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1.
Nature ; 579(7797): 152-157, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32076264

RESUMO

GPR52 is a class-A orphan G-protein-coupled receptor that is highly expressed in the brain and represents a promising therapeutic target for the treatment of Huntington's disease and several psychiatric disorders1,2. Pathological malfunction of GPR52 signalling occurs primarily through the heterotrimeric Gs protein2, but it is unclear how GPR52 and Gs couple for signal transduction and whether a native ligand or other activating input is required. Here we present the high-resolution structures of human GPR52 in three states: a ligand-free state, a Gs-coupled self-activation state and a potential allosteric ligand-bound state. Together, our structures reveal that extracellular loop 2 occupies the orthosteric binding pocket and operates as a built-in agonist, conferring an intrinsically high level of basal activity to GPR523. A fully active state is achieved when Gs is coupled to GPR52 in the absence of an external agonist. The receptor also features a side pocket for ligand binding. These insights into the structure and function of GPR52 could improve our understanding of other self-activated GPCRs, enable the identification of endogenous and tool ligands, and guide drug discovery efforts that target GPR52.


Assuntos
Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/metabolismo , Regulação Alostérica , Sítio Alostérico , Motivos de Aminoácidos , Sequência de Aminoácidos , Apoproteínas/agonistas , Apoproteínas/química , Apoproteínas/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Cristalografia por Raios X , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/ultraestrutura , Humanos , Ligantes , Modelos Moleculares , Receptores Acoplados a Proteínas-G/agonistas , Receptores Acoplados a Proteínas-G/ultraestrutura
2.
Nat Commun ; 11(1): 564, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992701

RESUMO

Detergents enable the purification of membrane proteins and are indispensable reagents in structural biology. Even though a large variety of detergents have been developed in the last century, the challenge remains to identify guidelines that allow fine-tuning of detergents for individual applications in membrane protein research. Addressing this challenge, here we introduce the family of oligoglycerol detergents (OGDs). Native mass spectrometry (MS) reveals that the modular OGD architecture offers the ability to control protein purification and to preserve interactions with native membrane lipids during purification. In addition to a broad range of bacterial membrane proteins, OGDs also enable the purification and analysis of a functional G-protein coupled receptor (GPCR). Moreover, given the modular design of these detergents, we anticipate fine-tuning of their properties for specific applications in structural biology. Seen from a broader perspective, this represents a significant advance for the investigation of membrane proteins and their interactions with lipids.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Detergentes/química , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/isolamento & purificação , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Escherichia coli , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/isolamento & purificação , Espectrometria de Massas , Lipídeos de Membrana , Modelos Moleculares , Peptídeo Hidrolases/química , Peptídeo Hidrolases/isolamento & purificação , Redobramento de Proteína , Solubilidade
3.
Nat Commun ; 11(1): 194, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31924782

RESUMO

Many drugs target the extracellular regions (ECRs) of cell-surface receptors. The large and alternatively-spliced ECRs of adhesion G protein-coupled receptors (aGPCRs) have key functions in diverse biological processes including neurodevelopment, embryogenesis, and tumorigenesis. However, their structures and mechanisms of action remain unclear, hampering drug development. The aGPCR Gpr126/Adgrg6 regulates Schwann cell myelination, ear canal formation, and heart development; and GPR126 mutations cause myelination defects in human. Here, we determine the structure of the complete zebrafish Gpr126 ECR and reveal five domains including a previously unknown domain. Strikingly, the Gpr126 ECR adopts a closed conformation that is stabilized by an alternatively spliced linker and a conserved calcium-binding site. Alternative splicing regulates ECR conformation and receptor signaling, while mutagenesis of the calcium-binding site abolishes Gpr126 function in vivo. These results demonstrate that Gpr126 ECR utilizes a multi-faceted dynamic approach to regulate receptor function and provide relevant insights for ECR-targeted drug design.


