RESUMO
Many neurotransmitter receptors activate G proteins through exchange of GDP for GTP. The intermediate nucleotide-free state has eluded characterization, due largely to its inherent instability. Here we characterize a G protein variant associated with a rare neurological disorder in humans. GαoK46E has a charge reversal that clashes with the phosphate groups of GDP and GTP. As anticipated, the purified protein binds poorly to guanine nucleotides yet retains wild-type affinity for G protein ßγ subunits. In cells with physiological concentrations of nucleotide, GαoK46E forms a stable complex with receptors and Gßγ, impeding effector activation. Further, we demonstrate that the mutant can be easily purified in complex with dopamine-bound D2 receptors, and use cryo-electron microscopy to determine the structure, including both domains of Gαo, without nucleotide or stabilizing nanobodies. These findings reveal the molecular basis for the first committed step of G protein activation, establish a mechanistic basis for a neurological disorder, provide a simplified strategy to determine receptor-G protein structures, and a method to detect high affinity agonist binding in cells.
Assuntos
Microscopia Crioeletrônica , Guanosina Difosfato , Guanosina Trifosfato , Mutação , Humanos , Células HEK293 , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/metabolismo , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D2/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Ligação Proteica , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/genéticaRESUMO
Opioid analgesics are widely used as a treatment option for pain management and relief. However, the misuse of opioid analgesics has contributed to the current opioid epidemic in the United States. Prescribed opioids such as morphine, codeine, oxycodone, and fentanyl are mu-opioid receptor (MOR) agonists primarily used in the clinic to treat pain or during medical procedures, but development of tolerance limits their utility for treatment of chronic pain. Here we explored the effects of biasing Gßγ signaling on tolerance development after chronic morphine treatment in vivo. We hypothesized that biasing Gßγ signaling with gallein could prevent activation of regulatory signaling pathways that result in tolerance to antinociceptive effects of MOR agonists. Gallein has been shown to bind to Gßγ and inhibit interactions of Gßγ with phospholipase-Cß3 (PLCß3) or G-protein-coupled receptor kinase 2 (GRK2) but not G-protein inwardly rectifying potassium (GIRK) channels. In mice, morphine-induced antinociception was evaluated in the 55°C warm water tail withdrawal assay. We used two paradigms for gallein treatment: administration during and after three times-daily morphine administration. Our results show that gallein cotreatment during repeated administration of morphine decreased opioid tolerance development and that gallein treatment in an opioid-tolerant state enhanced the potency of morphine. Mechanistically, our data suggest that PLCß3 is necessary for potentiating effects of gallein in an opioid-tolerant state but not in preventing the development of tolerance. These studies demonstrate that small molecules that target Gßγ signaling could reduce the need for large doses of opioid analgesics to treat pain by producing an opioid-sparing effect. SIGNIFICANCE STATEMENT: Biasing Gßγ signaling prevents tolerance to repeated morphine administration in vivo and potentiates the antinociceptive effects of morphine in an opioid-tolerant state. Mechanistically, phospholipase-Cß is necessary for potentiating effects of gallein in an opioid-tolerant state but not in preventing the development of tolerance. This study identifies a novel treatment strategy to decrease the development of tolerance to the analgesic effects of mu-opioid receptor agonists, which are necessary to improve pain treatment and decrease the incidence of opioid use disorder.
Assuntos
Analgésicos Opioides , Tolerância a Medicamentos , Subunidades beta da Proteína de Ligação ao GTP , Subunidades gama da Proteína de Ligação ao GTP , Camundongos Endogâmicos C57BL , Morfina , Nociceptividade , Transdução de Sinais , Animais , Morfina/farmacologia , Tolerância a Medicamentos/fisiologia , Transdução de Sinais/efeitos dos fármacos , Camundongos , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Masculino , Analgésicos Opioides/farmacologia , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Nociceptividade/efeitos dos fármacos , Receptores Opioides mu/metabolismo , Receptores Opioides mu/agonistas , Fosfolipase C beta/metabolismo , XantenosRESUMO
Chromaffin cells of the adrenal medulla transduce sympathetic nerve activity into stress hormone secretion. The two neurotransmitters principally responsible for coupling cell stimulation to secretion are acetylcholine and pituitary adenylate activating polypeptide (PACAP). In contrast to acetylcholine, PACAP evokes a persistent secretory response from chromaffin cells. However, the mechanisms by which PACAP acts are poorly understood. Here, it is shown that PACAP induces sustained increases in cytosolic Ca2+ which are disrupted when Ca2+ influx through L-type channels is blocked or internal Ca2+ stores are depleted. PACAP liberates stored Ca2+ via inositol trisphosphate receptors (IP3Rs) on the endoplasmic reticulum (ER), thereby functionally coupling Ca2+ mobilization to Ca2+ influx and supporting Ca2+-induced Ca2+-release. These Ca2+ influx and mobilization pathways are unified by an absolute dependence on phospholipase C epsilon (PLCε) activity. Thus, the persistent secretory response that is a defining feature of PACAP activity, in situ, is regulated by a signaling network that promotes sustained elevations in intracellular Ca2+ through multiple pathways.
