RESUMEN
Ligands of the Hedgehog (HH) pathway are paracrine signaling molecules that coordinate tissue development in metazoans. A remarkable feature of HH signaling is the repeated use of cholesterol in steps spanning ligand biogenesis, secretion, dispersal, and reception on target cells. A cholesterol molecule covalently attached to HH ligands is used as a molecular baton by transfer proteins to guide their secretion, spread, and reception. On target cells, a signaling circuit composed of a cholesterol transporter and sensor regulates transmission of HH signals across the plasma membrane to the cytoplasm. The repeated use of cholesterol in signaling supports the view that the HH pathway likely evolved by coopting ancient systems to regulate the abundance or organization of sterol-like lipids in membranes.
Asunto(s)
Colesterol , Proteínas Hedgehog , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Ligandos , Colesterol/metabolismo , Transducción de Señal , Esteroles/metabolismoRESUMEN
The human body contains thousands of metabolites derived from mammalian cells, the microbiota, food, and medical drugs. Many bioactive metabolites act through the engagement of G-protein-coupled receptors (GPCRs); however, technological limitations constrain current explorations of metabolite-GPCR interactions. Here, we developed a highly multiplexed screening technology called PRESTO-Salsa that enables simultaneous assessment of nearly all conventional GPCRs (>300 receptors) in a single well of a 96-well plate. Using PRESTO-Salsa, we screened 1,041 human-associated metabolites against the GPCRome and uncovered previously unreported endogenous, exogenous, and microbial GPCR agonists. Next, we leveraged PRESTO-Salsa to generate an atlas of microbiome-GPCR interactions across 435 human microbiome strains from multiple body sites, revealing conserved patterns of cross-tissue GPCR engagement and activation of CD97/ADGRE5 by the Porphyromonas gingivalis protease gingipain K. These studies thus establish a highly multiplexed bioactivity screening technology and expose a diverse landscape of human, diet, drug, and microbiota metabolome-GPCRome interactions.
Asunto(s)
Microbiota , Receptores Acoplados a Proteínas G , Animales , Humanos , Receptores Acoplados a Proteínas G/metabolismo , Metaboloma , Mamíferos/metabolismoRESUMEN
Receptor activity-modifying proteins (RAMPs) modulate the activity of many Family B GPCRs. We show that RAMP2 directly interacts with the glucagon receptor (GCGR), a Family B GPCR responsible for blood sugar homeostasis, and broadly inhibits receptor-induced downstream signaling. HDX-MS experiments demonstrate that RAMP2 enhances local flexibility in select locations in and near the receptor extracellular domain (ECD) and in the 6th transmembrane helix, whereas smFRET experiments show that this ECD disorder results in the inhibition of active and intermediate states of the intracellular surface. We determined the cryo-EM structure of the GCGR-Gs complex at 2.9 Å resolution in the presence of RAMP2. RAMP2 apparently does not interact with GCGR in an ordered manner; however, the receptor ECD is indeed largely disordered along with rearrangements of several intracellular hallmarks of activation. Our studies suggest that RAMP2 acts as a negative allosteric modulator of GCGR by enhancing conformational sampling of the ECD.
Asunto(s)
Glucagón , Receptores de Glucagón , Membrana Celular/metabolismo , Glucagón/metabolismo , Receptores de Glucagón/metabolismo , Proteína 2 Modificadora de la Actividad de Receptores/metabolismoRESUMEN
Intraflagellar transport (IFT) trains are massive molecular machines that traffic proteins between cilia and the cell body. Each IFT train is a dynamic polymer of two large complexes (IFT-A and -B) and motor proteins, posing a formidable challenge to mechanistic understanding. Here, we reconstituted the complete human IFT-A complex and obtained its structure using cryo-EM. Combined with AlphaFold prediction and genome-editing studies, our results illuminate how IFT-A polymerizes, interacts with IFT-B, and uses an array of ß-propeller and TPR domains to create "carriages" of the IFT train that engage TULP adaptor proteins. We show that IFT-Aâ TULP carriages are essential for cilia localization of diverse membrane proteins, as well as ICK-the key kinase regulating IFT train turnaround. These data establish a structural link between IFT-A's distinct functions, provide a blueprint for IFT-A in the train, and shed light on how IFT evolved from a proto-coatomer ancestor.