Assuntos
Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/metabolismo , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Processamento Alternativo , Animais , Sítios de Ligação , Cristalografia por Raios X , Desenho de Drogas , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Células de Schwann/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
5.
Curr Top Med Chem ; 19(26): 2378-2392, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31833462

RESUMO

G protein-coupled receptors (GPCRs) represent the largest family of proteins targeted by drug design and discovery efforts. Of these efforts, the development of GPCR agonists is highly desirable, due to their therapeutic robust utility in treating diseases caused by deficient receptor signaling. One of the challenges in designing potent and selective GPCR agonists lies in the inability to achieve combined high binding affinity and subtype selectivity, due to the high homology between orthosteric sites among GPCR subtypes. To combat this difficulty, researchers have begun to explore the utility of targeting topographically distinct and less conserved binding sites, namely "allosteric" sites. Pursuing these sites offers the benefit of achieving high subtype selectivity, however, it also can result in a decreased binding affinity and potency as compared to orthosteric agonists. Therefore, bitopic ligands comprised of an orthosteric agonist and an allosteric modulator connected by a spacer and allowing binding with both the orthosteric and allosteric sites within one receptor, have been developed. It may combine the high subtype selectivity of an allosteric modulator with the high binding affinity of an orthosteric agonist and provides desired advantages over orthosteric agonists or allosteric modulators alone. Herein, we review the recent advances in the development of bitopic agonists/activators for various GPCR targets and their novel therapeutic potentials.


Assuntos
Desenvolvimento de Medicamentos/métodos , Receptores Acoplados a Proteínas-G/agonistas , Sítio Alostérico , Sequência de Aminoácidos , Animais , Sítios de Ligação , Humanos , Ligantes , Receptores Acoplados a Proteínas-G/química , Homologia de Sequência de Aminoácidos
6.
Nat Struct Mol Biol ; 26(12): 1123-1131, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31740855

RESUMO

Classically, G-protein-coupled receptors (GPCRs) are thought to activate G protein from the plasma membrane and are subsequently desensitized by ß-arrestin (ß-arr). However, some GPCRs continue to signal through G protein from internalized compartments, mediated by a GPCR-G protein-ß-arr 'megaplex'. Nevertheless, the molecular architecture of the megaplex remains unknown. Here, we present its cryo-electron microscopy structure, which shows simultaneous engagement of human G protein and bovine ß-arr to the core and phosphorylated tail, respectively, of a single active human chimeric ß2-adrenergic receptor with the C-terminal tail of the arginine vasopressin type 2 receptor (ß2V2R). All three components adopt their canonical active conformations, suggesting that a single megaplex GPCR is capable of simultaneously activating G protein and ß-arr. Our findings provide a structural basis for GPCR-mediated sustained internalized G protein signaling.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais , beta-Arrestinas/metabolismo , Animais , Bovinos , Microscopia Crioeletrônica , Endossomos/metabolismo , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/ultraestrutura , Humanos , Modelos Moleculares , Conformação Proteica , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/metabolismo , Receptores Adrenérgicos beta 2/ultraestrutura , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/ultraestrutura , Receptores de Vasopressinas/química , Receptores de Vasopressinas/metabolismo , Receptores de Vasopressinas/ultraestrutura , beta-Arrestinas/química , beta-Arrestinas/ultraestrutura
8.
Nature ; 574(7779): 581-585, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31645725

