Allosteric interactions between agonists and antagonists within the adenosine A2A receptor-dopamine D2 receptor heterotetramer

Adenosine A_2A receptor (A_2AR)-dopamine D₂ receptor (D₂R) heteromers are key modulators of striatal neuronal function. It has been suggested that the psychostimulant effects of caffeine depend on its ability to block an allosteric modulation within the A_2AR-D₂R heteromer, by which adenosine decreases the affinity and intrinsic efficacy of dopamine at the D₂R. We describe novel unsuspected allosteric mechanisms within the heteromer by which not only A_2AR agonists, but also A_2AR antagonists, decrease the affinity and intrinsic efficacy of D₂R agonists and the affinity of D₂R antagonists. Strikingly, these allosteric modulations disappear on agonist and antagonist coadministration. This can be explained by a model that considers A_2AR-D₂R heteromers as heterotetramers, constituted by A_2AR and D₂R homodimers, as demonstrated by experiments with bioluminescence resonance energy transfer and bimolecular fluorescence and bioluminescence complementation. As predicted by the model, high concentrations of A_2AR antagonists behaved as A_2AR agonists and decreased D₂R function in the brain.Most evidence indicates that G protein-coupled receptors (GPCRs) form homodimers and heteromers. Homodimers seem to be a predominant species, and oligomeric entities can be viewed as multiples of dimers (1). It has been proposed that GPCR heteromers are constituted mainly by heteromers of homodimers (1, 2). Allosteric mechanisms determine a multiplicity of unique pharmacologic properties of GPCR homodimers and heteromers (1, 3). First, binding of a ligand to one of the receptors in the heteromer can modify the affinity of ligands for the other receptor (1, 3, 4). The most widely reproduced allosteric modulation of ligand-binding properties in a GPCR heteromer is the ability of adenosine A_2A receptor (A_2AR) agonists to decrease the affinity of dopamine D₂ receptor (D₂R) agonists in the A_2AR-D₂R heteromer (5). A_2AR-D₂R heteromers have been revealed both in transfected cells (6, 7), striatal neurons in culture (6, 8) and in situ, in mammalian striatum (9, 10), where they play an important role in the modulation of GABAergic striatopallidal neuronal function (9, 11).In addition to ligand-binding properties, unique properties for each GPCR oligomer emerge in relation to the varying intrinsic efficacy of ligands for different signaling pathways (1–3). Intrinsic efficacy refers to the power of the agonist to induce a functional response, independent of its affinity for the receptor. Thus, allosteric modulation of an agonist can potentially involve changes in affinity and/or intrinsic efficacy (1, 3). This principle can be observed in the A_2AR-D₂R heteromer, where a decrease in D₂R agonist affinity cannot alone explain the ability of an A_2AR agonist to abolish the decreased excitability of GABAergic striatopallidal neurons induced by high concentration of a D₂R agonist (9), which should overcome the decrease in affinity. Furthermore, a differential effect of allosteric modulations of different agonist-mediated signaling responses (i.e., functional selectivity) can occur within GPCR heteromers (1, 2, 8). Again, the A_2AR-D₂R heteromer provides a valuable example. A recent study has shown that different levels of intracellular Ca²⁺ exert different modulations of A_2AR-D₂R heteromer signaling (8). This depends on the ability of low and high Ca²⁺ to promote a selective interaction of the heteromer with different Ca²⁺-binding proteins, which differentially modulate allosteric interactions in the heteromer (8).It has been hypothesized that the allosteric interactions between A_2AR and D₂R agonists within the A_2AR-D₂R heteromer provide a mechanism responsible not only for the depressant effects of A_2AR agonists, but also for the psychostimulant effects of adenosine A_2AR antagonists and the nonselective adenosine receptor antagonist caffeine (9, 11, 12), with implications for several neuropsychiatric disorders (13). In fact, the same mechanism has provided the rationale for the use of A_2AR antagonists in patients with Parkinson’s disease (13, 14). The initial aim of the present study was to study in detail the ability of caffeine to counteract allosteric modulations between A_2AR and D₂R agonists (affinity and intrinsic efficacy) within the A_2AR-D₂R heteromer. Unexpectedly, when performing control radioligand-binding experiments, not only an A_2AR agonist, but also caffeine, significantly decreased D₂R agonist binding. However, when coadministered, the A_2AR agonist and caffeine co-counteracted their ability to modulate D₂R agonist binding. By exploring the molecular mechanisms behind these apparent inconsistencies, the present study provides new insight into the quaternary structure and function of A_2AR-D₂R heteromers.     

α2A adrenergic receptor promotes amyloidogenesis through disrupting APP-SorLA interaction

Accumulation of amyloid β (Aβ) peptides in the brain is the key pathogenic factor driving Alzheimer’s disease (AD). Endocytic sorting of amyloid precursor protein (APP) mediated by the vacuolar protein sorting (Vps10) family of receptors plays a decisive role in controlling the outcome of APP proteolytic processing and Aβ generation. Here we report for the first time to our knowledge that this process is regulated by a G protein-coupled receptor, the α_2A adrenergic receptor (α_2AAR). Genetic deficiency of the α_2AAR significantly reduces, whereas stimulation of this receptor enhances, Aβ generation and AD-related pathology. Activation of α_2AAR signaling disrupts APP interaction with a Vps10 family receptor, sorting-related receptor with A repeat (SorLA), in cells and in the mouse brain. As a consequence, activation of α_2AAR reduces Golgi localization of APP and concurrently promotes APP distribution in endosomes and cleavage by β secretase. The α_2AAR is a key component of the brain noradrenergic system. Profound noradrenergic dysfunction occurs consistently in patients at the early stages of AD. α_2AAR-promoted Aβ generation provides a novel mechanism underlying the connection between noradrenergic dysfunction and AD. Our study also suggests α_2AAR as a previously unappreciated therapeutic target for AD. Significantly, pharmacological blockade of the α_2AAR by a clinically used antagonist reduces AD-related pathology and ameliorates cognitive deficits in an AD transgenic model, suggesting that repurposing clinical α₂AR antagonists would be an effective therapeutic strategy for AD.Excess amyloid β (Aβ) peptides in the brain are a neuropathological hallmark of Alzheimer’s disease (AD) and are generally accepted as the key pathogenic factor of the disease (1). Aβ is generated by two sequential cleavages of amyloid precursor protein (APP) by β and γ secretase, whereas cleavage by α secretase within the Aβ domain precludes Aβ generation (2, 3). APP and the secretases undergo endocytic sorting into various organelles, such as the trans-Golgi network, the plasma membrane, and endosomes (2–6). The initial step of APP processing by α versus β secretase preferentially occurs in distinct compartments of the cell. Although α secretase-mediated cleavage of APP occurs on the plasma membrane, β secretase primarily interacts with and cleaves APP in endosomes (2–6). Therefore, endocytic sorting of APP into different membranous compartments, causing it to coreside or avoid a particular secretase, plays a decisive role in APP proteolytic processing. Consistent with this notion, abnormalities of the endocytic pathway have been found to precede Aβ deposition in late-onset AD (7).Retrograde sorting of APP from endosomes to trans-Golgi network mediated by the vacuolar protein sorting-10 (Vps10) family proteins and the retromer complex represents a critical mechanism to prevent amyloidogenic processing of APP (8–10) and has recently emerged as a potential target for therapeutic intervention (11). In particular, the sorting-related receptor with A repeat (SorLA) directs retrograde transport of APP to trans-Golgi network by binding to both APP and the retromer complex (12, 13) and retains APP in the Golgi (14), thus preventing its proteolytic processing. A connection between SorLA and AD was first revealed in patients with late-onset AD, in whom the levels of SorLA at the steady state are markedly reduced (15). Further human genetic studies identified variations of SORL1 (the gene encoding SorLA) resulting in a lower level of expression that are associated with late-onset sporadic AD (12, 16, 17). Moreover, nonsense and missense mutations of SORL1 cause autosomal dominant early-onset AD (18), supporting an etiological role of SorLA in AD. The function of SorLA in inhibiting Aβ production is confirmed by mouse genetic studies showing that loss of SorLA significantly increases Aβ levels in the brain (14) and enhances AD-related early pathology (19). Despite the importance of SorLA-dependent APP sorting in controlling Aβ metabolism and AD pathogenesis, how this process may be targeted by extracellular stimuli, such as neurotransmitters and hormones, to modulate amyloidogenesis remains largely unstudied.The α_2A adrenergic receptor (AR) belongs to the G protein-coupled receptor (GPCR) superfamily and is a crucial component of the brain noradrenergic (NA) system, controlling both NA input to the cerebral cortex and the resulting response in this brain region (20). Profound dysfunction of the NA system consistently occurs at the early stage of AD (21), raising the possibility of involvement of the α_2AAR in AD pathogenesis. Here we report for the first time to our knowledge that α_2AAR signaling regulates SorLA-dependent APP sorting and promotes amyloidogenic processing of APP by beta-site amyloid precursor protein cleaving enzyme (BACE) 1. The initial cleavage of APP by BACE1 is the rate-limiting factor of Aβ generation (22, 23). Furthermore, blockade of α_2AAR by a clinical antagonist reduces AD-related pathology and rescues cognitive deficits in an AD transgenic model, suggesting that repurposing clinical α₂AR antagonists would be a novel effective strategy for AD treatment.     