Asunto(s)
Cilios , Cinesinas , Humanos , Cilios/metabolismo , Transporte Biológico , Cinesinas/metabolismo , Dineínas/metabolismo , Proteínas de la Membrana/metabolismo , Transporte de Proteínas , Flagelos/metabolismoRESUMEN
ß-arrestins bind G protein-coupled receptors to terminate G protein signaling and to facilitate other downstream signaling pathways. Using single-molecule fluorescence resonance energy transfer imaging, we show that ß-arrestin is strongly autoinhibited in its basal state. Its engagement with a phosphopeptide mimicking phosphorylated receptor tail efficiently releases the ß-arrestin tail from its N domain to assume distinct conformations. Unexpectedly, we find that ß-arrestin binding to phosphorylated receptor, with a phosphorylation barcode identical to the isolated phosphopeptide, is highly inefficient and that agonist-promoted receptor activation is required for ß-arrestin activation, consistent with the release of a sequestered receptor C tail. These findings, together with focused cellular investigations, reveal that agonism and receptor C-tail release are specific determinants of the rate and efficiency of ß-arrestin activation by phosphorylated receptor. We infer that receptor phosphorylation patterns, in combination with receptor agonism, synergistically establish the strength and specificity with which diverse, downstream ß-arrestin-mediated events are directed.
Asunto(s)
Fosfopéptidos , Receptores Acoplados a Proteínas G , Fosfopéptidos/metabolismo , Fosforilación , Receptores Acoplados a Proteínas G/metabolismo , beta-Arrestina 1/metabolismo , beta-Arrestinas/metabolismoRESUMEN
Epidemiological studies reveal that marijuana increases the risk of cardiovascular disease (CVD); however, little is known about the mechanism. Δ9-tetrahydrocannabinol (Δ9-THC), the psychoactive component of marijuana, binds to cannabinoid receptor 1 (CB1/CNR1) in the vasculature and is implicated in CVD. A UK Biobank analysis found that cannabis was an risk factor for CVD. We found that marijuana smoking activated inflammatory cytokines implicated in CVD. In silico virtual screening identified genistein, a soybean isoflavone, as a putative CB1 antagonist. Human-induced pluripotent stem cell-derived endothelial cells were used to model Δ9-THC-induced inflammation and oxidative stress via NF-κB signaling. Knockdown of the CB1 receptor with siRNA, CRISPR interference, and genistein attenuated the effects of Δ9-THC. In mice, genistein blocked Δ9-THC-induced endothelial dysfunction in wire myograph, reduced atherosclerotic plaque, and had minimal penetration of the central nervous system. Genistein is a CB1 antagonist that attenuates Δ9-THC-induced atherosclerosis.
Asunto(s)
Cannabis , Enfermedades Cardiovasculares , Alucinógenos , Analgésicos , Animales , Agonistas de Receptores de Cannabinoides/farmacología , Dronabinol/farmacología , Células Endoteliales , Genisteína/farmacología , Genisteína/uso terapéutico , Inflamación/tratamiento farmacológico , Ratones , Receptor Cannabinoide CB1 , Receptores de CannabinoidesRESUMEN
Hedgehog pathway components and select G protein-coupled receptors (GPCRs) localize to the primary cilium, an organelle specialized for signal transduction. We investigated whether cells distinguish between ciliary and extraciliary GPCR signaling. To test whether ciliary and extraciliary cyclic AMP (cAMP) convey different information, we engineered optogenetic and chemogenetic tools to control the subcellular site of cAMP generation. Generating equal amounts of ciliary and cytoplasmic cAMP in zebrafish and mammalian cells revealed that ciliary cAMP, but not cytoplasmic cAMP, inhibited Hedgehog signaling. Modeling suggested that the distinct geometries of the cilium and cell body differentially activate local effectors. The search for effectors identified a ciliary pool of protein kinase A (PKA). Blocking the function of ciliary PKA, but not extraciliary PKA, activated Hedgehog signal transduction and reversed the effects of ciliary cAMP. Therefore, cells distinguish ciliary and extraciliary cAMP using functionally and spatially distinct pools of PKA, and different subcellular pools of cAMP convey different information.