RESUMO

The tricarboxylic acid cycle intermediate succinate is involved in metabolic processes and plays a crucial role in the homeostasis of mitochondrial reactive oxygen species1. The receptor responsible for succinate signalling, SUCNR1 (also known as GPR91), is a member of the G-protein-coupled-receptor family2 and links succinate signalling to renin-induced hypertension, retinal angiogenesis and inflammation3-5. Because SUCNR1 senses succinate as an immunological danger signal6-which has relevance for diseases including ulcerative colitis, liver fibrosis7, diabetes and rheumatoid arthritis3,8-it is of interest as a therapeutic target. Here we report the high-resolution crystal structure of rat SUCNR1 in complex with an intracellular binding nanobody in the inactive conformation. Structure-based mutagenesis and radioligand-binding studies, in conjunction with molecular modelling, identified key residues for species-selective antagonist binding and enabled the determination of the high-resolution crystal structure of a humanized rat SUCNR1 in complex with a high-affinity, human-selective antagonist denoted NF-56-EJ40. We anticipate that these structural insights into the architecture of the succinate receptor and its antagonist selectivity will enable structure-based drug discovery and will further help to elucidate the function of SUCNR1 in vitro and in vivo.


Assuntos
Compostos de Bifenilo/química , Compostos de Bifenilo/farmacologia , Piperazinas/química , Piperazinas/farmacologia , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Receptores Acoplados a Proteínas-G/química , Animais , Apoproteínas/antagonistas & inibidores , Apoproteínas/química , Apoproteínas/metabolismo , Cristalografia por Raios X , Humanos , Modelos Moleculares , Ratos , Receptores Acoplados a Proteínas-G/metabolismo , Receptores Purinérgicos P2Y1/química , Transdução de Sinais , Anticorpos de Domínio Único/química , Especificidade da Espécie , Ácido Succínico/metabolismo
9.
Pharmacol Rev ; 71(4): 503-519, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31515243

RESUMO

G protein-coupled receptors (GPCRs) remain one of the most successful targets of U.S. Food and Drug Administration-approved drugs. GPCR research has predominantly focused on the characterization of the intracellular interactome's contribution to GPCR function and pharmacology. However, emerging evidence uncovers a new dimension in the biology of GPCRs involving their extracellular and transcellular interactions that critically impact GPCR function and pharmacology. The seminal examples include a variety of adhesion GPCRs, such as ADGRLs/latrophilins, ADGRBs/brain angiogenesis inhibitors, ADGRG1/GPR56, ADGRG6/GPR126, ADGRE5/CD97, and ADGRC3/CELSR3. However, recent advances have indicated that class C GPCRs that contain large extracellular domains, including group III metabotropic glutamate receptors (mGluR4, mGluR6, mGluR7, mGluR8), γ-aminobutyric acid receptors, and orphans GPR158 and GPR179, can also participate in this form of transcellular regulation. In this review, we will focus on a variety of identified extracellular and transcellular GPCR-interacting partners, including teneurins, neurexins, integrins, fibronectin leucine-rich transmembranes, contactin-6, neuroligin, laminins, collagens, major prion protein, amyloid precursor protein, complement C1q-likes, stabilin-2, pikachurin, dystroglycan, complement decay-accelerating factor CD55, cluster of differentiation CD36 and CD90, extracellular leucine-rich repeat and fibronectin type III domain containing 1, and leucine-rich repeat, immunoglobulin-like domain and transmembrane domains. We provide an account on the diversity of extracellular and transcellular GPCR complexes and their contribution to key cellular and physiologic processes, including cell migration, axon guidance, cellular and synaptic adhesion, and synaptogenesis. Furthermore, we discuss models and mechanisms by which extracellular GPCR assemblies may regulate communication at cellular junctions. SIGNIFICANCE STATEMENT: G protein-coupled receptors (GPCRs) continue to be the prominent focus of pharmacological intervention for a variety of human pathologies. Although the majority of GPCR research has focused on the intracellular interactome, recent advancements have identified an extracellular dimension of GPCR modulation that alters accepted pharmacological principles of GPCRs. Herein, we describe known endogenous allosteric modulators acting on GPCRs both in cis and in trans.