Assuntos
Sinalização do Cálcio , Cálcio , Células Cromafins , Retículo Endoplasmático , Receptores de Inositol 1,4,5-Trifosfato , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Animais , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Retículo Endoplasmático/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Células Cromafins/metabolismo , Bovinos , Canais de Cálcio Tipo L/metabolismoRESUMO
An elevated level of lipoprotein(a), or Lp(a), in the bloodstream has been causally linked to the development of atherosclerotic cardiovascular disease and calcific aortic valve stenosis. Steady state levels of circulating lipoproteins are modulated by their rate of clearance, but the identity of the Lp(a) uptake receptor(s) has been controversial. In this study, we performed a genome-scale CRISPR screen to functionally interrogate all potential Lp(a) uptake regulators in HuH7 cells. Strikingly, the top positive and negative regulators of Lp(a) uptake in our screen were LDLR and MYLIP, encoding the LDL receptor and its ubiquitin ligase IDOL, respectively. We also found a significant correlation for other genes with established roles in LDLR regulation. No other gene products, including those previously proposed as Lp(a) receptors, exhibited a significant effect on Lp(a) uptake in our screen. We validated the functional influence of LDLR expression on HuH7 Lp(a) uptake, confirmed in vitro binding between the LDLR extracellular domain and purified Lp(a), and detected an association between loss-of-function LDLR variants and increased circulating Lp(a) levels in the UK Biobank cohort. Together, our findings support a central role for the LDL receptor in mediating Lp(a) uptake by hepatocytes.
RESUMO
BACKGROUND: Chronically elevated neurohumoral drive, and particularly elevated adrenergic tone leading to ß-adrenergic receptor (ß-AR) overstimulation in cardiac myocytes, is a key mechanism involved in the progression of heart failure. ß1-AR (ß1-adrenergic receptor) and ß2-ARs (ß2-adrenergic receptor) are the 2 major subtypes of ß-ARs present in the human heart; however, they elicit different or even opposite effects on cardiac function and hypertrophy. For example, chronic activation of ß1-ARs drives detrimental cardiac remodeling while ß2-AR signaling is protective. The underlying molecular mechanisms for cardiac protection through ß2-ARs remain unclear. METHODS: ß2-AR signaling mechanisms were studied in isolated neonatal rat ventricular myocytes and adult mouse ventricular myocytes using live cell imaging and Western blotting methods. Isolated myocytes and mice were used to examine the roles of ß2-AR signaling mechanisms in the regulation of cardiac hypertrophy. RESULTS: Here, we show that ß2-AR activation protects against hypertrophy through inhibition of phospholipaseCε signaling at the Golgi apparatus. The mechanism for ß2-AR-mediated phospholipase C inhibition requires internalization of ß2-AR, activation of Gi and Gßγ subunit signaling at endosome and ERK (extracellular regulated kinase) activation. This pathway inhibits both angiotensin II and Golgi-ß1-AR-mediated stimulation of phosphoinositide hydrolysis at the Golgi apparatus ultimately resulting in decreased PKD (protein kinase D) and histone deacetylase 5 phosphorylation and protection against cardiac hypertrophy. CONCLUSIONS: This reveals a mechanism for ß2-AR antagonism of the phospholipase Cε pathway that may contribute to the known protective effects of ß2-AR signaling on the development of heart failure.
Assuntos
Miócitos Cardíacos , Receptores Adrenérgicos beta 2 , Transdução de Sinais , Animais , Masculino , Camundongos , Ratos , Animais Recém-Nascidos , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Células Cultivadas , Endocitose , Complexo de Golgi/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos Cardíacos/metabolismo , Fosfoinositídeo Fosfolipase C/metabolismo , Proteína Quinase C/metabolismo , Ratos Sprague-Dawley , Receptores Adrenérgicos beta 2/metabolismoRESUMO
The chemokines of the immune system act as first responders by operating as chemoattractants, directing immune cells to specific locations of inflamed tissues. This promiscuous network is comprised of 50 ligands and 18 receptors where the ligands may interact with the receptors in various oligomeric states i.e., monomers, homodimers, and heterodimers. Chemokine receptors are G-protein coupled receptors (GPCRs) present in the membrane of immune cells. The migration of immune cells occurs in response to a concentration gradient of the ligands. Chemotaxis of neutrophils is directed by CXC-ligand (CXCL) activation of the membrane bound CXC chemokine receptor 2 (CXCR2). CXCR2 plays an important role in human health and is linked to disorders such as autoimmune disorders, inflammation, and cancer. Yet, despite their important role, little is known about the biophysical characteristics controlling ligand:ligand and ligand:receptor interaction essential for biological activity. In this work, we study the homodimers of three of the CXCR2 cognate ligands, CXCL1, CXCL5, and CXCL8. The ligands share high structural integrity but a low sequence identity. We show that the sequence diversity has evolved different binding affinities and stabilities for the CXC-ligands resulting in diverse agonist/antagonist behavior. Furthermore, CXC-ligands fold through a three-state mechanism, populating a folded monomeric state before associating into an active dimer.