Asunto(s)
Cilios/metabolismo , AMP Cíclico/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Citoplasma/metabolismo , Proteínas Hedgehog/antagonistas & inhibidores , Proteínas Hedgehog/metabolismo , Neuronas/metabolismo , Optogenética/métodos , Transducción de Señal/fisiología , Pez Cebra/metabolismoRESUMEN
Thermogenic adipocytes possess a therapeutically appealing, energy-expending capacity, which is canonically cold-induced by ligand-dependent activation of ß-adrenergic G protein-coupled receptors (GPCRs). Here, we uncover an alternate paradigm of GPCR-mediated adipose thermogenesis through the constitutively active receptor, GPR3. We show that the N terminus of GPR3 confers intrinsic signaling activity, resulting in continuous Gs-coupling and cAMP production without an exogenous ligand. Thus, transcriptional induction of Gpr3 represents the regulatory parallel to ligand-binding of conventional GPCRs. Consequently, increasing Gpr3 expression in thermogenic adipocytes is alone sufficient to drive energy expenditure and counteract metabolic disease in mice. Gpr3 transcription is cold-stimulated by a lipolytic signal, and dietary fat potentiates GPR3-dependent thermogenesis to amplify the response to caloric excess. Moreover, we find GPR3 to be an essential, adrenergic-independent regulator of human brown adipocytes. Taken together, our findings reveal a noncanonical mechanism of GPCR control and thermogenic activation through the lipolysis-induced expression of constitutively active GPR3.
Asunto(s)
Tejido Adiposo Pardo/metabolismo , Receptor de Androstano Constitutivo/metabolismo , Lipólisis , Receptores Acoplados a Proteínas G/metabolismo , Termogénesis , Adipocitos/metabolismo , Animales , Células COS , Células Cultivadas , Chlorocebus aethiops , Frío , Grasas de la Dieta/farmacología , Humanos , Ratones Endogámicos C57BL , Fenotipo , Receptores Acoplados a Proteínas G/genética , Transducción de Señal , Sistema Nervioso Simpático/metabolismo , Transcripción GenéticaRESUMEN
Human endocannabinoid systems modulate multiple physiological processes mainly through the activation of cannabinoid receptors CB1 and CB2. Their high sequence similarity, low agonist selectivity, and lack of activation and G protein-coupling knowledge have hindered the development of therapeutic applications. Importantly, missing structural information has significantly held back the development of promising CB2-selective agonist drugs for treating inflammatory and neuropathic pain without the psychoactivity of CB1. Here, we report the cryoelectron microscopy structures of synthetic cannabinoid-bound CB2 and CB1 in complex with Gi, as well as agonist-bound CB2 crystal structure. Of important scientific and therapeutic benefit, our results reveal a diverse activation and signaling mechanism, the structural basis of CB2-selective agonists design, and the unexpected interaction of cholesterol with CB1, suggestive of its endogenous allosteric modulating role.
Asunto(s)
Agonistas de Receptores de Cannabinoides/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Receptor Cannabinoide CB1/química , Receptor Cannabinoide CB2/química , Transducción de Señal , Regulación Alostérica , Sitio Alostérico , Animales , Células CHO , Agonistas de Receptores de Cannabinoides/química , Cannabinoides/química , Cannabinoides/farmacología , Línea Celular Tumoral , Colesterol/química , Colesterol/farmacología , Cricetinae , Cricetulus , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Humanos , Simulación de Dinámica Molecular , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB2/metabolismo , Células Sf9 , SpodopteraRESUMEN
The cannabinoid receptor CB2 is predominately expressed in the immune system, and selective modulation of CB2 without the psychoactivity of CB1 has therapeutic potential in inflammatory, fibrotic, and neurodegenerative diseases. Here, we report the crystal structure of human CB2 in complex with a rationally designed antagonist, AM10257, at 2.8 Å resolution. The CB2-AM10257 structure reveals a distinctly different binding pose compared with CB1. However, the extracellular portion of the antagonist-bound CB2 shares a high degree of conformational similarity with the agonist-bound CB1, which led to the discovery of AM10257's unexpected opposing functional profile of CB2 antagonism versus CB1 agonism. Further structural analysis using mutagenesis studies and molecular docking revealed the molecular basis of their function and selectivity for CB2 and CB1. Additional analyses of our designed antagonist and agonist pairs provide important insight into the activation mechanism of CB2. The present findings should facilitate rational drug design toward precise modulation of the endocannabinoid system.