Reprogramming of G protein-coupled receptor recycling and signaling by a kinase switch

The postendocytic recycling of signaling receptors is subject to multiple requirements. Why this is so, considering that many other proteins can recycle without apparent requirements, is a fundamental question. Here we show that cells can leverage these requirements to switch the recycling of the beta-2 adrenergic receptor (B2AR), a prototypic signaling receptor, between sequence-dependent and bulk recycling pathways, based on extracellular signals. This switch is determined by protein kinase A-mediated phosphorylation of B2AR on the cytoplasmic tail. The phosphorylation state of B2AR dictates its partitioning into spatially and functionally distinct endosomal microdomains mediating bulk and sequence-dependent recycling, and also regulates the rate of B2AR recycling and resensitization. Our results demonstrate that G protein-coupled receptor recycling is not always restricted to the sequence-dependent pathway, but may be reprogrammed as needed by physiological signals. Such flexible reprogramming might provide a versatile method for rapidly modulating cellular responses to extracellular signaling.How proteins are sorted in the endocytic pathway is a fundamental question in cell biology. This is especially relevant for signaling receptors, given that relatively small changes in rates of receptor sorting into the recycling pathway can cause significant changes in surface receptors, and hence in cellular sensitivity (1–3). Our knowledge of receptor signaling and trafficking comes mainly from studying examples such as the beta-2 adrenergic receptor (B2AR), a prototypical member of G protein-coupled receptor (GPCR) family, the largest family of signaling receptors (2–5). B2AR activation initiates surface receptor removal and transport to endosomes, causing cellular desensitization (6, 7). The rate and extent of resensitization is then determined by B2AR surface recycling (1–3, 8, 9).Interestingly, the recycling of signaling receptors is functionally distinct from the recycling of constitutively cycling proteins like the transferrin receptor (TfR) (1, 6, 10, 11). TfR recycles by “bulk” geometric sorting, largely independent of specific cytoplasmic sequences (12, 13). B2AR recycling, in contrast, requires a specific PSD95-Dlg1-zo-1 domain (PDZ)-ligand sequence on its C-terminal tail, which links the receptor to the actin cytoskeleton (14, 15). Recent work has identified physically and biochemically distinct microdomains on early endosomes that mediate B2AR recycling independent of TfR (14–16). Although the exact mechanisms of B2AR sorting into these domains remain under investigation, this sorting clearly requires specific sequence elements on B2AR (1, 10, 11, 17). Importantly, why signaling receptor sorting is subject to such specialized requirements, considering that cargo like TfR apparently can recycle without specific sequence requirements, is not clear (1, 12–16). One possibility is that these requirements allow signaling pathways to regulate and redirect receptor trafficking between different pathways as needed (17–19). Although this is an attractive idea, whether and how physiological signals regulate receptor sorting remain poorly understood (7, 19).Here we show that adrenergic signaling can switch B2AR recycling between the sequence-dependent and bulk recycling pathways. Adrenergic activation, via protein kinase A (PKA)-mediated B2AR phosphorylation on the cytoplasmic tail, restricts B2AR to spatially defined PDZ- and actin-dependent endosomal microdomains. Dephosphorylation of B2AR switches B2AR to the bulk (PDZ-independent) recycling pathway, causing faster recycling of B2AR and increased cellular sensitivity. Our results suggest that cells may leverage sequence requirements for rapid adaptive reprogramming of signaling receptor trafficking and cellular sensitivity.     

Caffeine acts through neuronal adenosine A2A receptors to prevent mood and memory dysfunction triggered by chronic stress

The consumption of caffeine (an adenosine receptor antagonist) correlates inversely with depression and memory deterioration, and adenosine A_2A receptor (A_2AR) antagonists emerge as candidate therapeutic targets because they control aberrant synaptic plasticity and afford neuroprotection. Therefore we tested the ability of A_2AR to control the behavioral, electrophysiological, and neurochemical modifications caused by chronic unpredictable stress (CUS), which alters hippocampal circuits, dampens mood and memory performance, and enhances susceptibility to depression. CUS for 3 wk in adult mice induced anxiogenic and helpless-like behavior and decreased memory performance. These behavioral changes were accompanied by synaptic alterations, typified by a decrease in synaptic plasticity and a reduced density of synaptic proteins (synaptosomal-associated protein 25, syntaxin, and vesicular glutamate transporter type 1), together with an increased density of A_2AR in glutamatergic terminals in the hippocampus. Except for anxiety, for which results were mixed, CUS-induced behavioral and synaptic alterations were prevented by (i) caffeine (1 g/L in the drinking water, starting 3 wk before and continued throughout CUS); (ii) the selective A_2AR antagonist KW6002 (3 mg/kg, p.o.); (iii) global A_2AR deletion; and (iv) selective A_2AR deletion in forebrain neurons. Notably, A_2AR blockade was not only prophylactic but also therapeutically efficacious, because a 3-wk treatment with the A_2AR antagonist {"type":"entrez-protein","attrs":{"text":"SCH58261","term_id":"1052882304","term_text":"SCH58261"}}SCH58261 (0.1 mg/kg, i.p.) reversed the mood and synaptic dysfunction caused by CUS. These results herald a key role for synaptic A_2AR in the control of chronic stress-induced modifications and suggest A_2AR as candidate targets to alleviate the consequences of chronic stress on brain function.Repeated stress elicits neurochemical and morphological changes that negatively affect brain functioning (1, 2). Thus, repeated stress is a trigger or a risk factor for neuropsychiatric disorders, namely depression, in both humans and animal models (2, 3). Given the absence of effective therapeutic tools, novel strategies to manage the impact of chronic stress are needed, and analyzing particular lifestyles can provide important leads. Notably, caffeine consumption increases in stressful conditions (4) and correlates inversely with the incidence of depression (5, 6) and the risk of suicide (7, 8). However, the molecular targets operated by caffeine to afford these beneficial effects have not been defined.Caffeine is the most widely consumed psychoactive drug. The only molecular targets for caffeine at nontoxic doses are the main adenosine receptors in the brain, namely the inhibitory A₁ receptors (A₁R) and the facilitatory A_2A receptors (A_2AR) (9). A_2AR blockade affords robust protection against noxious brain conditions (10), an effect that might result from the ability of neuronal A_2AR to control aberrant plasticity (11, 12) and synaptotoxicity (13–15) or from A_2AR’s impact on astrocytes (16) or microglia (17). The protection provided by A_2AR blockade prompts the hypothesis that A_2AR antagonism may underlie the beneficial effects of caffeine on chronic stress, in accordance with the role of synaptic (18, 19) or glial dysfunction (20) in mood disorders. Thus, A_2AR antagonists prolonged escape behavior in two screening tests for antidepressant activity (21–23) and prevented maternal separation-induced long-term cognitive impact (12). We combined pharmacological and tissue-selective A_2AR transgenic mice (24, 25) to test if neuronal A_2AR controlled the modifications caused by chronic unpredictable stress (CUS).     