Assuntos
Interleucina-8 , Receptores de Interleucina-8B , Humanos , Receptores de Interleucina-8B/genética , Ligantes , Interleucina-8/metabolismo , Quimiocinas/metabolismo , Quimiocina CXCL1 , Fatores Quimiotáticos/metabolismo , QuimiotaxiaRESUMO
Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) infects up to a quarter of the world's population. Although immune responses can control Mtb infection, 5%-10% of infected individuals can progress to active TB disease (progressors). A myriad of host factors regulate disease progression in TB and a better understanding of immune correlates of protection and disease is pivotal for the development of new therapeutics. Comparison of human whole blood transcriptomic metadata with that of macaque TB progressors and Mtb-infected diversity outbred mice (DO) led to the identification of differentially regulated gene (DEG) signatures, associated with TB progression or control. The current study assessed the function of Phospholipase C epsilon (PLCÆ1), the top downregulated gene across species in TB progressors, using a gene-specific knockout mouse model of Mtb infection and in vitro Mtb-infected bone marrow-derived macrophages. PLCÆ1 gene expression was downregulated in TB progressors across species. PLCε1 deficiency in the mouse model resulted in increased susceptibility to Mtb infection, coincident accumulation of lung myeloid cells, and reduced ability to mount antibacterial responses. However, PLCε1 was not required for the activation and accumulation of T cells in mice. Our results suggest an important early role for PLCÆ1 in shaping innate immune response to TB and may represent a putative target for host-directed therapy.
Assuntos
Mycobacterium tuberculosis , Fosfoinositídeo Fosfolipase C , Tuberculose , Humanos , Camundongos , Animais , Ativação de Macrófagos , Imunidade InataRESUMO
Highly homologous members of the Gαi family, Gαi1-3, have distinct tissue distributions and physiological functions, yet their biochemical and functional properties are very similar. We recently identified PDZ-RhoGEF (PRG) as a novel Gαi1 effector that is poorly activated by Gαi2. In a proteomic proximity labeling screen we observed a strong preference for Gαi1 relative to Gαi2 with respect to engagement of a broad range of potential targets. We investigated the mechanistic basis for this selectivity using PRG as a representative target. Substitution of either the helical domain (HD) from Gαi1 into Gαi2 or substitution of a single amino acid, A230 in Gαi2 with the corresponding D in Gαi1, largely rescues PRG activation and interactions with other potential Gαi targets. Molecular dynamics simulations combined with Bayesian network models revealed that in the GTP bound state, separation at the HD-Ras-like domain (RLD) interface is more pronounced in Gαi2 than Gαi1. Mutation of A230 to D in Gαi2 stabilizes HD-RLD interactions via ionic interactions with R145 in the HD which in turn modify the conformation of Switch III. These data support a model where D229 in Gαi1 interacts with R144 and stabilizes a network of interactions between HD and RLD to promote protein target recognition. The corresponding A230 in Gαi2 is unable to stabilize this network leading to an overall lower efficacy with respect to target interactions. This study reveals distinct mechanistic properties that could underly differential biological and physiological consequences of activation of Gαi1 or Gαi2 by G protein-coupled receptors.
Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP , Transdução de Sinais , Humanos , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Simulação de Dinâmica Molecular , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/metabolismo , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/genética , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/química , Células HEK293 , Domínios Proteicos , Estabilidade Proteica , Ligação ProteicaRESUMO
Chromaffin cells of the adrenal medulla transduce sympathetic nerve activity into stress hormone secretion. The two neurotransmitters principally responsible for coupling cell stimulation to secretion are acetylcholine and pituitary adenylate activating polypeptide (PACAP). In contrast to acetylcholine, PACAP evokes a persistent secretory response from chromaffin cells. However, the mechanisms by which PACAP acts are poorly understood. Here, it is shown that PACAP induces sustained increases in cytosolic Ca 2+ which are disrupted when Ca 2+ influx through L-type channels is blocked or internal Ca 2+ stores are depleted. PACAP liberates stored Ca 2+ via inositol trisphosphate receptors (IP3Rs) on the endoplasmic reticulum (ER), thereby functionally coupling Ca 2+ mobilization to Ca 2+ influx and supporting Ca 2+ -induced Ca 2+ -release. These Ca 2+ influx and mobilization pathways are unified by an absolute dependence on phospholipase C epsilon (PLCε) activity. Thus, the persistent secretory response that is a defining feature of PACAP activity, in situ , is regulated by a signaling network that promotes sustained elevations in intracellular Ca 2+ through multiple pathways.