Asunto(s)
Receptor Cannabinoide CB2/metabolismo , Receptor Cannabinoide CB2/ultraestructura , Animales , Antagonistas de Receptores de Cannabinoides/farmacología , Cannabinoides/farmacología , Diseño de Fármacos , Endocannabinoides , Humanos , Ligandos , Simulación del Acoplamiento Molecular , Unión Proteica , Receptor Cannabinoide CB1/antagonistas & inhibidores , Receptor Cannabinoide CB2/química , Receptores de Cannabinoides/química , Receptores de Cannabinoides/metabolismo , Receptores de Cannabinoides/ultraestructura , Receptores Acoplados a Proteínas G/metabolismo , Células Sf9 , Relación Estructura-ActividadRESUMEN
The crystal structure of the ß2-adrenergic receptor (ß2AR) bound to the G protein adenylyl cyclase stimulatory G protein (Gs) captured the complex in a nucleotide-free state (ß2AR-Gsempty). Unfortunately, the ß2AR-Gsempty complex does not provide a clear explanation for G protein coupling specificity. Evidence from several sources suggests the existence of a transient complex between the ß2AR and GDP-bound Gs protein (ß2AR-GsGDP) that may represent an intermediate on the way to the formation of ß2AR-Gsempty and may contribute to coupling specificity. Here we present a structure of the ß2AR in complex with the carboxyl terminal 14 amino acids from Gαs along with the structure of the GDP-bound Gs heterotrimer. These structures provide evidence for an alternate interaction between the ß2AR and Gs that may represent an intermediate that contributes to Gs coupling specificity.
Asunto(s)
Adenilil Ciclasas/química , Subunidades alfa de la Proteína de Unión al GTP Gs/química , Modelos Moleculares , Receptores Adrenérgicos beta 2/química , Humanos , Relación Estructura-ActividadRESUMEN
G protein-coupled receptor (GPCR) signaling is the primary method eukaryotes use to respond to specific cues in their environment. However, the relationship between stimulus and response for each GPCR is difficult to predict due to diversity in natural signal transduction architecture and expression. Using genome engineering in yeast, we constructed an insulated, modular GPCR signal transduction system to study how the response to stimuli can be predictably tuned using synthetic tools. We delineated the contributions of a minimal set of key components via computational and experimental refactoring, identifying simple design principles for rationally tuning the dose response. Using five different GPCRs, we demonstrate how this enables cells and consortia to be engineered to respond to desired concentrations of peptides, metabolites, and hormones relevant to human health. This work enables rational tuning of cell sensing while providing a framework to guide reprogramming of GPCR-based signaling in other systems.
Asunto(s)
Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Expresión Génica/efectos de los fármacos , Ingeniería Genética , Humanos , Feromonas/farmacología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The angiotensin II (AngII) type 1 receptor (AT1R) is a critical regulator of cardiovascular and renal function and is an important model for studies of G-protein-coupled receptor (GPCR) signaling. By stabilizing the receptor with a single-domain antibody fragment ("nanobody") discovered using a synthetic yeast-displayed library, we determined the crystal structure of active-state human AT1R bound to an AngII analog with partial agonist activity. The nanobody binds to the receptor's intracellular transducer pocket, stabilizing the large conformational changes characteristic of activated GPCRs. The peptide engages the AT1R through an extensive interface spanning from the receptor core to its extracellular face and N terminus, remodeling the ligand-binding cavity. Remarkably, the mechanism used to propagate conformational changes through the receptor diverges from other GPCRs at several key sites, highlighting the diversity of allosteric mechanisms among GPCRs. Our structure provides insight into how AngII and its analogs stimulate full or biased signaling, respectively.
Asunto(s)
Receptor de Angiotensina Tipo 1/metabolismo , Anticuerpos de Dominio Único/farmacología , Angiotensina II , Bloqueadores del Receptor Tipo 1 de Angiotensina II/metabolismo , Arrestinas/metabolismo , Células HEK293 , Humanos , Fragmentos de Inmunoglobulinas/farmacología , Conformación Proteica , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/efectos de los fármacos , Anticuerpos de Dominio Único/metabolismo , beta-Arrestinas/metabolismoRESUMEN
The activation of G proteins by G protein-coupled receptors (GPCRs) underlies the majority of transmembrane signaling by hormones and neurotransmitters. Recent structures of GPCR-G protein complexes obtained by crystallography and cryoelectron microscopy (cryo-EM) reveal similar interactions between GPCRs and the alpha subunit of different G protein isoforms. While some G protein subtype-specific differences are observed, there is no clear structural explanation for G protein subtype-selectivity. All of these complexes are stabilized in the nucleotide-free state, a condition that does not exist in living cells. In an effort to better understand the structural basis of coupling specificity, we used time-resolved structural mass spectrometry techniques to investigate GPCR-G protein complex formation and G-protein activation. Our results suggest that coupling specificity is determined by one or more transient intermediate states that serve as selectivity filters and precede the formation of the stable nucleotide-free GPCR-G protein complexes observed in crystal and cryo-EM structures.
