety of physiological and pathological roles via activating six types of G-protein-coupled LPA receptors (LPA1-6). Our preliminary study found that LPA1 is highly expressed in lung cancer tissues compared with paracancerous tissues, but the role of LPA1 in lung carcinoma is unclear. This study aimed to elucidate the association between LPA1 and lung tumour behaviour at the cellular and animal model levels. We found that LPA promoted the migration, proliferation and colony formation of a lung cancer cell line (A549). LPA1 and LPA3 are preferentially expressed in A549 cells, and both Ki16425 (LPA1 and LPA3 antagonist) and ono7300243 (LPA1 antagonist) completely blocked the LPA-induced actions. These results were further verified by experiments of the LPA1/3 overexpression and LPA1 knockdown A549 cells. Furthermore, LPA1 overexpression and knockdown A549 cells were used to assess the in vivo tumour-bearing animal model and the mechanism underlying LPA-induced actions. In the animal model, A549 cell-derived tumour volume was significantly increased by LPA1 overexpression and significantly decreased by LPA1 knockdown respectively, suggesting that LPA1 is a regulator of in vivo tumour formation. Our results also indicated that the LPA1/Gi/MAP kinase/NF-κB pathway is involved in LPA-induced oncogenic actions in A549 cells. Thus, targeting LPA1 may be a novel strategy for treating lung carcinoma.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Receptores de Ácidos Lisofosfatídicos/antagonistas & inibidores , Células A549 , Animais , Antineoplásicos/uso terapêutico , Movimento Celular/efeitos dos fármacos , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Isoxazóis/farmacologia , Isoxazóis/uso terapêutico , Neoplasias Pulmonares/patologia , Lisofosfolipídeos/metabolismo , Masculino , Camundongos , NF-kappa B/metabolismo , Propionatos/farmacologia , Propionatos/uso terapêutico , Receptores de Ácidos Lisofosfatídicos/genética , Receptores de Ácidos Lisofosfatídicos/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Mol Pharmacol ; 97(1): 2-8, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31704717

RESUMO

The thyrotropin (TSH) receptor (TSHR) signals via G proteins of all four classes and ß-arrestin 1. Stimulation of TSHR leads to increasing cAMP production that has been reported as a monotonic dose-response curve that plateaus at high TSH doses. In HEK 293 cells overexpressing TSHRs (HEK-TSHR cells), we found that TSHR activation exhibits an "inverted U-shaped dose-response curve" with increasing cAMP production at low doses of TSH and decreased cAMP production at high doses (>1 mU/ml). Since protein kinase A inhibition by H-89 and knockdown of ß-arrestin 1 or ß-arrestin 2 did not affect the decreased cAMP production at high TSH doses, we studied the roles of TSHR downregulation and of Gi/Go proteins. A high TSH dose (100 mU/ml) caused a 33% decrease in cell-surface TSHR. However, because inhibiting TSHR downregulation with combined expression of a dominant negative dynamin 1 and ß-arrestin 2 knockdown had no effect, we concluded that downregulation is not involved in the biphasic cAMP response. Pertussis toxin, which inhibits activation of Gi/Go, abolished the biphasic response with no statistically significant difference in cAMP levels at 1 and 100 mU/ml TSH. Concordantly, co-knockdown of Gi/Go proteins increased cAMP levels stimulated by 100 mU/ml TSH from 55% to 73% of the peak level. These data show that biphasic regulation of cAMP production is mediated by Gs and Gi/Go at low and high TSH doses, respectively, which may represent a mechanism to prevent overstimulation in TSHR-expressing cells. SIGNIFICANCE STATEMENT: We demonstrate biphasic regulation of TSH-mediated cAMP production involving coupling of the TSH receptor (TSHR) to Gs at low TSH doses and to Gi/o at high TSH doses. We suggest that this biphasic cAMP response allows the TSHR to mediate responses at lower levels of TSH and that decreased cAMP production at high doses may represent a mechanism to prevent overstimulation of TSHR-expressing cells. This mechanism could prevent chronic stimulation of thyroid gland function.


Assuntos
AMP Cíclico/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Receptores da Tireotropina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tireotropina/administração & dosagem , Relação Dose-Resposta a Droga , Regulação para Baixo , Dinamina I/genética , Dinamina I/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/antagonistas & inibidores , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Toxina Pertussis/administração & dosagem , Receptores da Tireotropina/genética , Transdução de Sinais/genética , beta-Arrestina 2/genética , beta-Arrestina 2/metabolismo
17.
Artigo em Inglês | MEDLINE | ID: mdl-31875697

