RESUMO
The conversion of phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 3,4,5-triphosphate by phosphoinositide 3-kinase γ (PI3Kγ) is critical for neutrophil chemotaxis and cancer metastasis. PI3Kγ is activated by Gßγ heterodimers released from G protein-coupled receptors responding to extracellular signals. Here we determined cryo-electron microscopy structures of Sus scrofa PI3Kγ-human Gßγ complexes in the presence of substrates/analogs, revealing two Gßγ binding sites: one on the p110γ helical domain and another on the p101 C-terminal domain. Comparison with PI3Kγ alone reveals conformational changes in the kinase domain upon Gßγ binding that are similar to Ras·GTP-induced changes. Assays of variants perturbing the Gßγ binding sites and interdomain contacts altered by Gßγ binding suggest that Gßγ recruits the enzyme to membranes and allosterically regulates activity via both sites. Studies of zebrafish neutrophil migration align with these findings, paving the way for in-depth investigation of Gßγ-mediated activation mechanisms in this enzyme family and drug development for PI3Kγ.
Assuntos
Classe Ib de Fosfatidilinositol 3-Quinase , Microscopia Crioeletrônica , Subunidades beta da Proteína de Ligação ao GTP , Subunidades gama da Proteína de Ligação ao GTP , Animais , Classe Ib de Fosfatidilinositol 3-Quinase/metabolismo , Classe Ib de Fosfatidilinositol 3-Quinase/química , Humanos , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/química , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/química , Sítios de Ligação , Peixe-Zebra , Ligação Proteica , Neutrófilos/metabolismo , Modelos Moleculares , Ativação Enzimática , Conformação Proteica , Regulação AlostéricaRESUMO
GPR158 is an orphan G proteincoupled receptor (GPCR) highly expressed in the brain, where it controls synapse formation and function. GPR158 has also been implicated in depression, carcinogenesis, and cognition. However, the structural organization and signaling mechanisms of GPR158 are largely unknown. We used single-particle cryoelectron microscopy (cryo-EM) to determine the structures of human GPR158 alone and bound to an RGS signaling complex. The structures reveal a homodimeric organization stabilized by a pair of phospholipids and the presence of an extracellular Cache domain, an unusual ligand-binding domain in GPCRs. We further demonstrate the structural basis of GPR158 coupling to RGS7-Gß5. Together, these results provide insights into the unusual biology of orphan receptors and the formation of GPCR-RGS complexes.
Assuntos
Subunidades beta da Proteína de Ligação ao GTP/química , Proteínas RGS/química , Receptores Acoplados a Proteínas G/química , Sítios de Ligação , Microscopia Crioeletrônica , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Humanos , Ligantes , Modelos Moleculares , Fosfolipídeos/química , Ligação Proteica , Conformação Proteica , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Multimerização Proteica , Subunidades Proteicas/química , Proteínas RGS/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de SinaisRESUMO
Smoothened (SMO), a class Frizzled G protein-coupled receptor (class F GPCR), transduces the Hedgehog signal across the cell membrane. Sterols can bind to its extracellular cysteine-rich domain (CRD) and to several sites in the seven transmembrane helices (7-TMs) of SMO. However, the mechanism by which sterols regulate SMO via multiple sites is unknown. Here we determined the structures of SMO-Gi complexes bound to the synthetic SMO agonist (SAG) and to 24(S),25-epoxycholesterol (24(S),25-EC). A novel sterol-binding site in the extracellular extension of TM6 was revealed to connect other sites in 7-TMs and CRD, forming an intramolecular sterol channel from the middle side of 7-TMs to CRD. Additional structures of two gain-of-function variants, SMOD384R and SMOG111C/I496C, showed that blocking the channel at its midpoints allows sterols to occupy the binding sites in 7-TMs, thereby activating SMO. These data indicate that sterol transport through the core of SMO is a major regulator of SMO-mediated signaling.