Single-molecule view of basal activity and activation mechanisms of the G protein-coupled receptor β2AR

Binding of extracellular ligands to G protein-coupled receptors (GPCRs) initiates transmembrane signaling by inducing conformational changes on the cytoplasmic receptor surface. Knowledge of this process provides a platform for the development of GPCR-targeting drugs. Here, using a site-specific Cy3 fluorescence probe in the human β₂-adrenergic receptor (β₂AR), we observed that individual receptor molecules in the native-like environment of phospholipid nanodiscs undergo spontaneous transitions between two distinct conformational states. These states are assigned to inactive and active-like receptor conformations. Individual receptor molecules in the apo form repeatedly sample both conformations, with a bias toward the inactive conformation. Experiments in the presence of drug ligands show that binding of the full agonist formoterol shifts the conformational distribution in favor of the active-like conformation, whereas binding of the inverse agonist ICI-118,551 favors the inactive conformation. Analysis of single-molecule dwell-time distributions for each state reveals that formoterol increases the frequency of activation transitions, while also reducing the frequency of deactivation events. In contrast, the inverse agonist increases the frequency of deactivation transitions. Our observations account for the high level of basal activity of this receptor and provide insights that help to rationalize, on the molecular level, the widely documented variability of the pharmacological efficacies among GPCR-targeting drugs.G protein-coupled receptors (GPCRs) mediate a multitude of physiological functions and are the targets for a myriad of drugs (1), many of which elicit different functional outcomes through the same receptor (2). It remains to be rationalized at the molecular level why some drugs stimulate the signaling activity of a GPCR (full or partial agonists), whereas others either repress the receptor (inverse agonists) or have no effect on the intrinsic signaling activity (neutral antagonists). Moreover, the existence of a high basal activity of some GPCRs (3) suggests that the conformational transitions leading to activation may occur spontaneously, even in the absence of ligands, which in turn raises questions about the mechanistic roles of GPCR ligands. Understanding the mechanisms and pathways of receptor activation or deactivation, and how these are linked to the binding of ligands with different chemical structures and pharmacological efficacies, will aid in design of new GPCR-targeted drugs with tailored pharmacological responses and fewer side effects. To attain these goals, new methods are required to visualize the conformational dynamics of GPCRs in the presence and absence of drugs.The β₂-adrenergic receptor (β₂AR) has been extensively investigated in crystals (4, 5), by NMR in solution (6–11), by bulk fluorescence spectroscopy in solution (12–14) and in cells (2), by single-molecule fluorescence spectroscopy (15–17), and by molecular dynamics simulations (18). Despite the availability of high-resolution crystal structures of β₂AR in inactive (4) and active (5) conformations, it remains unknown how ligands regulate transitions between the two states and why β₂AR exhibits a significant level of ligand-independent, basal signaling activity. To address these questions, we use single-molecule fluorescence spectroscopy to monitor activation-linked conformational transitions of individual receptor molecules in real time over extended time periods. Our results highlight the intrinsically dynamic character of β₂AR and provide insights into the mechanism of receptor activation and the roles of β₂AR ligands.     

Presynaptic serotonin 2A receptors modulate thalamocortical plasticity and associative learning

Higher-level cognitive processes strongly depend on a complex interplay between mediodorsal thalamus nuclei and the prefrontal cortex (PFC). Alteration of thalamofrontal connectivity has been involved in cognitive deficits of schizophrenia. Prefrontal serotonin (5-HT)_2A receptors play an essential role in cortical network activity, but the mechanism underlying their modulation of glutamatergic transmission and plasticity at thalamocortical synapses remains largely unexplored. Here, we show that 5-HT_2A receptor activation enhances NMDA transmission and gates the induction of temporal-dependent plasticity mediated by NMDA receptors at thalamocortical synapses in acute PFC slices. Expressing 5-HT_2A receptors in the mediodorsal thalamus (presynaptic site) of 5-HT_2A receptor-deficient mice, but not in the PFC (postsynaptic site), using a viral gene-delivery approach, rescued the otherwise absent potentiation of NMDA transmission, induction of temporal plasticity, and deficit in associative memory. These results provide, to our knowledge, the first physiological evidence of a role of presynaptic 5-HT_2A receptors located at thalamocortical synapses in the control of thalamofrontal connectivity and the associated cognitive functions.The prefrontal cortex (PFC) is a brain region critical for many high-level cognitive processes, such as executive functions, attention, and working and contextual memories (1). Pyramidal neurons located in layer V of the PFC integrate excitatory glutamatergic inputs originating from both cortical and subcortical areas. The latter include the mediodorsal thalamus (MD) nuclei, which project densely to the medial PFC (mPFC) and are part of the neuronal network underlying executive control and working memory (2–4). Disruption of this network has been involved in cognitive symptoms of psychiatric disorders, such as schizophrenia (3, 5). These symptoms severely compromise the quality of life of patients and remain poorly controlled by currently available antipsychotics (3, 6).The PFC is densely innervated by serotonin (5-hydroxytryptamine, 5-HT) neurons originating from the dorsal and median raphe nuclei and numerous lines of evidence indicate a critical role of 5-HT in the control of emotional and cognitive functions depending on PFC activity (7, 8). The modulatory action of 5-HT reflects its complex pattern of effects on cortical network activity, depending on the 5-HT receptor subtypes involved, and on receptor localization in pyramidal neurons, GABAergic interneurons or nerve terminals of afferent neurons.Among the 14 5-HT receptor subtypes, the 5-HT_2A receptor is a Gq protein-coupled receptor (9, 10) particularly enriched in the mPFC, with a predominant expression in apical dendrites of layer V pyramidal neurons (11–14). Moreover, a low proportion of 5-HT_2A receptors was detected presynaptically on thalamocortical fibers (12, 15–17).Activation of 5-HT_2A receptors exerts complex effects upon the activity of the PFC network (18). The most prominent one is an increase in pyramidal neuron excitability, which likely results from the inhibition of slow calcium-activated after hyperpolarization current (19). 5-HT_2A receptor stimulation also increases the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) in pyramidal neurons (19–22). The prevailing view is that postsynaptic 5-HT_2A receptors expressed on pyramidal neurons located in layer V are key modulators of glutamatergic PFC network activity (14, 21–24). However, the role of presynaptic 5-HT_2A receptors located on thalamic afferents in the modulation of glutamatergic transmission at thalamocortical synapses remains unexplored.Here, we addressed this issue by combining electrophysiological recordings in acute PFC slices with viral infections to specifically rescue the expression of 5-HT_2A receptors at the presynaptic site (MD) or postsynaptic site (PFC) in 5-HT_2A receptor-deficient (5-HT_2A^−/−) mice (25). We focused our study on NMDA transmission in line with previous findings indicating that many symptoms of schizophrenia might arise from modifications in PFC connectivity involving glutamatergic transmission at NMDA receptors (26, 27). To our knowledge, we provide the first direct evidence that stimulation of presynaptic 5-HT_2A receptors at thalamocortical synapses gates the induction of spike timing-dependent long-term depression (t-LTD) by facilitating the activation of presynaptic NMDA receptors at these synapses. In line with the role of t-LTD in associative learning (28), these studies were extended by behavioral experiments to explore the role of presynaptic 5-HT_2A receptors at thalamocortical synapses in several paradigms of episodic-like memory.     