Asunto(s)
Proteínas de Unión al GTP/química , Complejos Multienzimáticos/química , Receptores Acoplados a Proteínas G/química , Animales , Bovinos , Microscopía por Crioelectrón , Cristalografía por Rayos X , Humanos , Complejos Multienzimáticos/ultraestructura , Estructura Cuaternaria de Proteína , RatasRESUMEN
Although human genetic studies have implicated many susceptible genes associated with plasma lipid levels, their physiological and molecular functions are not fully characterized. Here we demonstrate that orphan G protein-coupled receptor 146 (GPR146) promotes activity of hepatic sterol regulatory element binding protein 2 (SREBP2) through activation of the extracellular signal-regulated kinase (ERK) signaling pathway, thereby regulating hepatic very low-density lipoprotein (VLDL) secretion, and subsequently circulating low-density lipoprotein cholesterol (LDL-C) and triglycerides (TG) levels. Remarkably, GPR146 deficiency reduces plasma cholesterol levels substantially in both wild-type and LDL receptor (LDLR)-deficient mice. Finally, aortic atherosclerotic lesions are reduced by 90% and 70%, respectively, in male and female LDLR-deficient mice upon GPR146 depletion. Taken together, these findings outline a regulatory role for the GPR146/ERK axis in systemic cholesterol metabolism and suggest that GPR146 inhibition could be an effective strategy to reduce plasma cholesterol levels and atherosclerosis.
Asunto(s)
Aterosclerosis/metabolismo , Hipercolesterolemia/metabolismo , Receptores Acoplados a Proteínas G/deficiencia , Animales , Aterosclerosis/sangre , Secuencia de Bases , Colesterol/sangre , Dependovirus/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Ayuno , Femenino , Hepatocitos/metabolismo , Humanos , Hipercolesterolemia/sangre , Lipoproteínas VLDL/metabolismo , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , ARN Interferente Pequeño/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de LDL/metabolismo , Transducción de Señal , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismo , Triglicéridos/sangre , Regulación hacia ArribaRESUMEN
"Biased" G protein-coupled receptor (GPCR) agonists preferentially activate pathways mediated by G proteins or ß-arrestins. Here, we use double electron-electron resonance spectroscopy to probe the changes that ligands induce in the conformational distribution of the angiotensin II type I receptor. Monitoring distances between 10 pairs of nitroxide labels distributed across the intracellular regions enabled mapping of four underlying sets of conformations. Ligands from different functional classes have distinct, characteristic effects on the conformational heterogeneity of the receptor. Compared to angiotensin II, the endogenous agonist, agonists with enhanced Gq coupling more strongly stabilize an "open" conformation with an accessible transducer-binding site. ß-arrestin-biased agonists deficient in Gq coupling do not stabilize this open conformation but instead favor two more occluded conformations. These data suggest a structural mechanism for biased ligand action at the angiotensin receptor that can be exploited to rationally design GPCR-targeting drugs with greater specificity of action.
Asunto(s)
Angiotensinas/metabolismo , Receptor de Angiotensina Tipo 1/metabolismo , Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Antagonistas de Receptores de Angiotensina/metabolismo , Arrestinas/metabolismo , Línea Celular , Humanos , Ligandos , Conformación Proteica , Receptores de Angiotensina/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Espectroscopía de Pérdida de Energía de Electrones/métodos , beta-Arrestinas/metabolismoRESUMEN
G protein-coupled receptors (GPCRs) mediate the majority of cellular responses to external stimuli. Upon activation by a ligand, the receptor binds to a partner heterotrimeric G protein and promotes exchange of GTP for GDP, leading to dissociation of the G protein into α and ßγ subunits that mediate downstream signals. GPCRs can also activate distinct signaling pathways through arrestins. Active states of GPCRs form by small rearrangements of the ligand-binding, or orthosteric, site that are amplified into larger conformational changes. Molecular understanding of the allosteric coupling between ligand binding and G protein or arrestin interaction is emerging from structures of several GPCRs crystallized in inactive and active states, spectroscopic data, and computer simulations. The coupling is loose, rather than concerted, and agonist binding does not fully stabilize the receptor in an active conformation. Distinct intermediates whose populations are shifted by ligands of different efficacies underlie the complex pharmacology of GPCRs.