Assuntos
Colesterol/análogos & derivados , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Transdução de Sinais , Receptor Smoothened/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Colesterol/química , Colesterol/metabolismo , Cicloexilaminas/química , Cicloexilaminas/farmacologia , Subunidades alfa de Proteínas de Ligação ao GTP/química , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades beta da Proteína de Ligação ao GTP/química , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/química , Subunidades gama da Proteína de Ligação ao GTP/genética , Regulação da Expressão Gênica , Células HEK293 , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Anticorpos de Cadeia Única , Receptor Smoothened/agonistas , Receptor Smoothened/química , Receptor Smoothened/genética , Tiofenos/química , Tiofenos/farmacologiaRESUMO
Signaling by the G-protein-coupled receptors (GPCRs) plays fundamental role in a vast number of essential physiological functions. Precise control of GPCR signaling requires action of regulators of G protein signaling (RGS) proteins that deactivate heterotrimeric G proteins. RGS proteins are elaborately regulated and comprise multiple domains and subunits, yet structural organization of these assemblies is poorly understood. Here, we report a crystal structure and dynamics analyses of the multisubunit complex of RGS7, a major regulator of neuronal signaling with key roles in controlling a number of drug target GPCRs and links to neuropsychiatric disease, metabolism, and cancer. The crystal structure in combination with molecular dynamics and mass spectrometry analyses reveals unique organizational features of the complex and long-range conformational changes imposed by its constituent subunits during allosteric modulation. Notably, several intermolecular interfaces in the complex work in synergy to provide coordinated modulation of this key GPCR regulator.
Assuntos
Proteínas de Transporte/química , Subunidades beta da Proteína de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Simulação de Dinâmica Molecular , Neurônios/metabolismo , Proteínas RGS/química , Sequência de Aminoácidos , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cristalografia por Raios X , Medição da Troca de Deutério , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Espectrometria de Massas , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Conformação Proteica , Multimerização Proteica , Proteínas RGS/genética , Proteínas RGS/metabolismo , Homologia de Sequência de Aminoácidos , Células Sf9 , SpodopteraRESUMO
De novo germline mutations in GNB1 have been associated with a neurodevelopmental phenotype. To date, 28 patients with variants classified as pathogenic have been reported. We add 18 patients with de novo mutations to this cohort, including a patient with mosaicism for a GNB1 mutation who presented with a milder phenotype. Consistent with previous reports, developmental delay in these patients was moderate to severe, and more than half of the patients were non-ambulatory and nonverbal. The most observed substitution affects the p.Ile80 residue encoded in exon 6, with 28% of patients carrying a variant at this residue. Dystonia and growth delay were observed more frequently in patients carrying variants in this residue, suggesting a potential genotype-phenotype correlation. In the new cohort of 18 patients, 50% of males had genitourinary anomalies and 61% of patients had gastrointestinal anomalies, suggesting a possible association of these findings with variants in GNB1. In addition, cutaneous mastocytosis, reported once before in a patient with a GNB1 variant, was observed in three additional patients, providing further evidence for an association to GNB1. We will review clinical and molecular data of these new cases and all previously reported cases to further define the phenotype and establish possible genotype-phenotype correlations.
Assuntos
Subunidades beta da Proteína de Ligação ao GTP/genética , Estudos de Associação Genética , Mutação/genética , Adolescente , Criança , Pré-Escolar , Estudos de Coortes , Epilepsia/genética , Feminino , Subunidades beta da Proteína de Ligação ao GTP/química , Humanos , Masculino , Sistema Nervoso/crescimento & desenvolvimento , Fenótipo , Gravidez , Estrutura Terciária de ProteínaRESUMO
G protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by mediating a GDP to GTP exchange in the Gα subunit. This leads to dissociation of the heterotrimer into Gα-GTP and Gßγ dimer. The Gα-GTP and Gßγ dimer each regulate a variety of downstream pathways to control various aspects of human physiology. Dysregulated Gßγ-signaling is a central element of various neurological and cancer-related anomalies. However, Gßγ also serves as a negative regulator of Gα that is essential for G protein inactivation, and thus has the potential for numerous side effects when targeted therapeutically. Here we report a llama-derived nanobody (Nb5) that binds tightly to the Gßγ dimer. Nb5 responds to all combinations of ß-subtypes and γ-subtypes and competes with other Gßγ-regulatory proteins for a common binding site on the Gßγ dimer. Despite its inhibitory effect on Gßγ-mediated signaling, Nb5 has no effect on Gαq-mediated and Gαs-mediated signaling events in living cells.
Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Anticorpos de Domínio Único/metabolismo , Sítios de Ligação , Dimerização , Subunidades alfa de Proteínas de Ligação ao GTP/química , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades beta da Proteína de Ligação ao GTP/química , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/química , Subunidades gama da Proteína de Ligação ao GTP/genética , Guanosina Trifosfato/metabolismo , Humanos , Ligação Proteica , Transdução de Sinais , Anticorpos de Domínio Único/químicaRESUMO
G protein ßγ subunit (Gßγ) is a major signal transducer and controls processes ranging from cell migration to gene transcription. Despite having significant subtype heterogeneity and exhibiting diverse cell- and tissue-specific expression levels, Gßγ is often considered a unified signaling entity with a defined functionality. However, the molecular and mechanistic basis of Gßγ's signaling specificity is unknown. Here, we demonstrate that Gγ subunits, bearing the sole plasma membrane (PM)-anchoring motif, control the PM affinity of Gßγ and thereby differentially modulate Gßγ effector signaling in a Gγ-specific manner. Both Gßγ signaling activity and the migration rate of macrophages are strongly dependent on the PM affinity of Gγ. We also found that the type of C-terminal prenylation and five to six pre-CaaX motif residues at the PM-interacting region of Gγ control the PM affinity of Gßγ. We further show that the overall PM affinity of the Gßγ pool of a cell type is a strong predictor of its Gßγ signaling-activation efficacy. A kinetic model encompassing multiple Gγ types and parameterized for empirical Gßγ behaviors not only recapitulated experimentally observed signaling of Gßγ, but also suggested a Gγ-dependent, active-inactive conformational switch for the PM-bound Gßγ, regulating effector signaling. Overall, our results unveil crucial aspects of signaling and cell migration regulation by Gγ type-specific PM affinities of Gßγ.
Assuntos
Sinalização do Cálcio , Membrana Celular/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Macrófagos/metabolismo , Modelos Biológicos , Animais , Membrana Celular/química , Movimento Celular , Biologia Computacional , Subunidades beta da Proteína de Ligação ao GTP/antagonistas & inibidores , Subunidades beta da Proteína de Ligação ao GTP/química , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/antagonistas & inibidores , Subunidades gama da Proteína de Ligação ao GTP/química , Subunidades gama da Proteína de Ligação ao GTP/genética , Meia-Vida , Células HeLa , Humanos , Cinética , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Ativação de Macrófagos , Macrófagos/citologia , Macrófagos/imunologia , Camundongos , Domínios e Motivos de Interação entre Proteínas , Prenilação de Proteína , Transporte Proteico , Células RAW 264.7 , Interferência de RNA , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Thrombosis is caused by the activation of platelets at the site of ruptured atherosclerotic plaques. This activation involves engagement of G protein-coupled receptors (GPCR) on platelets that promote their aggregation. Although it is known that protein kinases and phosphatases modulate GPCR signaling, how serine/threonine phosphatases integrate with G protein signaling pathways is less understood. Because the subcellular localization and substrate specificity of the catalytic subunit of protein phosphatase 1 (PP1c) is dictated by PP1c-interacting proteins, here we sought to identify new PP1c interactors. GPCRs signal via the canonical heterotrimeric Gα and Gßγ subunits. Using a yeast two-hybrid screen, we discovered an interaction between PP1cα and the heterotrimeric G protein Gß1 subunit. Co-immunoprecipitation studies with epitope-tagged PP1c and Gß1 revealed that Gß1 interacts with the PP1c α, ß, and γ1 isoforms. Purified PP1c bound to recombinant Gß1-GST protein, and PP1c co-immunoprecipitated with Gß1 in unstimulated platelets. Thrombin stimulation of platelets induced the dissociation of the PP1c-Gß1 complex, which correlated with an association of PP1c with phospholipase C ß3 (PLCß3), along with a concomitant dephosphorylation of the inhibitory Ser1105 residue in PLCß3. siRNA-mediated depletion of GNB1 (encoding Gß1) in murine megakaryocytes reduced protease-activated receptor 4, activating peptide-induced soluble fibrinogen binding. Thrombin-induced aggregation was decreased in PP1cα-/- murine platelets and in human platelets treated with a small-molecule inhibitor of Gßγ. Finally, disruption of PP1c-Gß1 complexes with myristoylated Gß1 peptides containing the PP1c binding site moderately decreased thrombin-induced human platelet aggregation. These findings suggest that Gß1 protein enlists PP1c to modulate GPCR signaling in platelets.