RESUMO

In mouse ileal myocytes, muscarinic receptor-mediated cationic current (mIcat) occurs mainly through synergism of M2 and M3 subtypes involving Gi/o-type GTP-binding proteins and phospholipase C (PLC). We have further studied the M2/M3 synergistic pathway. Carbachol-induced mIcat was markedly depressed by YM-254890, a Gq/11 protein inhibitor. However, the mIcat was unaffected by heparin, calphostin C, or chelerythrine, suggesting that mIcat activation does not involve signaling molecules downstream of phosphatidylinositol 4,5-bisphosphate (PIP2) breakdown. M2-knockout (KO) mice displayed a reduced mIcat (~10% of wild-type mIcat) because of the lack of M2-Gi/o signaling. The impaired mIcat was insensitive to neuropeptide Y possessing a Gi/o-stimulating activity. M3-KO mice also displayed a reduced mIcat (~6% of wild-type mIcat) because of the lack of M3-Gq/11 signaling, and the mIcat was insensitive to prostaglandin F2α possessing a Gq/11-stimulating activity. These results suggest the importance of Gq/11/PLC-hydrolyzed PIP2 breakdown itself in mIcat activation and also support the idea that the M2/M3 synergistic pathway represents a signaling complex consisting of M2-Gi/o and M3-Gq/11-PLC systems in which both G proteins are special for this pathway but not general in receptor coupling.


Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Mucosa Intestinal/metabolismo , Miócitos de Músculo Liso/metabolismo , Receptor Muscarínico M2/metabolismo , Receptor Muscarínico M3/metabolismo , Animais , Agonistas Colinérgicos/farmacologia , Relação Dose-Resposta a Droga , Feminino , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/agonistas , Cobaias , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Peptídeos Cíclicos/farmacologia , Receptor Muscarínico M2/agonistas , Receptor Muscarínico M3/agonistas
19.
Nat Commun ; 10(1): 5774, 2019 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-31852903

RESUMO

Translation initiation is a major rate-limiting step for protein synthesis. However, recent studies strongly suggest that the efficiency of protein synthesis is additionally regulated by multiple factors that impact the elongation phase. To assess the influence of early elongation on protein synthesis, we employed a library of more than 250,000 reporters combined with in vitro and in vivo protein expression assays. Here we report that the identity of the amino acids encoded by codons 3 to 5 impact protein yield. This effect is independent of tRNA abundance, translation initiation efficiency, or overall mRNA structure. Single-molecule measurements of translation kinetics revealed pausing of the ribosome and aborted protein synthesis on codons 4 and 5 of distinct amino acid and nucleotide compositions. Finally, introduction of preferred sequence motifs only at specific codon positions improves protein synthesis efficiency for recombinant proteins. Collectively, our data underscore the critical role of early elongation events in translational control of gene expression.


Assuntos
Códon/genética , Elongação Traducional da Cadeia Peptídica/genética , Ribossomos/metabolismo , Aminoácidos/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Biblioteca Gênica , Genes Reporter/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Nucleotídeos/metabolismo , Iniciação Traducional da Cadeia Peptídica , Proteínas RGS/genética , Proteínas RGS/metabolismo , RNA de Transferência/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Imagem Individual de Molécula
20.
Int J Mol Sci ; 20(21)2019 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-31653051

RESUMO

The neurotransmitter molecule acetylcholine is capable of activating five muscarinic acetylcholine receptors, M1 through M5, which belong to the superfamily of G-protein-coupled receptors (GPCRs). These five receptors share high sequence and structure homology; however, the M1, M3, and M5 receptor subtypes signal preferentially through the Gαq/11 subset of G proteins, whereas the M2 and M4 receptor subtypes signal through the Gαi/o subset of G proteins, resulting in very different intracellular signaling cascades and physiological effects. The structural basis for this innate ability of the M1/M3/M5 set of receptors and the highly homologous M2/M4 set of receptors to couple to different G proteins is poorly understood. In this study, we used molecular dynamics (MD) simulations coupled with thermodynamic analyses of M1 and M2 receptors coupled to both Gαi and Gαq to understand the structural basis of the M1 receptor's preference for the Gαq protein and the M2 receptor's preference for the Gαi protein. The MD studies showed that the M1 and M2 receptors can couple to both Gα proteins such that the M1 receptor engages with the two Gα proteins in slightly different orientations and the M2 receptor engages with the two Gα proteins in the same orientation. Thermodynamic studies of the free energy of binding of the receptors to the Gα proteins showed that the M1 and M2 receptors bind more strongly to their cognate Gα proteins compared to their non-cognate ones, which is in line with previous experimental studies on the M3 receptor. A detailed analysis of receptor-G protein interactions showed some cognate-complex-specific interactions for the M2:Gαi complex; however, G protein selectivity determinants are spread over a large overlapping subset of residues. Conserved interaction between transmembrane helices 5 and 6 far away from the G-protein-binding receptor interface was found only in the two cognate complexes and not in the non-cognate complexes. An analysis of residues implicated previously in G protein selectivity, in light of the cognate and non-cognate structures, shaded a more nuanced role of those residues in affecting G protein selectivity. The simulation of both cognate and non-cognate receptor-G protein complexes fills a structural gap due to difficulties in determining non-cognate complex structures and provides an enhanced framework to probe the mechanisms of G protein selectivity exhibited by most GPCRs.


Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Simulação de Dinâmica Molecular , Receptores Muscarínicos/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/química , Humanos , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Receptores Muscarínicos/química , Termodinâmica
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