Assuntos
Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Receptores Acoplados a Proteínas-G/metabolismo , Animais , Moléculas de Adesão Celular/metabolismo , Membrana Celular/metabolismo , Humanos , Ligantes , Terapia de Alvo Molecular , Receptores Acoplados a Proteínas-G/química
10.
Pharmacol Rev ; 71(4): 571-595, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31551350

RESUMO

Endogenous ions play important roles in the function and pharmacology of G-protein coupled receptors (GPCRs). Historically the evidence for ionic modulation of GPCR function dates to 1973 with studies of opioid receptors, where it was demonstrated that physiologic concentrations of sodium allosterically attenuated agonist binding. This Na+-selective effect was distinct from effects of other monovalent and divalent cations, with the latter usually counteracting sodium's negative allosteric modulation of binding. Since then, numerous studies documenting the effects of mono- and divalent ions on GPCR function have been published. While ions can act selectively and nonselectively at many sites in different receptors, the discovery of the conserved sodium ion site in class A GPCR structures in 2012 revealed the unique nature of Na+ site, which has emerged as a near-universal site for allosteric modulation of class A GPCR structure and function. In this review, we synthesize and highlight recent advances in the functional, biophysical, and structural characterization of ions bound to GPCRs. Taken together, these findings provide a molecular understanding of the unique roles of Na+ and other ions as GPCR allosteric modulators. We will also discuss how this knowledge can be applied to the redesign of receptors and ligand probes for desired functional and pharmacological profiles. SIGNIFICANCE STATEMENT: The function and pharmacology of GPCRs strongly depend on the presence of mono and divalent ions in experimental assays and in living organisms. Recent insights into the molecular mechanism of this ion-dependent allosterism from structural, biophysical, biochemical, and computational studies provide quantitative understandings of the pharmacological effects of drugs in vitro and in vivo and open new avenues for the rational design of chemical probes and drug candidates with improved properties.


Assuntos
Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Receptores Acoplados a Proteínas-G/metabolismo , Sódio/metabolismo , Sítio Alostérico , Ânions/química , Ânions/metabolismo , Sítios de Ligação , Cátions Bivalentes/química , Cátions Bivalentes/metabolismo , Cátions Monovalentes/química , Cátions Monovalentes/metabolismo , Cloretos/química , Cloretos/metabolismo , Cristalografia por Raios X , Humanos , Ligantes , Conformação Proteica , Receptores Acoplados a Proteínas-G/química , Sódio/química , Relação Estrutura-Atividade , Zinco/química , Zinco/metabolismo
11.
Molecules ; 24(19)2019 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-31561452

RESUMO

The identification and three step synthesis of 3-O-protocatechuoylceanothic acid, a novel and natural GPR120 agonist, is described. This ceanothane-type triterpenoid was identified from the components of Ziziphus jujuba roots and was found to be a new GPR120 agonist with a novel structure. We synthetically converted ceanothic acid, which does not have GPR120 agonist activity, into 3-O-protocatechuoylceanothic acid in three steps. In addition, we present the corrected NMR spectrum of 3-O-protocatechuoylceanothic acid based on our synthesis.


Assuntos
Receptores Acoplados a Proteínas-G/química , Triterpenos/síntese química , Triterpenos/farmacologia , Animais , Células CHO , Técnicas de Química Sintética , Cricetulus , Humanos , Ligantes , Estrutura Molecular , Receptores Acoplados a Proteínas-G/agonistas , Proteínas Recombinantes , Relação Estrutura-Atividade
12.
Int J Mol Sci ; 20(17)2019 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-31470676

RESUMO

G protein-coupled receptors (GPCRs) are critical drug targets. GPCRs convey signals from the extracellular to the intracellular environment through G proteins. Some ligands that bind to GPCRs activate different downstream signaling pathways. G protein activation, or -arrestin biased signaling, involves ligands binding to receptors and stabilizing conformations that trigger a specific pathway. -arrestin biased signaling has become a hot target for structure-based drug discovery. However, challenges include that there are few crystal structures available in the Protein Data Bank and that GPCRs are highly dynamic. Hence, molecular dynamics (MD) simulations are especially valuable for obtaining detailed mechanistic information, including identification of allosteric sites and understanding modulators' interactions with receptors and ligands. Here, we highlight recent MD simulation studies and enhanced sampling methods used to study biased G protein-coupled receptor signaling and their conformational dynamics as well as applications to drug discovery.