Assuntos
Plaquetas/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Megacariócitos/metabolismo , Modelos Moleculares , Fosfolipase C beta/metabolismo , Proteína Fosfatase 1/metabolismo , Transdução de Sinais , Substituição de Aminoácidos , Animais , Plaquetas/enzimologia , Células da Medula Óssea/citologia , Células da Medula Óssea/enzimologia , Células da Medula Óssea/metabolismo , Células Cultivadas , Cruzamentos Genéticos , Feminino , Subunidades beta da Proteína de Ligação ao GTP/química , Subunidades beta da Proteína de Ligação ao GTP/genética , Proteínas Heterotriméricas de Ligação ao GTP/antagonistas & inibidores , Proteínas Heterotriméricas de Ligação ao GTP/química , Proteínas Heterotriméricas de Ligação ao GTP/genética , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Masculino , Megacariócitos/citologia , Megacariócitos/enzimologia , Camundongos Knockout , Camundongos Transgênicos , Mutagênese Sítio-Dirigida , Fosfolipase C beta/química , Fosfolipase C beta/genética , Agregação Plaquetária , Mutação Puntual , Domínios e Motivos de Interação entre Proteínas , Proteína Fosfatase 1/química , Proteína Fosfatase 1/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
The enzyme adenylyl cyclase (AC) plays a pivotal role in a variety of signal transduction pathways inside the cell, where it catalyzes the cyclization of adenosine triphosphate (ATP) into the second-messenger cyclic adenosine monophosphate (cAMP). Among other roles, AC regulates processes involved in neural plasticity, innervation of smooth muscles of the heart and the endocrine system of the pancreas. The functional diversity of AC is manifested in its different isoforms, each having a specific regulation pattern. There is an increasing amount of data available concerning the regulatory properties of AC isoforms, however little is known about the interactions on a structural level. Here, we conducted a comparative electrostatic analysis of the catalytic domains of all nine transmembrane AC isoforms with the aim of detecting, verifying and predicting the binding sites of molecular regulators on AC. The results provide support for the positioning of the binding site of the inhibitory protein Gi α at a pseudo-symmetric position to the stimulatory Gs α binding site. They also provide a structural interpretation of the Gßγ interaction with ACs 2, 4, and 7 and suggest a new binding site for RGS2. Comparison of the small molecule binding sites on AC shows that overall they have high electrostatic similarity, but regions of electrostatic differences are identified. These could provide a basis for the development of novel compounds with isoform-specific modulatory effects on AC. Proteins 2016; 84:1844-1858. © 2016 Wiley Periodicals, Inc.
Assuntos
Trifosfato de Adenosina/química , Inibidores de Adenilil Ciclases/química , Adenilil Ciclases/química , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades beta da Proteína de Ligação ao GTP/química , Subunidades gama da Proteína de Ligação ao GTP/química , Proteínas RGS/química , Motivos de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Ligantes , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Estrutura Secundária de Proteína , Bibliotecas de Moléculas Pequenas/química , Eletricidade Estática , Relação Estrutura-AtividadeRESUMO
Whole-exome sequencing of 13 individuals with developmental delay commonly accompanied by abnormal muscle tone and seizures identified de novo missense mutations enriched within a sub-region of GNB1, a gene encoding the guanine nucleotide-binding protein subunit beta-1, Gß. These 13 individuals were identified among a base of 5,855 individuals recruited for various undiagnosed genetic disorders. The probability of observing 13 or more de novo mutations by chance among 5,855 individuals is very low (p = 7.1 × 10(-21)), implicating GNB1 as a genome-wide-significant disease-associated gene. The majority of these 13 mutations affect known Gß binding sites, which suggests that a likely disease mechanism is through the disruption of the protein interface required for Gα-Gßγ interaction (resulting in a constitutively active Gßγ) or through the disruption of residues relevant for interaction between Gßγ and certain downstream effectors (resulting in reduced interaction with the effectors). Strikingly, 8 of the 13 individuals recruited here for a neurodevelopmental disorder have a germline de novo GNB1 mutation that overlaps a set of five recurrent somatic tumor mutations for which recent functional studies demonstrated a gain-of-function effect due to constitutive activation of G protein downstream signaling cascades for some of the affected residues.
Assuntos
Deficiências do Desenvolvimento/etiologia , Subunidades beta da Proteína de Ligação ao GTP/genética , Mutação em Linhagem Germinativa/genética , Deficiência Intelectual/etiologia , Hipotonia Muscular/etiologia , Convulsões/etiologia , Adolescente , Adulto , Criança , Pré-Escolar , Deficiências do Desenvolvimento/patologia , Exoma/genética , Feminino , Subunidades beta da Proteína de Ligação ao GTP/química , Humanos , Lactente , Deficiência Intelectual/patologia , Masculino , Hipotonia Muscular/patologia , Fenótipo , Conformação Proteica , Convulsões/patologia , Transdução de Sinais , Adulto JovemRESUMO
Class IB phosphoinositide 3-kinases γ (PI3Kγ) are second-messenger-generating enzymes downstream of signalling cascades triggered by G-protein-coupled receptors (GPCRs). PI3Kγ variants have one catalytic p110γ subunit that can form two different heterodimers by binding to one of a pair of non-catalytic subunits, p87 or p101. Growing experimental data argue for a different regulation of p87-p110γ and p101-p110γ allowing integration into distinct signalling pathways. Pharmacological tools enabling distinct modulation of the two variants are missing. The ability of an anti-p110γ monoclonal antibody [mAb(A)p110γ] to block PI3Kγ enzymatic activity attracted us to characterize this tool in detail using purified proteins. In order to get insight into the antibody-p110γ interface, hydrogen-deuterium exchange coupled to MS (HDX-MS) measurements were performed demonstrating binding of the monoclonal antibody to the C2 domain in p110γ, which was accompanied by conformational changes in the helical domain harbouring the Gßγ-binding site. We then studied the modulation of phospholipid vesicles association of PI3Kγ by the antibody. p87-p110γ showed a significantly reduced Gßγ-mediated phospholipid recruitment as compared with p101-p110γ. Concomitantly, in the presence of mAb(A)p110γ, Gßγ did not bind to p87-p110γ. These data correlated with the ability of the antibody to block Gßγ-stimulated lipid kinase activity of p87-p110γ 30-fold more potently than p101-p110γ. Our data argue for differential regulatory functions of the non-catalytic subunits and a specific Gßγ-dependent regulation of p101 in PI3Kγ activation. In this scenario, we consider the antibody as a valuable tool to dissect the distinct roles of the two PI3Kγ variants downstream of GPCRs.