Luminescence color switching of supramolecular assemblies of discrete molecular decanuclear gold(I) sulfido complexes

A series of discrete decanuclear gold(I) μ₃-sulfido complexes with alkyl chains of various lengths on the aminodiphosphine ligands, [Au₁₀{Ph₂PN(C_nH_2n+1)PPh₂}₄(μ₃-S)₄](ClO₄)₂, has been synthesized and characterized. These complexes have been shown to form supramolecular nanoaggregate assemblies upon solvent modulation. The photoluminescence (PL) colors of the nanoaggregates can be switched from green to yellow to red by varying the solvent systems from which they are formed. The PL color variation was investigated and correlated with the nanostructured morphological transformation from the spherical shape to the cube as observed by transmission electron microscopy and scanning electron microscopy. Such variations in PL colors have not been observed in their analogous complexes with short alkyl chains, suggesting that the long alkyl chains would play a key role in governing the supramolecular nanoaggregate assembly and the emission properties of the decanuclear gold(I) sulfido complexes. The long hydrophobic alkyl chains are believed to induce the formation of supramolecular nanoaggregate assemblies with different morphologies and packing densities under different solvent systems, leading to a change in the extent of Au(I)–Au(I) interactions, rigidity, and emission properties.Gold(I) complexes are one of the fascinating classes of complexes that reveal photophysical properties that are highly sensitive to the nuclearity of the metal centers and the metal–metal distances (1–59). In a certain sense, they bear an analogy or resemblance to the interesting classes of metal nanoparticles (NPs) (60–69) and quantum dots (QDs) (70–76) in that the properties of the nanostructured materials also show a strong dependence on their sizes and shapes. Interestingly, while the optical and spectroscopic properties of metal NPs and QDs show a strong dependence on the interparticle distances, those of polynuclear gold(I) complexes are known to mainly depend on the nuclearity and the internuclear separations of gold(I) centers within the individual molecular complexes or clusters, with influence of the intermolecular interactions between discrete polynuclear molecular complexes relatively less explored (34–38), and those of polynuclear gold(I) clusters not reported. Moreover, while studies on polynuclear gold(I) complexes or clusters are known (34–54), less is explored of their hierarchical assembly and nanostructures as well as the influence of intercluster aggregation on the optical properties (34–38). Among the gold(I) complexes, polynuclear gold(I) chalcogenido complexes represent an important and interesting class (44–51). While directed supramolecular assembly of discrete Au₁₂ (52), Au₁₆ (53), Au₁₈ (51), and Au₃₆ (54) metallomacrocycles as well as trinuclear gold(I) columnar stacks (34–38) have been reported, there have been no corresponding studies on the supramolecular hierarchical assembly of polynuclear gold(I) chalcogenido clusters.Based on our interests and experience in the study of gold(I) chalcogenido clusters (44–46, 51), it is believed that nanoaggegrates with interesting luminescence properties and morphology could be prepared by the judicious design of the gold(I) chalcogenido clusters. As demonstrated by our previous studies on the aggregation behavior of square-planar platinum(II) complexes (77–80) where an enhancement of the solubility of the metal complexes via introduction of solubilizing groups on the ligands and the fine control between solvophobicity and solvophilicity of the complexes would have a crucial influence on the factors governing supramolecular assembly and the formation of aggregates (80), introduction of long alkyl chains as solubilizing groups in the gold(I) sulfido clusters may serve as an effective way to enhance the solubility of the gold(I) clusters for the construction of supramolecular assemblies of novel luminescent nanoaggegrates.Herein, we report the preparation and tunable spectroscopic properties of a series of decanuclear gold(I) μ₃-sulfido complexes with alkyl chains of different lengths on the aminophosphine ligands, [Au₁₀{Ph₂PN(C_nH_2n+1)PPh₂}₄(μ₃-S)₄](ClO₄)₂ [n = 8 (1), 12 (2), 14 (3), 18 (4)] and their supramolecular assembly to form nanoaggregates. The emission colors of the nanoaggregates of 2−4 can be switched from green to yellow to red by varying the solvent systems from which they are formed. These results have been compared with their short alkyl chain-containing counterparts, 1 and a related [Au₁₀{Ph₂PN(C₃H₇)PPh₂}₄(μ₃-S)₄](ClO₄)₂ (45). The present work demonstrates that polynuclear gold(I) chalcogenides, with the introduction of appropriate functional groups, can serve as building blocks for the construction of novel hierarchical nanostructured materials with environment-responsive properties, and it represents a rare example in which nanoaggregates have been assembled with the use of discrete molecular metal clusters as building blocks.     

Heteromerization of chemokine (C-X-C motif) receptor 4 with α1A/B-adrenergic receptors controls α1-adrenergic receptor function

Recent evidence suggests that chemokine (C-X-C motif) receptor 4 (CXCR4) contributes to the regulation of blood pressure through interactions with α₁-adrenergic receptors (ARs) in vascular smooth muscle. The underlying molecular mechanisms, however, are unknown. Using proximity ligation assays to visualize single-molecule interactions, we detected that α_1A/B-ARs associate with CXCR4 on the cell surface of rat and human vascular smooth muscle cells (VSMC). Furthermore, α_1A/B-AR could be coimmunoprecipitated with CXCR4 in a HeLa expression system and in human VSMC. A peptide derived from the second transmembrane helix of CXCR4 induced chemical shift changes in the NMR spectrum of CXCR4 in membranes, disturbed the association between α_1A/B-AR and CXCR4, and inhibited Ca²⁺ mobilization, myosin light chain (MLC) 2 phosphorylation, and contraction of VSMC upon α₁-AR activation. CXCR4 silencing reduced α_1A/B-AR:CXCR4 heteromeric complexes in VSMC and abolished phenylephrine-induced Ca²⁺ fluxes and MLC2 phosphorylation. Treatment of rats with CXCR4 agonists (CXCL12, ubiquitin) reduced the EC₅₀ of the phenylephrine-induced blood pressure response three- to fourfold. These observations suggest that disruption of the quaternary structure of α_1A/B-AR:CXCR4 heteromeric complexes by targeting transmembrane helix 2 of CXCR4 and depletion of the heteromeric receptor complexes by CXCR4 knockdown inhibit α₁-AR–mediated function in VSMC and that activation of CXCR4 enhances the potency of α₁-AR agonists. Our findings extend the current understanding of the molecular mechanisms regulating α₁-AR and provide an example of the importance of G protein-coupled receptor (GPCR) heteromerization for GPCR function. Compounds targeting the α_1A/B-AR:CXCR4 interaction could provide an alternative pharmacological approach to modulate blood pressure.Chemokine (C-X-C motif) receptor 4 (CXCR4) is a G protein-coupled receptor (GPCR) that is essential during development. Animals lacking CXCR4 are not viable and demonstrate defects of the hematopoietic and cardiovascular system (1). After birth, CXCR4 is expressed in many tissues, including the heart and vasculature, and fulfills multiple functions in the immune system, such as regulation of leukocyte trafficking, stem cell mobilization, and homing (2, 3). Moreover, CXCR4 is involved in various disease processes, such as HIV infection, cancer metastasis, and tissue repair (3–5).In addition to these established functions, recent observations suggest that CXCR4 also contributes to the regulation of hemodynamics and blood pressure. Treatment with the CXCR4 antagonists AMD3100 and AMD3465 reduced blood pressure in experimental models of pulmonary arterial and systemic hypertension (6, 7). We have shown previously that AMD3100 reduces hemodynamic stability and blood pressure during the cardiovascular stress response to traumatic and hemorrhagic shock, whereas selective activation of CXCR4 with the noncognate agonist ubiquitin improves hemodynamic stability and increases systemic blood pressure after traumatic, hemorrhagic, and endotoxic shock (8–13). Because in vivo pharmacological targeting of CXCR4 did not affect myocardial function, these findings suggested that effects of CXCR4 on hemodynamics and blood pressure are mediated via modulation of vascular function (9). Accordingly, we observed that CXCR4 activation enhances and sensitizes vasoconstriction of isolated mesenteric arteries and veins in response to α₁-adrenergic receptor (AR) activation with phenylephrine (PE) (9). As these effects were independent of the vascular endothelium, interactions between CXCR4 and α₁-AR in vascular smooth muscle likely constitute the physiological basis for these observations (9). The molecular mechanisms underlying interactions between CXCR4 and α₁-AR in vascular smooth muscle, however, remain unknown.Crosstalk between GPCRs is a widely recognized principle that expands the physiological repertoire of GPCR-mediated signaling events and functions (14–19). Receptor crosstalk can be attributed to a variety of molecular mechanisms, including receptor hetero-oligomerization (14–23). The formation of homo- and/or hetero-oligomeric complexes among GPCRs is thought to be important for many aspects of GPCR function (22–24).CXCR4 has been shown to associate with multiple chemokine receptors in various expression systems (3, 25–28). ARs are also known to be able to form heteromeric receptor complexes (29–35), and recent evidence suggests that AR may also be able to form heteromeric complexes with chemokine receptors (36–38). Thus, we studied whether α₁-AR and CXCR4 may interact on the cell surface of vascular smooth muscle cells through the formation of heteromeric receptor complexes.Here, we provide evidence that heteromeric receptor complexes between α_1A-AR and CXCR4 and between α_1B-AR and CXCR4 are constitutively expressed in rat and human vascular smooth muscle cells (VSMC). We show that disruption of the quaternary structure of the heteromeric receptor complex by targeting transmembrane helix (TM) 2 of CXCR4 and depletion of heteromeric receptor complexes by CXCR4 knockdown inhibit α₁-AR agonist-induced key signaling events and contraction of VSMC. Furthermore, we show that treatment with CXCR4 agonists increases the potency of the α₁-AR agonist PE to increase blood pressure in vivo. Our observations suggest that α₁-AR function in VSMC is controlled through the formation of heteromeric α_1A/B-AR:CXCR4 complexes.     