Assuntos
Descoberta de Drogas/métodos , Proteínas de Ligação ao GTP/química , Simulação de Dinâmica Molecular , Domínios Proteicos , Receptores Acoplados a Proteínas-G/química , Sítios de Ligação , Proteínas de Ligação ao GTP/metabolismo , Humanos , Ligantes , Ligação Proteica , Receptores Acoplados a Proteínas-G/metabolismo
13.
J Exp Clin Cancer Res ; 38(1): 335, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31370872

RESUMO

BACKGROUND: The chemical carcinogen 3-methylcholanthrene (3MC) binds to the aryl hydrocarbon receptor (AHR) that regulates the expression of cytochrome P450 (CYP) enzymes as CYP1B1, which is involved in the oncogenic activation of environmental pollutants as well as in the estrogen biosynthesis and metabolism. 3MC was shown to induce estrogenic responses binding to the estrogen receptor (ER) α and stimulating a functional interaction between AHR and ERα. Recently, the G protein estrogen receptor (GPER) has been reported to mediate certain biological responses induced by endogenous estrogens and environmental compounds eliciting an estrogen-like activity. METHODS: Molecular dynamics and docking simulations were performed to evaluate the potential of 3MC to interact with GPER. SkBr3 breast cancer cells and cancer-associated fibroblasts (CAFs) derived from breast tumor patients were used as model system. Real-time PCR and western blotting analysis were performed in order to evaluate the activation of transduction mediators as well as the mRNA and protein levels of CYP1B1 and cyclin D1. Co-immunoprecipitation studies were performed in order to explore the potential of 3MC to trigger the association of GPER with AHR and EGFR. Luciferase assays were carried out to determine the activity of CYP1B1 promoter deletion constructs upon 3MC exposure, while the nuclear shuttle of AHR induced by 3MC was assessed through confocal microscopy. Cell proliferation stimulated by 3MC was determined as biological counterpart of the aforementioned experimental assays. The statistical analysis was performed by ANOVA. RESULTS: We first ascertained by docking simulations the ability of 3MC to interact with GPER. Thereafter, we established that 3MC activates the EGFR/ERK/c-Fos transduction signaling through both AHR and GPER in SkBr3 cells and CAFs. Then, we found that these receptors are involved in the up-regulation of CYP1B1 and cyclin D1 as well as in the stimulation of growth responses induced by 3MC. CONCLUSIONS: In the present study we have provided novel insights regarding the molecular mechanisms by which 3MC may trigger a physical and functional interaction between AHR and GPER, leading to the stimulation of both SkBr3 breast cancer cells and CAFs. Altogether, our results indicate that 3MC may engage both GPER and AHR transduction pathways toward breast cancer progression.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Neoplasias da Mama/metabolismo , Fibroblastos Associados a Câncer/efeitos dos fármacos , Fibroblastos Associados a Câncer/metabolismo , Metilcolantreno/farmacologia , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores Estrogênicos/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Fibroblastos Associados a Câncer/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citocromo P-450 CYP1B1/genética , Citocromo P-450 CYP1B1/metabolismo , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Metilcolantreno/química , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Transporte Proteico , Receptores de Hidrocarboneto Arílico/química , Receptores Estrogênicos/química , Receptores Acoplados a Proteínas-G/química , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade
14.
Chemistry ; 25(68): 15483-15487, 2019 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-31461184