Assuntos
Anticorpos Monoclonais Murinos/química , Classe Ib de Fosfatidilinositol 3-Quinase , Subunidades beta da Proteína de Ligação ao GTP , Subunidades gama da Proteína de Ligação ao GTP , Animais , Classe Ib de Fosfatidilinositol 3-Quinase/química , Classe Ib de Fosfatidilinositol 3-Quinase/genética , Classe Ib de Fosfatidilinositol 3-Quinase/metabolismo , Medição da Troca de Deutério , Subunidades beta da Proteína de Ligação ao GTP/química , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/química , Subunidades gama da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Células Sf9 , SpodopteraRESUMO
Evaluating sites of protein-protein interactions can be an arduous task involving extensive mutagenesis work and attempts to express and purify individual proteins in sufficient quantities. Peptide mapping is a useful alternative to traditional methods as it allows rapid detection of regions and/or individual residues important for binding, and it can be readily applied to numerous proteins at once. Here we describe the use of the ResPep SL SPOT method to evaluate protein-protein binding interactions such as that between G-protein ßγ subunits and SNARE proteins, identifying both regions of interest and subsequently individual residues which can then be manipulated in further biochemical assays to confirm their validity.
Assuntos
Subunidades beta da Proteína de Ligação ao GTP/química , Peptídeos/química , Mapeamento de Interação de Proteínas/métodos , Proteínas SNARE/química , Alanina/química , Mutagênese , Ligação ProteicaRESUMO
G-protein signaling depends on the ability of the individual subunits of the G-protein heterotrimer to assemble into a functional complex. Formation of the G-protein ßγ (Gßγ) dimer is particularly challenging because it is an obligate dimer in which the individual subunits are unstable on their own. Recent studies have revealed an intricate chaperone system that brings Gß and Gγ together. This system includes cytosolic chaperonin containing TCP-1 (CCT; also called TRiC) and its cochaperone phosducin-like protein 1 (PhLP1). Two key intermediates in the Gßγ assembly process, the Gß-CCT and the PhLP1-Gß-CCT complexes, were isolated and analyzed by a hybrid structural approach using cryo-electron microscopy, chemical cross-linking coupled with mass spectrometry, and unnatural amino acid cross-linking. The structures show that Gß interacts with CCT in a near-native state through interactions of the Gγ-binding region of Gß with the CCTγ subunit. PhLP1 binding stabilizes the Gß fold, disrupting interactions with CCT and releasing a PhLP1-Gß dimer for assembly with Gγ. This view provides unique insight into the interplay between CCT and a cochaperone to orchestrate the folding of a protein substrate.