Giant ankyrin-G stabilizes somatodendritic GABAergic synapses through opposing endocytosis of GABAA receptors

GABA_A-receptor-based interneuron circuitry is essential for higher order function of the human nervous system and is implicated in schizophrenia, depression, anxiety disorders, and autism. Here we demonstrate that giant ankyrin-G (480-kDa ankyrin-G) promotes stability of somatodendritic GABAergic synapses in vitro and in vivo. Moreover, giant ankyrin-G forms developmentally regulated and cell-type-specific micron-scale domains within extrasynaptic somatodendritic plasma membranes of pyramidal neurons. We further find that giant ankyrin-G promotes GABAergic synapse stability through opposing endocytosis of GABA_A receptors, and requires a newly described interaction with GABARAP, a GABA_A receptor-associated protein. We thus present a new mechanism for stabilization of GABAergic interneuron synapses and micron-scale organization of extrasynaptic membrane that provides a rationale for studies linking ankyrin-G genetic variation with psychiatric disease and abnormal neurodevelopment.Interneurons that release γ-aminobutyric acid (GABA) are a major source of inhibitory signaling in vertebrate nervous systems, and play important roles in cognition, mood, and behavior (1, 2). Many of these inhibitory interneurons release GABA, which binds to ionotropic ligand-gated GABA_A receptors located at GABAergic synapses and at extrasynaptic sites, and these GABA_A receptors are sites of action for benzodiazepine and barbiturates (3). GABA_A receptors are dynamic, with continuous exchange between synaptic and extrasynaptic sites in the plane of the membrane, as well as endocytic trafficking between the cell surface and intracellular compartments (3–6). GABA_A receptor cell surface expression is believed to be required for formation of GABAergic synapses based on studies with heterogeneously-expressed GABA_A receptors (7). However, the role of GABA_A receptors in preserving GABAergic synapses has not yet been described in a native neuronal environment.GABAergic synapses localize to both the axon initial segment (AIS) as well as somatodendritic sites of target neurons (2, 8, 9). In the cerebellum, basket and stellar interneurons project specific axon terminals to the AISs of Purkinje cells, forming GABAergic “pinceau” synapses (10). Formation of these pinceau synapses depends on a steep gradient of the cell adhesion molecule neurofascin, which is enriched at the AIS (11, 12). Both GABAergic pinceau synapses and the neurofascin gradient are missing in mice with cerebellar knockout out of the membrane adaptor ankyrin-G (11, 13). Ankyrin-G coordinates multiple proteins at AISs including voltage-gated sodium channels (VGSC), KCNQ2/3 channels, 186-kDa neurofascin, and beta-4 spectrin (14). A role of ankyrin-G in stabilizing GABAergic synapses outside of the the AIS of cerebellar neurons has not been explored.Assembly of AISs as well as their GABAergic synapses requires giant ankyrin-G, which contains a 7.8-kb alternatively spliced nervous system-specific exon found only in vertebrates (14). In addition to ANK repeats and a beta-spectrin-binding domain, giant ankyrin-G (480-kDa ankyrin-G) contains 2,600 residues configured as an extended fibrous polypeptide (14–17). Giant ankyrin-G has been assumed to be confined to AISs and nodes of Ranvier and a general role for ankyrin-G in GABAergic synapse stability at other cellular sites has not been entertained (14, 15, 18).Here we report that giant ankyrin-G is present in extrasynaptic microdomains on the somatodendritic surfaces of hippocampal and cortical neurons, and describe a giant ankyrin-G–based mechanism required for cell surface expression of GABA_A receptors and for maintaining somatodendritic GABAergic synapses. We find that somatodendritic giant ankyrin-G inhibits GABA_A receptor endocytosis through an interaction with the GABA_A receptor-associated protein (GABARAP). This previously unidentified role for giant ankyrin-G provides a newly resolved step in the formation of GABA_A-receptor-mediated circuitry in the cerebral cortex as well as a rationale for recent linkage of human mutations in the giant ankyrin exon with autism and severe cognitive dysfunction (19).     

Tonic GABAA conductance bidirectionally controls interneuron firing pattern and synchronization in the CA3 hippocampal network

The spiking output of interneurons is key for rhythm generation in the brain. However, what controls interneuronal firing remains incompletely understood. Here we combine dynamic clamp experiments with neural network simulations to understand how tonic GABA_A conductance regulates the firing pattern of CA3 interneurons. In baseline conditions, tonic GABA_A depolarizes these cells, thus exerting an excitatory action while also reducing the excitatory postsynaptic potential (EPSP) amplitude through shunting. As a result, the emergence of weak tonic GABA_A conductance transforms the interneuron firing pattern driven by individual EPSPs into a more regular spiking mode determined by the cell intrinsic properties. The increased regularity of spiking parallels stronger synchronization of the local network. With further increases in tonic GABA_A conductance the shunting inhibition starts to dominate over excitatory actions and thus moderates interneuronal firing. The remaining spikes tend to follow the timing of suprathreshold EPSPs and thus become less regular again. The latter parallels a weakening in network synchronization. Thus, our observations suggest that tonic GABA_A conductance can bidirectionally control brain rhythms through changes in the excitability of interneurons and in the temporal structure of their firing patterns.Rhythmic activity paces signal transfer within brain circuits. Brain rhythms are believed to depend heavily on the networks of inhibitory interneurons (1–4). In addition to synaptic inputs, interneuron excitability in the hippocampus is determined by tonic GABA_A conductance (5, 6), which could thus contribute to hippocampal rhythmogenesis. Indeed, GABA transaminase inhibitor vigabatrin increases the ambient GABA concentration, enhancing the power of the theta-rhythm in rats (7). In mice expressing GFP under the GAD67 promoter the reduced levels of ambient GABA correlate with a decreased power of kainate-induced oscillations in vitro (8). The latter decrease is reversed by a GABA uptake inhibitor, guvacine, which raises ambient GABA. GABA release by astrocytes also increases the gamma oscillation power in hippocampal area CA1 in vivo (9). Intriguingly, in hippocampal slices of animals lacking δ subunit-containing GABA_A receptors (which mediate tonic conductance in many local cell types including interneurons) the average frequency of cholinergically induced gamma oscillations is increased, whereas the oscillation power tends to drop (10). However, cellular mechanisms underlying such phenomena remain poorly understood.One possible explanation is the influence of tonic GABA_A conductance on the firing pattern of interneurons. Activation of GABA_A receptors inhibits most neurons, through either membrane hyperpolarization or shunting or both (11). In the adult brain, a depolarizing action of GABA has also been reported in various cell types, including hippocampal interneurons (3, 12–15). GABAergic depolarization can prompt spike generation, thus countering the shunting effects (14, 16). Therefore, experimental evidence indicates that the net effect of GABA_A receptor activation combines the excitatory action of depolarization and the inhibitory consequences of shunting, with the latter prevailing when the GABA_A receptor conductance is sufficiently strong. As a result, increasing the tonic GABA_A signaling can have a biphasic effect on individual hippocampal interneurons: excitatory at weak conductances and inhibitory at strong (14). Here we find that weak tonic GABA_A conductance favors a more regular firing pattern of interneurons, thus facilitating synchronization of the CA3 network. In contrast, strong GABA_A conductance makes the firing pattern more dependent on the stochastic excitatory synaptic input, thus reducing network synchrony.     