RESUMO

Photorelease of caged compounds is among the most powerful experimental approaches for studying cellular functions on fast timescales. However, its full potential has yet to be exploited, as the number of caged small molecules available for cell biological studies has been limited by synthetic challenges. Addressing this problem, a straightforward, one-step procedure for efficiently synthesizing caged compounds was developed. An in situ generated benzylic coumarin triflate reagent was used to specifically functionalize carboxylate and phosphate moieties in the presence of free hydroxy groups, generating various caged lipid metabolites, including a number of GPCR ligands. By combining the photo-caged ligands with the respective receptors, an easily implementable experimental platform for the optical control and analysis of GPCR-mediated signal transduction in living cells was developed. Ultimately, the described synthetic strategy allows rapid generation of photo-caged small molecules and thus greatly facilitates the analysis of their biological roles in live cell microscopy assays.


Assuntos
Cumarínicos/química , Lipídeos/síntese química , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Humanos , Ligantes , Lipídeos/química , Receptores Acoplados a Proteínas-G/química , Transdução de Sinais/fisiologia
15.
BMC Evol Biol ; 19(1): 143, 2019 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-31299890

RESUMO

BACKGROUND: Our laboratory identified ADGRL4/ELTD1, an orphan GPCR belonging to the adhesion GPCR (aGPCR) family, as a novel regulator of angiogenesis and a potential anti-cancer therapeutic target. Little is known about how ADGRL4/ELTD1 (and aGPCRs in general) function, a problem compounded by a lack of known ligands or means of activation. With this in mind, we turned to computational evolutionary biology with the aim of better understanding ADGRL4/ELTD1. RESULTS: We identified ADGRL4/ELTD1 as a highly conserved early angiogenic gene which emerged in the first true vertebrates (bony fish) approximately 435 million years ago (mya), evolving alongside key angiogenic genes VEGFR2 and DLL4. We identified 3 evolutionary ADGRL4/ELTD1 variants based on EGF domain deletions with variant 2 first emerging 101 mya (95% CI 96-105) in Afrotheria and 82 mya (95% CI 76-89) in Primates. Additionally, conservation mapping across all orthologues reveals highest level conservation in EGF Ca binding domain 1, suggesting that this motif plays an essential role, as well as specific regions of the GAIN domain, GPS motif and 7TM domain, suggesting possible activation mechanisms and ligand binding positions. Additionally, we found that ADGRL4/ELTD1 (a member aGPCR family 1) is possibly ancestral to members of aGPCR family 2. CONCLUSION: This work establishes ADGRL4/ELTD1's evolution, sheds light on its possible activation and ligand binding zones, and establishes the first temporal references for the emergence of ADGRL4/ELTD1 variants during vertebrate evolution. Our approach is applicable to the greater aGPCR family and opens up new avenues for future experimental work.


Assuntos
Sequência Conservada , Evolução Molecular , Neovascularização Fisiológica , Receptores Acoplados a Proteínas-G/genética , Vertebrados/genética , Animais , Adesão Celular , Sistemas de Liberação de Medicamentos , Peixes/genética , Humanos , Neovascularização Fisiológica/genética , Filogenia , Domínios Proteicos , Receptores Acoplados a Proteínas-G/química , Deleção de Sequência , Fatores de Tempo
16.
Cell Prolif ; 52(5): e12661, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31318114