Assuntos
Proteínas de Transporte/química , Chaperonina com TCP-1/química , Subunidades beta da Proteína de Ligação ao GTP/química , Subunidades gama da Proteína de Ligação ao GTP/química , Proteínas do Tecido Nervoso/química , Multimerização Proteica , Aminoácidos/metabolismo , Animais , Benzofenonas , Proteínas de Transporte/ultraestrutura , Chaperonina com TCP-1/ultraestrutura , Reagentes de Ligações Cruzadas/metabolismo , Microscopia Crioeletrônica , Subunidades beta da Proteína de Ligação ao GTP/ultraestrutura , Subunidades gama da Proteína de Ligação ao GTP/ultraestrutura , Humanos , Espectrometria de Massas , Modelos Moleculares , Proteínas do Tecido Nervoso/ultraestrutura , Fenilalanina/análogos & derivados , Estrutura Secundária de ProteínaRESUMO
PLEKHG2 is a Dbl family Rho guanine nucleotide exchange factor (RhoGEF) whose gene was originally identified as being upregulated in a leukemia mouse model and was later shown to be activated by heterotrimeric G protein ßγ (Gßγ) subunits. However, its function and activation mechanisms remain elusive. Here we show that, compared to its expression in primary human T cells, its expression is upregulated in several leukemia cell lines, including Jurkat T cells. Downregulation of PLEKHG2 in Jurkat T cells by small interfering RNAs (siRNAs) specifically inhibited Gßγ-stimulated Rac and Cdc42, but not RhoA, activation. Consequently, suppressing PLEKHG2 expression blocked actin polymerization and SDF1α-stimulated lymphocyte migration. Additional studies indicate that Gßγ likely activates PLEKHG2, in part by binding the N terminus of PLEKHG2 to release an autoinhibition imposed by its C terminus, which interacts with a region encompassing the catalytic Dbl homology (DH) domain. As a result, overexpressing either the N terminus or the C terminus of PLEKHG2 blocked Gßγ-stimulated Rac and Cdc42 activation and prevented Jurkat T cells from forming membrane protrusions and migrating. Together, our studies have provided the first evidence for the endogenous function of PLEKHG2, which may serve as a key Gßγ-stimulated RhoGEF that regulates lymphocyte chemotaxis via Rac and Cdc42 activation and actin polymerization.
Assuntos
Quimiotaxia de Leucócito , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Células Jurkat/fisiologia , Multimerização Proteica , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Actinas/metabolismo , Substituição de Aminoácidos , Sítios de Ligação , Quimiocina CXCL12/metabolismo , Ativação Enzimática , Subunidades beta da Proteína de Ligação ao GTP/química , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/química , Subunidades gama da Proteína de Ligação ao GTP/genética , Fatores de Troca do Nucleotídeo Guanina/química , Células HEK293 , Células HL-60 , Humanos , Modelos Moleculares , Ligação Proteica , Domínios e Motivos de Interação entre ProteínasRESUMO
G protein-coupled receptors (GPCRs) participate in ubiquitous transmembrane signal transduction processes by activating heterotrimeric G proteins. In the current "canonical" model of GPCR signaling, arrestins terminate receptor signaling by impairing receptor-G-protein coupling and promoting receptor internalization. However, parathyroid hormone receptor type 1 (PTHR), an essential GPCR involved in bone and mineral metabolism, does not follow this conventional desensitization paradigm. ß-Arrestins prolong G protein (G(S))-mediated cAMP generation triggered by PTH, a process that correlates with the persistence of arrestin-PTHR complexes on endosomes and which is thought to be associated with prolonged physiological calcemic and phosphate responses. This presents an inescapable paradox for the current model of arrestin-mediated receptor-G-protein decoupling. Here we show that PTHR forms a ternary complex that includes arrestin and the Gßγ dimer in response to PTH stimulation, which in turn causes an accelerated rate of G(S) activation and increases the steady-state levels of activated G(S), leading to prolonged generation of cAMP. This work provides the mechanistic basis for an alternative model of GPCR signaling in which arrestins contribute to sustaining the effect of an agonist hormone on the receptor.
Assuntos
Arrestinas/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Receptor Tipo 1 de Hormônio Paratireóideo/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Arrestinas/química , AMP Cíclico/biossíntese , Transferência Ressonante de Energia de Fluorescência , Subunidades beta da Proteína de Ligação ao GTP/química , Subunidades gama da Proteína de Ligação ao GTP/química , Células HEK293 , Humanos , Cinética , Modelos Biológicos , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Hormônio Paratireóideo/metabolismo , Hormônio Paratireóideo/farmacologia , Receptor Tipo 1 de Hormônio Paratireóideo/química , Receptores Acoplados a Proteínas G/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , beta-ArrestinasRESUMO
BACKGROUND: Gßγ interaction with GlyR is an important determinant in ethanol potentiation of this channel. RESULTS: A small peptide, RQH(C7), can inhibit ethanol potentiation of GlyR currents. CONCLUSION: Results with RQH(C7) indicate that ethanol mediated potentiation of GlyR is in part by Gßγ activation. SIGNIFICANCE: Molecular interaction between Gßγ and GlyR could be used as a target for pharmacological modification of ethanol effects. Previous studies indicate that ethanol can modulate glycine receptors (GlyR), in part, through Gßγ interaction with basic residues in the intracellular loop. In this study, we show that a seven-amino acid peptide (RQH(C7)), which has the primary structure of a motif in the large intracellular loop of GlyR (GlyR-IL), was able to inhibit the ethanol-elicited potentiation of this channel from 47 ± 2 to 16 ± 4%, without interfering with the effect of Gßγ on GIRK (G protein activated inwardly rectifying potassium channel) activation. RQH(C7) displayed a concentration-dependent effect on ethanol action in evoked and synaptic currents. A fragment of GlyR-IL without the basic amino acids did not interact with Gßγ or inhibit ethanol potentiation of GlyR. In silico analysis using docking and molecular dynamics allowed to identify a region of ~350Å(2) involving aspartic acids 186, 228, and 246 in Gßγ where we propose that RQH(C7) binds and exerts its blocking action on the effect of ethanol in GlyR.