Asunto(s)
Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Regulación Alostérica , Sitios de Unión , Secuencia Conservada , Cristalografía por Rayos X , Metabolismo Energético , Humanos , Ligandos , Modelos Moleculares , Conformación Proteica , Receptores Acoplados a Proteínas G/genéticaRESUMEN
Signaling across cellular membranes, the 826 human G protein-coupled receptors (GPCRs) govern a wide range of vital physiological processes, making GPCRs prominent drug targets. X-ray crystallography provided GPCR molecular architectures, which also revealed the need for additional structural dynamics data to support drug development. Here, nuclear magnetic resonance (NMR) spectroscopy with the wild-type-like A2A adenosine receptor (A2AAR) in solution provides a comprehensive characterization of signaling-related structural dynamics. All six tryptophan indole and eight glycine backbone 15N-1H NMR signals in A2AAR were individually assigned. These NMR probes provided insight into the role of Asp522.50 as an allosteric link between the orthosteric drug binding site and the intracellular signaling surface, revealing strong interactions with the toggle switch Trp 2466.48, and delineated the structural response to variable efficacy of bound drugs across A2AAR. The present data support GPCR signaling based on dynamic interactions between two semi-independent subdomains connected by an allosteric switch at Asp522.50.
Asunto(s)
Regulación Alostérica , Receptor de Adenosina A2A/química , Transducción de Señal , Agonistas del Receptor de Adenosina A2/química , Agonistas del Receptor de Adenosina A2/farmacología , Sitio Alostérico , Animales , Simulación del Acoplamiento Molecular , Pichia , Unión Proteica , Receptor de Adenosina A2A/metabolismo , Células Sf9 , SpodopteraRESUMEN
The phosphorylation of agonist-occupied G-protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) functions to turn off G-protein signaling and turn on arrestin-mediated signaling. While a structural understanding of GPCR/G-protein and GPCR/arrestin complexes has emerged in recent years, the molecular architecture of a GPCR/GRK complex remains poorly defined. We used a comprehensive integrated approach of cross-linking, hydrogen-deuterium exchange mass spectrometry (MS), electron microscopy, mutagenesis, molecular dynamics simulations, and computational docking to analyze GRK5 interaction with the ß2-adrenergic receptor (ß2AR). These studies revealed a dynamic mechanism of complex formation that involves large conformational changes in the GRK5 RH/catalytic domain interface upon receptor binding. These changes facilitate contacts between intracellular loops 2 and 3 and the C terminus of the ß2AR with the GRK5 RH bundle subdomain, membrane-binding surface, and kinase catalytic cleft, respectively. These studies significantly contribute to our understanding of the mechanism by which GRKs regulate the function of activated GPCRs. PAPERCLIP.
Asunto(s)
Quinasa 5 del Receptor Acoplado a Proteína-G/química , Mamíferos/metabolismo , Receptores Adrenérgicos beta 2/química , Animales , Camélidos del Nuevo Mundo , Bovinos , Quinasa 5 del Receptor Acoplado a Proteína-G/genética , Quinasa 5 del Receptor Acoplado a Proteína-G/metabolismo , Humanos , Espectrometría de Masas , Microscopía Electrónica , Modelos Moleculares , Simulación de Dinámica Molecular , Unión Proteica , Ratas , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismoRESUMEN
G-protein-coupled receptors (GPCRs) play critical roles in regulating physiological processes ranging from neurotransmission to cardiovascular function. Current methods for tracking GPCR signaling suffer from low throughput, modification or overexpression of effector proteins, and low temporal resolution. Here, we show that peroxidase-catalyzed proximity labeling can be combined with isobaric tagging and mass spectrometry to enable quantitative, time-resolved measurement of GPCR agonist response in living cells. Using this technique, termed "GPCR-APEX," we track activation and internalization of the angiotensin II type 1 receptor and the ß2 adrenoceptor. These receptors co-localize with a variety of G proteins even before receptor activation, and activated receptors are largely sequestered from G proteins upon internalization. Additionally, the two receptors show differing internalization kinetics, and we identify the membrane protein LMBRD2 as a potential regulator of ß2 adrenoceptor signaling, underscoring the value of a dynamic view of receptor function.