Covalent agonists for studying G protein-coupled receptor activation

Structural studies on G protein-coupled receptors (GPCRs) provide important insights into the architecture and function of these important drug targets. However, the crystallization of GPCRs in active states is particularly challenging, requiring the formation of stable and conformationally homogeneous ligand-receptor complexes. Native hormones, neurotransmitters, and synthetic agonists that bind with low affinity are ineffective at stabilizing an active state for crystallogenesis. To promote structural studies on the pharmacologically highly relevant class of aminergic GPCRs, we here present the development of covalently binding molecular tools activating G_s-, G_i-, and G_q-coupled receptors. The covalent agonists are derived from the monoamine neurotransmitters noradrenaline, dopamine, serotonin, and histamine, and they were accessed using a general and versatile synthetic strategy. We demonstrate that the tool compounds presented herein display an efficient covalent binding mode and that the respective covalent ligand-receptor complexes activate G proteins comparable to the natural neurotransmitters. A crystal structure of the β₂-adrenoreceptor in complex with a covalent noradrenaline analog and a conformationally selective antibody (nanobody) verified that these agonists can be used to facilitate crystallogenesis.One of the major obstacles to the investigation of the structural basis of G protein-coupled receptor (GPCR) activation is the flexibility of their seven-transmembrane core, particularly in the active state (1), and the resulting biochemical instability of the solubilized protein (2, 3). Protein crystallography, the most powerful tool for the study of GPCR structure, requires the formation of stable and conformationally homogeneous ligand-receptor complexes (4). High-affinity agonists with dissociation constants in the low to subnanomolar range and low off-rates facilitate stabilization of the protein throughout the process of expression, purification, and crystallogenesis (2); however, endogenous neurotransmitters usually show poor binding affinity. Low binding affinity with rapid association and dissociation rates leads to conformational heterogeneity that prevents the formation of diffraction-quality crystals. The rapid dissociation rate of agonists also makes it difficult to generate active-state stabilizing proteins, such as the camelid antibodies (nanobodies) that have been used to obtain active-state structures of the β₂-adrenergic receptor (β₂AR) (5) and M2 muscarinic receptor (6).To prevent ligand dissociation, irreversible ligation of electrophilic moieties like halomethylketones, isothiocyanates, Michael acceptors, or aziridinium groups of small-molecule ligands with a suitably positioned nucleophilic residue in the receptor has been used (7–16). However, irreversible ligands often suffer from incomplete cross-linking (15) and reduced receptor activation when covalent binding leads to loss of agonist efficacy (10, 16). Furthermore, their highly electrophilic nature and the abundance of nucleophilic groups in biological systems may lead to a low coupling selectivity (7, 8).Disulfide-based cross-linking approaches (17, 18) offer the advantage that the covalent binding of disulfide-containing compounds is chemoselective for cysteine and enforced by the affinity of the ligand-pharmacophore rather than by the electrophilicity of the cross-linking function (19). We refer to the described ligands as covalent rather than irreversible agonists because cleavage may be promoted by reducing agents and the disulfide transfer process is a reversible chemical reaction in general.Structural information on the target protein facilitates the development of covalent ligand-receptor pairs. Mutation of H93^2.64 in the β₂AR to cysteine introduced an anchor for the disulfide-based covalent agonist FAUC50, which does not perturb ligand binding or the activation of the receptor, and thus enabled, to our knowledge, the first agonist-bound GPCR structure (20). Taking advantage of the high structural homology among aminergic GPCRs, we reasoned that the introduction of cysteine into position X^2.64 should also result in a covalently binding receptor mutant for other aminergic GPCRs.We here report a methodology to generate disulfide-based covalent ligand-receptor pairs to promote structural and functional studies on GPCRs. We demonstrate that even the low-affinity endogenous agonists noradrenaline, dopamine, and serotonin can be converted into efficient covalently binding molecular tools for the β₂AR, the dopamine D₂ receptor (D₂R), and the 5-hydroxytryptamine 2A (5-HT_2A) serotonergic subtype representing G_s-, G_i-, and G_q-coupled GPCRs, respectively. Analogous studies were conducted starting from histamine and the receptor subtype H₁. We applied this strategy to obtain an active-state crystal structure of the β₂AR^H93C and a covalent (nor)adrenaline analog.     

Structural basis for membrane recruitment and allosteric activation of cytohesin family Arf GTPase exchange factors

Membrane recruitment of cytohesin family Arf guanine nucleotide exchange factors depends on interactions with phosphoinositides and active Arf GTPases that, in turn, relieve autoinhibition of the catalytic Sec7 domain through an unknown structural mechanism. Here, we show that Arf6-GTP relieves autoinhibition by binding to an allosteric site that includes the autoinhibitory elements in addition to the PH domain. The crystal structure of a cytohesin-3 construct encompassing the allosteric site in complex with the head group of phosphatidyl inositol 3,4,5-trisphosphate and N-terminally truncated Arf6-GTP reveals a large conformational rearrangement, whereby autoinhibition can be relieved by competitive sequestration of the autoinhibitory elements in grooves at the Arf6/PH domain interface. Disposition of the known membrane targeting determinants on a common surface is compatible with multivalent membrane docking and subsequent activation of Arf substrates, suggesting a plausible model through which membrane recruitment and allosteric activation could be structurally integrated.Guanine nucleotide exchange factors (GEFs) activate GTPases by catalyzing exchange of GDP for GTP (1). Because many GEFs are recruited to membranes through interactions with phospholipids, active GTPases, or other membrane-associated proteins (1–5), GTPase activation can be restricted or amplified by spatial–temporal overlap of GEFs with binding partners. GEF activity can also be controlled by autoregulatory mechanisms, which may depend on membrane recruitment (6–11). Structural relationships between these mechanisms are poorly understood.Arf GTPases function in trafficking and cytoskeletal dynamics (5, 12, 13). Membrane partitioning of a myristoylated (myr) N-terminal amphipathic helix primes Arfs for activation by Sec7 domain GEFs (14–17). Cytohesins comprise a metazoan Arf GEF family that includes the mammalian proteins cytohesin-1 (Cyth1), ARNO (Cyth2), and Grp1 (Cyth3). The Drosophila homolog steppke functions in insulin-like growth factor signaling, whereas Cyth1 and Grp1 have been implicated in insulin signaling and Glut4 trafficking, respectively (18–20). Cytohesins share a modular architecture consisting of heptad repeats, a Sec7 domain with exchange activity for Arf1 and Arf6, a PH domain that binds phosphatidyl inositol (PI) polyphosphates, and a C-terminal helix (CtH) that overlaps with a polybasic region (PBR) (21–28). The overlapping CtH and PBR will be referred to as the CtH/PBR. The phosphoinositide specificity of the PH domain is influenced by alternative splicing, which generates diglycine (2G) and triglycine (3G) variants differing by insertion of a glycine residue in the β1/β2 loop (29). Despite similar PI(4,5)P₂ (PIP₂) affinities, the 2G variant has 30-fold higher affinity for PI(3,4,5)P₃ (PIP₃) (30). In both cases, PIP₃ is required for plasma membrane (PM) recruitment (23, 26, 31–33), which is promoted by expression of constitutively active Arf6 or Arl4d and impaired by PH domain mutations that disrupt PIP₃ or Arf6 binding, or by CtH/PBR mutations (8, 34–36).Cytohesins are autoinhibited by the Sec7-PH linker and CtH/PBR, which obstruct substrate binding (8). Autoinhibition can be relieved by Arf6-GTP binding in the presence of the PIP₃ head group (8). Active myr-Arf1 and myr-Arf6 also stimulate exchange activity on PIP₂-containing liposomes (37). Whether this effect is due to relief of autoinhibition per se or enhanced membrane recruitment is not yet clear. Phosphoinositide recognition by PH domains, catalysis of nucleotide exchange by Sec7 domains, and autoinhibition in cytohesins are well characterized (8, 16, 17, 30, 38–43). How Arf-GTP binding relieves autoinhibition and promotes membrane recruitment is unknown. Here, we determine the structural basis for relief of autoinhibition and investigate potential mechanistic relationships between allosteric regulation, phosphoinositide binding, and membrane targeting.     