Assuntos
Etanol/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Peptídeos/metabolismo , Receptores de Glicina/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/química , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/genética , Células HEK293 , Humanos , Cinética , Peptídeos/química , Ligação Proteica , Receptores de Glicina/química , Receptores de Glicina/genéticaRESUMO
G-protein-regulated PI3Kγ (phosphoinositide 3-kinase γ) plays a crucial role in inflammatory and allergic processes. PI3Kγ, a dimeric protein formed by the non-catalytic p101 and catalytic p110γ subunits, is stimulated by receptor-released Gßγ complexes. We have demonstrated previously that Gßγ stimulates both monomeric p110γ and dimeric p110γ/p101 lipid kinase activity in vitro. In order to identify the Gß residues responsible for the Gßγ-PI3Kγ interaction, we examined Gß1 mutants for their ability to stimulate lipid and protein kinase activities and to recruit PI3Kγ to lipid vesicles. Our findings revealed different interaction profiles of Gß residues interacting with p110γ or p110γ/p101. Moreover, p101 was able to rescue the stimulatory activity of Gß1 mutants incapable of modulating monomeric p110γ. In addition to the known adaptor function of p101, in the present paper we show a novel regulatory role of p101 in the activation of PI3Kγ.
Assuntos
Classe Ib de Fosfatidilinositol 3-Quinase/fisiologia , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Animais , Células Cultivadas , Classe Ib de Fosfatidilinositol 3-Quinase/química , Classe Ib de Fosfatidilinositol 3-Quinase/genética , Classe Ib de Fosfatidilinositol 3-Quinase/metabolismo , Resistência a Medicamentos/genética , Resistência a Medicamentos/fisiologia , Ativação Enzimática/genética , Subunidades beta da Proteína de Ligação ao GTP/química , Subunidades beta da Proteína de Ligação ao GTP/genética , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Isoenzimas/fisiologia , Metabolismo dos Lipídeos/genética , Metabolismo dos Lipídeos/fisiologia , Modelos Moleculares , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteólise/efeitos dos fármacos , Spodoptera , Transfecção , Tripsina/metabolismo , Tripsina/farmacologiaRESUMO
BACKGROUND: Short-term presynaptic inhibition mediated by G protein-coupled receptors involves a direct interaction between G proteins and the vesicle release machinery. Recent studies implicate the C terminus of the vesicle-associated protein SNAP-25 as a molecular binding target of Gßγ that transiently reduces vesicular release. However, it is not known whether SNAP-25 is a target for molecular modifications expressing long-term changes in transmitter release probability. METHODOLOGY/PRINCIPAL FINDINGS: This study utilized two-photon laser scanning microscopy for real-time imaging of action potential-evoked [Ca(2+)] increases, in single Schaffer collateral presynaptic release sites in in vitro hippocampal slices, plus simultaneous recording of Schaffer collateral-evoked synaptic potentials. We used electroporation to infuse small peptides through CA3 cell bodies into presynaptic Schaffer collateral terminals to selectively study the presynaptic effect of scavenging the G-protein Gßγ. We demonstrate here that the C terminus of SNAP-25 is necessary for expression of LTD, but not long-term potentiation (LTP), of synaptic strength. Using type A botulinum toxin (BoNT/A) to enzymatically cleave the 9 amino acid C-terminus of SNAP-25 eliminated the ability of low frequency synaptic stimulation to induce LTD, but not LTP, even if release probability was restored to pre-BoNT/A levels by elevating extracellular [Ca(2+)]. Presynaptic electroporation infusion of the 14-amino acid C-terminus of SNAP-25 (Ct-SNAP-25), to scavenge Gßγ, reduced both the transient presynaptic inhibition produced by the group II metabotropic glutamate receptor stimulation, and LTD. Furthermore, presynaptic infusion of mSIRK, a second, structurally distinct Gßγ scavenging peptide, also blocked the induction of LTD. While Gßγ binds directly to and inhibit voltage-dependent Ca(2+) channels, imaging of presynaptic [Ca(2+)] with Mg-Green revealed that low-frequency stimulation only transiently reduced presynaptic Ca(2+) influx, an effect not altered by infusion of Ct-SNAP-25. CONCLUSIONS/SIGNIFICANCE: The C-terminus of SNAP-25, which links synaptotagmin I to the SNARE complex, is a binding target for Gßγ necessary for both transient transmitter-mediated presynaptic inhibition, and the induction of presynaptic LTD.