RESUMO

OBJECTIVES: Circular RNAs (circRNAs) are non-coding RNAs, some of which are thought to be involved in gastric cancer development. Here, we examined the functions of circRNA hsa_circ_006100 in gastric cancer cells and an animal model of gastric cancer. MATERIALS AND METHODS: The expression of hsa_circ_006100, miR-195 and various functional genes was determined by quantitative RT-PCR. Cell viability, clone formation, apoptosis and cell migration/invasion abilities were analysed by the CCK-8 assay, crystal violet staining, Hoechst staining and Transwell assay, respectively. A tumour model was established by subcutaneously injecting tumour cells into nude mice. Levels of protein expression were analysed by Western blotting and immunohistochemistry. RESULTS: A bioinformatics analysis showed that miR-195 was negatively co-expressed with hsa_circ_006100. Patients with a high hsa_circ_006100 level or low miR-195 level had tumours with a high TNM stage, poor cellular differentiation and lymph node metastasis. miR-195 was targeted and inhibited by hsa_circ_006100. Overexpression of hsa_circ_006100 enhanced cellular viability and proliferation, while miR-195 suppressed hsa_circ_006100-enhanced cell growth and induced apoptosis in MGC-803 and AGS cells. Forced hsa_circ_006100 expression promoted the migration and invasion of MGC-803 and AGS cells, while those activities were inhibited by miR-195. Mechanistically, GPRC5A was predicted as a target of miR-195 and was upregulated in gastric cancer. A miR-195 inhibitor restored cell viability, proliferation, migration and invasion, and repressed apoptosis via GPRC5A. In vivo studies showed that knockdown of hsa_circ_006100 delayed tumour growth, reduced PCNA expression and upregulated miR-195 and BCL-2 expression which was restored by miR-195 inhibition due to GPRC5A/EGFR signalling, and changed the EMT phenotype in vivo. CONCLUSIONS: Hsa_circ_006100 functions as an oncogene in gastric cancer and exerts its effects via miR-195/GPRC5A signalling.


Assuntos
MicroRNAs/metabolismo , RNA/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Neoplasias Gástricas/patologia , Animais , Antagomirs/metabolismo , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Metástase Linfática , Masculino , Camundongos , Camundongos Nus , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Pessoa de Meia-Idade , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA/antagonistas & inibidores , RNA/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/genética , Transdução de Sinais , Neoplasias Gástricas/metabolismo
17.
J Chem Theory Comput ; 15(8): 4673-4686, 2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31265271

RESUMO

The time step of atomistic molecular dynamics (MD) simulations is determined by the fastest motions in the system and is typically limited to 2 fs. An increasingly popular approach is to increase the mass of the hydrogen atoms to ∼3 amu and decrease the mass of the parent atom by an equivalent amount. This approach, known as hydrogen-mass repartitioning (HMR), permits time steps up to 4 fs with reasonable simulation stability. While HMR has been applied in many published studies to date, it has not been extensively tested for membrane-containing systems. Here, we compare the results of simulations of a variety of membranes and membrane-protein systems run using a 2 fs time step and a 4 fs time step with HMR. For pure membrane systems, we find almost no difference in structural properties, such as area-per-lipid, electron density profiles, and order parameters, although there are differences in kinetic properties such as the diffusion constant. Conductance through a porin in an applied field, partitioning of a small peptide, hydrogen-bond dynamics, and membrane mixing show very little dependence on HMR and the time step. We also tested a 9 Å cutoff as compared to the standard CHARMM cutoff of 12 Å, finding significant deviations in many properties tested. We conclude that HMR is a valid approach for membrane systems, but a 9 Å cutoff is not.


Assuntos
Membrana Celular/química , Hidrogênio/química , Bicamadas Lipídicas/química , Proteínas de Membrana/química , Simulação de Dinâmica Molecular , Difusão , Humanos , Movimento (Física) , Peptídeos/química , Fosfatidilcolinas/química , Multimerização Proteica , Receptores Acoplados a Proteínas-G/química , Termodinâmica
18.
Nat Struct Mol Biol ; 26(7): 535-544, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31270468

RESUMO

Metabotropic receptors are responsible for so-called 'slow synaptic transmission' and mediate the effects of hundreds of peptide and non-peptide neurotransmitters and neuromodulators. Over the past decade or so, a revolution in membrane-protein structural determination has clarified the molecular determinants responsible for the actions of these receptors. This Review focuses on the G protein-coupled receptors (GPCRs) that are targets of neuropsychiatric drugs and shows how insights into the structure and function of these important synaptic proteins are accelerating understanding of their actions. Notably, elucidating the structure and function of GPCRs should enhance the structure-guided discovery of novel chemical tools with which to manipulate and understand these synaptic proteins.