Heterodimeric coiled-coil interactions of human GABAB receptor

Metabotropic GABA_B receptor is a G protein-coupled receptor that mediates inhibitory neurotransmission in the CNS. It functions as an obligatory heterodimer of GABA_B receptor 1 (GBR1) and GABA_B receptor 2 (GBR2) subunits. The association between GBR1 and GBR2 masks an endoplasmic reticulum (ER) retention signal in the cytoplasmic region of GBR1 and facilitates cell surface expression of both subunits. Here, we present, to our knowledge, the first crystal structure of an intracellular coiled-coil heterodimer of human GABA_B receptor. We found that polar interactions buried within the hydrophobic core determine the specificity of heterodimer pairing. Disruption of the hydrophobic coiled-coil interface with single mutations in either subunit impairs surface expression of GBR1, confirming that the coiled-coil interaction is required to inactivate the adjacent ER retention signal of GBR1. The coiled-coil assembly buries an internalization motif of GBR1 at the heterodimer interface. The ER retention signal of GBR1 is not part of the core coiled-coil structure, suggesting that it is sterically shielded by GBR2 upon heterodimer formation.The major inhibitory neurotransmitter in the CNS is GABA. Metabotropic GABA_B receptor is a G protein-coupled receptor (GPCR) that mediates slow synaptic inhibition (1, 2). It constitutes an important drug target for many neurological disorders, including epilepsy, spasticity, anxiety, and nociception (1, 2).Formation of a functional GABA_B receptor requires the heterodimeric assembly of GABA_B receptor 1 (GBR1) and GABA_B receptor 2 (GBR2) subunits (3–7). Both consist of an N-terminal extracellular domain, a seven-helix transmembrane domain, and a C-terminal intracellular domain. The intracellular domain of each subunit contains a stretch of coiled-coil sequence, and interaction between the coiled-coil helices is partly responsible for GABA_B receptor heterodimerization (5, 8).The intracellular region of GABA_B receptor hosts elements that control receptor trafficking (9). Specifically, GBR1 has a di-leucine internalization signal (EKSRLL) (9) and an endoplasmic reticulum (ER) retention signal (RSRR) (9–11) located within or near its coiled-coil domain (9). GBR1 is trapped within the ER when expressed alone (12) but can reach the cell surface upon association with GBR2 (9, 11). Mutation or removal of the ER retention signal in GBR1 results in plasma membrane expression of GBR1 (9–11). Furthermore, interaction between the coiled-coil domains of GBR1 and GBR2 masks this ER retention signal to facilitate the cell surface expression of both subunits (9–11). Although mutation of the di-leucine motif itself is not sufficient to release GBR1 from intracellular retention, it enhances cell surface expression of various GBR1 mutants that lack the ER retention signal (9).The coiled-coil domain of GBR1 associates with a number of intracellular proteins involved in trafficking, including the coat protein complex I (COPI) (13), the scaffolding protein 14-3-3 (13, 14), the GPCR interacting scaffolding protein GISP (15), and the guanidine exchange factor msec7-1 (16). In particular, COPI specifically recognizes the ER retention signal sequence of GBR1 and is involved in the intracellular retention of GBR1 (13). The msec7-1 protein increases the cell surface expression of GABA_B receptor by binding to the di-leucine internalization motif (16).Despite its important role in GABA_B receptor assembly and trafficking, the atomic details of the coiled-coil interaction between subunits are not known. In this study, we present the crystal structure of a GBR1/GBR2 coiled-coil heterodimer and identify specific contacts at the heterodimer interface that control the surface expression of GBR1.     

Structural dynamics and energetics underlying allosteric inactivation of the cannabinoid receptor CB1

G protein-coupled receptors (GPCRs) are surprisingly flexible molecules that can do much more than simply turn on G proteins. Some even exhibit biased signaling, wherein the same receptor preferentially activates different G-protein or arrestin signaling pathways depending on the type of ligand bound. Why this behavior occurs is still unclear, but it can happen with both traditional ligands and ligands that bind allosterically outside the orthosteric receptor binding pocket. Here, we looked for structural mechanisms underlying these phenomena in the marijuana receptor CB₁. Our work focused on the allosteric ligand Org 27569, which has an unusual effect on CB₁—it simultaneously increases agonist binding, decreases G-protein activation, and induces biased signaling. Using classical pharmacological binding studies, we find that Org 27569 binds to a unique allosteric site on CB₁ and show that it can act alone (without need for agonist cobinding). Through mutagenesis studies, we find that the ability of Org 27569 to bind is related to how much receptor is in an active conformation that can couple with G protein. Using these data, we estimated the energy differences between the inactive and active states. Finally, site-directed fluorescence labeling studies show the CB₁ structure stabilized by Org 27569 is different and unique from that stabilized by antagonist or agonist. Specifically, transmembrane helix 6 (TM6) movements associated with G-protein activation are blocked, but at the same time, helix 8/TM7 movements are enhanced, suggesting a possible mechanism for the ability of Org 27569 to induce biased signaling.Classically, our understanding of G protein-coupled receptor (GPCR) signaling presumed that the receptor formed one unique, active receptor structure in response to agonist binding. We now know that this paradigm is too simple. A growing body of evidence shows that GPCRs can adopt different active conformations depending on the type of signal (ligand) bound, making it unlikely that only one GPCR structure is present at any given moment (1, 2). These different ligand-dependent conformations could explain why a wide range of activities can be observed for some GPCRs, such as coupling to multiple different G-protein types or signaling through non–G-protein signaling partners, such as the protein arrestin (3). This phenomenon—diverse ligands bound to the same receptor selectively eliciting different signaling pathways—is referred to as functional selectivity or biased signaling.What are these different receptor conformations, and why might they result in biased signaling? One possibility is that they involve different orientations of transmembrane helix 5 (TM5) and TM6 in the cytoplasmic face. An outward movement of TM6 is critical for G-protein activation, because it exposes a hydrophobic binding site and enables formation of the ternary complex of receptor, ligand, and G protein (4–9). Newer evidence suggests that there is likely some plasticity in TM6 movement during activation, with differences in either the magnitude or probability of the movement explaining varying degrees of G-protein signaling (3, 10, 11).Some types of biased signaling may also arise when TM7 and its attached helix 8 (H8) adopt different conformations in the cytoplasmic face, because movements in this region have been detected during receptor activation (12–14). However, H8/TM7 movement does not seem to be required for G-protein activation (15), and this region does not contact the G protein in the recent ternary complex structure (7). For these reasons, H8/TM7 movements may not be directly involved in G-protein binding but rather, may play a role in the binding of arrestin and/or kinase, thus triggering arrestin-centric signaling pathways (14, 16).The mechanism(s) through which allosteric molecules alter GPCR structure is also an unresolved question and an area of increasing interest (17–19) for which novel approaches are being developed (20) because of the potential that these ligands offer for new treatment paradigms (21). Allosteric ligands for several GPCRs have now been identified, including ligands for the cannabinoid, muscarinic, and μ-opioid.To gain more information about the structural changes accompanying both biased signaling and allosteric modulation of GPCRs, we have been studying the effects of an unusual allosteric ligand on CB₁, the marijuana GPCR. The use of this ligand, called Org 27569, provides a unique way to detect previously unidentified GPCR conformations for several reasons. First, because it binds allosterically, Org 27569 likely uses a different mechanism to act on CB₁. It also enables well-characterized radioactively labeled orthosteric CB₁ ligands to be used. Second, Org 27569 exhibits a number of unusual effects—it increases agonist binding to the receptor while simultaneously inhibiting G-protein activation (10, 22), and inducing biased signaling (23–25). Thus, it is hard to imagine how these different effects could occur unless the CB₁–Org 27569 bound state adopts a unique and different conformation. A cartoon representation of CB₁ and the putative Org 27569 binding site is shown in Fig. 1.Open in a separate windowFig. 1.A 2D cartoon model of CB₁ showing the approximate location of orthosteric and allosteric binding sites and the various mutations used. The traditional (orthosteric) ligand binding site is depicted as a dashed white oval, and the (proposed) allosteric ligand Org 27569 binding site is depicted as a dashed purple circle (50). Key point mutations in CB₁ include a CAM (I348Y^6.40; green) or CIM (Y294A^5.58; red). These mutations presumably cause their effect by altering interactions with a highly conserved Arg (R) in TM3 (black square). Radioligand binding studies used CB₁-Gα_i, a full-length human CB₁ receptor fused to the G-protein Gα_i (tan). SDFL studies used a minimal cysteine receptor (θ) with a truncated N terminus (Δ87) and a truncated C terminus (Δ417) to which the 1D4 epitope tag (black boxes) is attached to enable purification and unique reactive cysteines introduced at either TM6 (A342C^6.34; blue) or H8/TM7 (L404C^8.50; orange) to enable labeling with the fluorophore bimane (Inset). The different C-terminal modifications following residue 417 are indicated as X, where the sequence is either X₁ (for CB₁-Gα_i) or X₂ (for θ).Recently, we reported that, although Org 27569 stabilizes CB₁ interactions with the agonist, it simultaneously blocks the TM6 movements required for G-protein activation discussed above (10), thus explaining the negative effect of Org 27569 on G-protein signaling. These conclusions were based on site-directed fluorescence labeling (SDFL) studies of purified CB₁ that showed that, although Org 27569 induces CB₁ to adopt a high-affinity agonist binding conformation, it is not the high-affinity agonist binding conformation traditionally associated with the formation of the ternary complex with G protein (4).Here, we set out to characterize the conformation and energetics underlying this unique Org 27569 trapped state and identify a mechanism for the unusual effects discussed above using a combination of classical pharmacology, mathematical modeling, and SDFL studies. One goal was to determine if Org 27569 could act on the receptor in the absence of an orthosteric ligand. Another goal was to explore the linkage between Org 27569 binding and TM6 movements in CB₁ by asking the question: because Org 27569 binding blocks TM6 movement, does impairing TM6 movement inhibit Org 27569 binding? We did this by creating and testing two different CB₁ mutants: one constitutively active mutant (CAM) and one constitutively inactive mutant (CIM). In these mutants, TM6 movement was either enhanced (CAM) or impaired (CIM). Radioligand binding studies were then performed on these mutants in the presence of Org 27569 to test the above hypothesis and assess the energetics underlying Org 27569 binding. [All radioligand binding and efficacy measurements, unless otherwise stated, were done in the absence of sodium, a well-known negative allosteric modulator of GPCR activity, to enhance basal activity and reduce allosteric variables. This fact could contribute to the relatively high R*/R ratios that we observe for WT CB₁ along with the use of G-protein chimeras in our measurements (because G proteins can allosterically modulate receptor affinity).]Finally, we used SDFL to probe the structural differences between active, inactive, and Org 27569-bound CB₁, with the goal of identifying other structural changes in the receptor that might explain the mechanism of allosteric modulation and biased signaling, specifically focusing on movements at TM6 as well as H8/TM7. Our results are intriguing—they show that Org 27569 binding stabilizes a different receptor conformation, one that may be related to its ability to induce biased signaling.     