Assuntos
Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Depressão Sináptica de Longo Prazo/fisiologia , Proteína 25 Associada a Sinaptossoma/metabolismo , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Sequência de Aminoácidos , Animais , Toxinas Botulínicas Tipo A/farmacologia , Cálcio/metabolismo , Cálcio/farmacologia , Subunidades beta da Proteína de Ligação ao GTP/química , Subunidades gama da Proteína de Ligação ao GTP/química , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Técnicas In Vitro , Potenciação de Longa Duração/efeitos dos fármacos , Potenciação de Longa Duração/fisiologia , Depressão Sináptica de Longo Prazo/efeitos dos fármacos , Microscopia Confocal/métodos , Inibição Neural/efeitos dos fármacos , Inibição Neural/fisiologia , Fármacos Neuromusculares/farmacologia , Peptídeos/metabolismo , Peptídeos/farmacologia , Ligação Proteica , Células Piramidais/efeitos dos fármacos , Células Piramidais/metabolismo , Células Piramidais/fisiologia , Ratos , Ratos Sprague-Dawley , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologiaRESUMO
Previous NMR experiments on unbound G protein ßγ heterodimer suggested that particular residues in the binding interface are mobile on the nanosecond timescale. In this work we performed nanosecond-timescale molecular dynamics simulations to investigate conformational changes and dynamics of Gßγ in the presence of several binding partners: a high-affinity peptide (SIGK), phosducin, and the GDP-bound α subunit. In these simulations, the high mobility of GßW99 was reduced by SIGK, and it appeared that a tyrosine might stabilize GßW99 by hydrophobic or aromatic stacking interactions in addition to hydrogen bonds. Simulations of the phosducin-Gßγ complex showed that the mobility of GßW99 was restricted, consistent with inferences from NMR. However, large-scale conformational changes of Gßγ due to binding, which were hypothesized in the NMR study, were not observed in the simulations, most likely due to their short (nanosecond) duration. A pocket consisting of hydrophobic amino acids on Gα appears to restrict GßW99 mobility in the crystal structure of the Gαßγ? heterotrimer. The simulation trajectories are consistent with this idea. However, local conformational changes of residues GßW63, GßW211, GßW297, GßW332, and GßW339 were detected during the MD simulations. As expected, the magnitude of atomic fluctuations observed in simulations was greater for α than for the ßγ subunits, suggesting that α has greater flexibility. These observations support the notion that to maintain the high mobility of GßW99 observed by solution NMR requires that the Gß-α interface must open up on time scale longer than can be observed in nanosecond scale simulations.
Assuntos
Subunidades beta da Proteína de Ligação ao GTP/química , Subunidades gama da Proteína de Ligação ao GTP/química , Proteínas do Olho/química , Reguladores de Proteínas de Ligação ao GTP/química , Guanosina Difosfato/química , Ligantes , Simulação de Dinâmica Molecular , Peptídeos/química , Fosfoproteínas/química , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Água/químicaRESUMO
G protein-coupled receptors transduce signals through heterotrimeric G protein Gα and Gßγ subunits, both of which interact with downstream effectors to regulate cell function. Gßγ signaling has been implicated in the pathophysiology of several diseases, suggesting that Gßγ could be an important pharmaceutical target. Previously, we used a combination of virtual and manual screening to find small molecules that bind to a protein-protein interaction "hot spot" on Gßγ and block regulation of physiological effectors. One of the most potent and effective compounds from this screen was selenocystamine. In this study, we investigated the mechanism of action of selenocystamine and found that selenocysteamine forms a covalent complex with Gßγ by a reversible redox mechanism. Mass spectrometry and site-directed mutagenesis suggest that selenocysteamine preferentially modifies GßCys204, but also a second undefined site. The high potency of selenocystamine in Gßγ inhibition seems to arise from both high reactivity of the diselenide group and binding to a specific site on Gß. Using structural information about the "hot spot," we developed a strategy to selectively target redox reversible compounds to a specific site on Gßγ using peptide carriers such as SIGCAFKILGY(-cysteamine) [SIGC(-cysteamine)]. Mass spectrometry and site-directed mutagenesis indicate that SIGC(-cysteamine) specifically and efficiently leads to cysteamine (half-cystamine) modification of a single site on Gß, likely GßCys204, and inhibits Gßγ more than a hundred times more potently than cystamine. These data support the concept that covalent modifiers can be specifically targeted to the Gßγ "hot spot" through rational incorporation into molecules that noncovalently bind to Gßγ.