Assuntos
Descoberta de Drogas , Receptores Acoplados a Proteínas-G/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Animais , Fármacos do Sistema Nervoso Central/química , Fármacos do Sistema Nervoso Central/farmacologia , Desenho de Drogas , Descoberta de Drogas/métodos , Humanos , Transtornos Mentais/tratamento farmacológico , Modelos Moleculares , Terapia de Alvo Molecular , Neurotransmissores/metabolismo , Receptores Acoplados a Proteínas-G/agonistas , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Receptores Acoplados a Proteínas-G/química , Receptores de Glutamato Metabotrópico/agonistas , Receptores de Glutamato Metabotrópico/antagonistas & inibidores , Receptores de Glutamato Metabotrópico/química , Receptores de Glutamato Metabotrópico/metabolismo
19.
Nat Commun ; 10(1): 3289, 2019 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-31337749

RESUMO

Allosteric modulators are highly desirable as drugs, particularly for G-protein-coupled receptor (GPCR) targets, because allosteric drugs can achieve selectivity between closely related receptors. The mechanisms by which allosteric modulators achieve selectivity remain elusive, however, particularly given recent structures that reveal similar allosteric binding sites across receptors. Here we show that positive allosteric modulators (PAMs) of the M1 muscarinic acetylcholine receptor (mAChR) achieve exquisite selectivity by occupying a dynamic pocket absent in existing crystal structures. This cryptic pocket forms far more frequently in molecular dynamics simulations of the M1 mAChR than in those of other mAChRs. These observations reconcile mutagenesis data that previously appeared contradictory. Further mutagenesis experiments validate our prediction that preventing cryptic pocket opening decreases the affinity of M1-selective PAMs. Our findings suggest opportunities for the design of subtype-specific drugs exploiting cryptic pockets that open in certain receptors but not in other receptors with nearly identical static structures.


Assuntos
Receptor Muscarínico M1/química , Receptores Acoplados a Proteínas-G/química , Regulação Alostérica , Sítio Alostérico , Cristalografia por Raios X , Desenho de Drogas , Ligantes , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida
20.
Oncogene ; 38(38): 6491-6506, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31337866

RESUMO

Oncodriver genes are usually identified when mutations recur in multiple tumours. Different drivers often converge in the activation or repression of key cancer-relevant pathways. However, as many pathways contain multiple members of the same gene family, individual mutations might be overlooked, as each family member would necessarily have a lower mutation frequency and thus not identified as significant in any one-gene-at-a-time analysis. Here, we looked for mutated, functional sequence positions in gene families that were mutually exclusive (in patients) with another gene in the same pathway, which identified both known and new candidate oncodrivers. For instance, many inactivating mutations in multiple G-protein (particularly Gi/o) coupled receptors, are mutually exclusive with Gαs oncogenic activating mutations, both of which ultimately enhance cAMP signalling. By integrating transcriptomics and interaction data, we show that the Gs pathway is upregulated in multiple cancer types, even those lacking known GNAS activating mutations. This suggests that cancer cells may develop alternative strategies to activate adenylate cyclase signalling in multiple cancer types. Our study provides a mechanistic interpretation for several rare somatic mutations in multi-gene oncodrivers, and offers possible explanations for known and potential off-label cancer treatments, suggesting new therapeutic opportunities.


Assuntos
Mutação , Neoplasias/genética , Oncogenes/genética , Receptores Acoplados a Proteínas-G/genética , Cromograninas/genética , Biologia Computacional , Epistasia Genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Frequência do Gene , Redes Reguladoras de Genes/fisiologia , Genes Supressores de Tumor , Células HEK293 , Humanos , Modelos Moleculares , Família Multigênica/genética , Mutação/fisiologia , Neoplasias/mortalidade , Neoplasias/patologia , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais/genética , Análise de Sobrevida , Fatores de Transcrição/genética
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