Transition paths,diffusive processes,and preequilibria of protein folding

Fundamental relationships between the thermodynamics and kinetics of protein folding were investigated using chain models of natural proteins with diverse folding rates by extensive comparisons between the distribution of conformations in thermodynamic equilibrium and the distribution of conformations sampled along folding trajectories. Consistent with theory and single-molecule experiment, duration of the folding transition paths exhibits only a weak correlation with overall folding time. Conformational distributions of folding trajectories near the overall thermodynamic folding/unfolding barrier show significant deviations from preequilibrium. These deviations, the distribution of transition path times, and the variation of mean transition path time for different proteins can all be rationalized by a diffusive process that we modeled using simple Monte Carlo algorithms with an effective coordinate-independent diffusion coefficient. Conformations in the initial stages of transition paths tend to form more nonlocal contacts than typical conformations with the same number of native contacts. This statistical bias, which is indicative of preferred folding pathways, should be amenable to future single-molecule measurements. We found that the preexponential factor defined in the transition state theory of folding varies from protein to protein and that this variation can be rationalized by our Monte Carlo diffusion model. Thus, protein folding physics is different in certain fundamental respects from the physics envisioned by a simple transition-state picture. Nonetheless, transition state theory can be a useful approximate predictor of cooperative folding speed, because the height of the overall folding barrier is apparently a proxy for related rate-determining physical properties.Protein folding is an intriguing phenomenon at the interface of physics and biology. In the early days of folding kinetics studies, folding was formulated almost exclusively in terms of mass-action rate equations connecting the folded, unfolded, and possibly, one or a few intermediate states (1, 2). With the advent of site-directed mutagenesis, the concept of free energy barriers from transition state theory (TST) (3) was introduced to interpret mutational data (4), and subsequently, it was adopted for the Φ-value analysis (5). Since the 1990s, the availability of more detailed experimental data (6), in conjunction with computational development of coarse-grained chain models, has led to an energy landscape picture of folding (7–15). This perspective emphasizes the diversity of microscopic folding trajectories, and it conceptualizes folding as a diffusive process (16–25) akin to the theory of Kramers (26).For two-state-like folding, the transition path (TP), i.e., the sequence of kinetic events that leads directly from the unfolded state to the folded state (27, 28), constitutes only a tiny fraction of a folding trajectory that spends most of the time diffusing, seemingly unproductively, in the vicinity of the free energy minimum of the unfolded state. The development of ultrafast laser spectroscopy (29, 30) and single-molecule (27, 28, 31) techniques have made it possible to establish upper bounds on the transition path time (t_TP) ranging from <200 and <10 μs by earlier (27) and more recent (28), respectively, direct single-molecule FRET to <2 μs (30) by bulk relaxation measurements. Consistent with these observations, recent extensive atomic simulations have also provided estimated t_TP values of the order of ∼1 μs (32, 33). These advances offer exciting prospects of characterizing the productive events along folding TPs.It is timely, therefore, to further the theoretical investigation of TP-related questions (19). To this end, we used coarse-grained C_α models (14) to perform extensive simulations of the folding trajectories of small proteins with 56- to 86-aa residues. These tractable models are useful, because despite significant progress, current atomic models cannot provide the same degree of sampling coverage for proteins of comparable sizes (32, 33). In addition to structural insights, this study provides previously unexplored vantage points to compare the diffusion and TST pictures of folding. Deviations of folding behaviors from TST predictions are not unexpected, because TST is mostly applicable to simple gas reactions; however, the nature and extent of the deviations have not been much explored. Our explicit-chain simulation data conform well to the diffusion picture but not as well to TST. In particular, the preexponential factors of the simulated folding rates exhibit a small but appreciable variation that depends on native topology. These findings and others reported below underscore the importance of single-molecule measurements (13, 27, 28, 31, 34, 35) in assessing the merits of proposed scenarios and organizing principles of folding (7–25, 36, 37).     

Electrochemical tuning of vertically aligned MoS2 nanofilms and its application in improving hydrogen evolution reaction

The ability to intercalate guest species into the van der Waals gap of 2D layered materials affords the opportunity to engineer the electronic structures for a variety of applications. Here we demonstrate the continuous tuning of layer vertically aligned MoS₂ nanofilms through electrochemical intercalation of Li⁺ ions. By scanning the Li intercalation potential from high to low, we have gained control of multiple important material properties in a continuous manner, including tuning the oxidation state of Mo, the transition of semiconducting 2H to metallic 1T phase, and expanding the van der Waals gap until exfoliation. Using such nanofilms after different degree of Li intercalation, we show the significant improvement of the hydrogen evolution reaction activity. A strong correlation between such tunable material properties and hydrogen evolution reaction activity is established. This work provides an intriguing and effective approach on tuning electronic structures for optimizing the catalytic activity.Layer-structured 2D materials are an interesting family of materials with strong covalent bonding within molecular layers and weak van der Waals interaction between layers. Beyond intensively studied graphene-related materials (1–4), there has been recent strong interest in other layered materials whose vertical thickness can be thinned down to less than few nanometers and horizontal width can also be reduced to nanoscale (5–9). The strong interest is driven by their interesting physical and chemical properties (2, 10) and their potential applications in transistors, batteries, topological insulators, thermoelectrics, artificial photosynthesis, and catalysis (4, 11–25).One of the unique properties of 2D layered materials is their ability to intercalate guest species into their van der Waals gaps, opening up the opportunities to tune the properties of materials. For example, the spacing between the 2D layers could be increased by intercalation such as lithium (Li) intercalated graphite or molybdenum disulfide (MoS₂) and copper intercalated bismuth selenide (26–29). The electronic structures of the host lattice, such as the charge density, anisotropic transport, oxidation state, and phase transition, may also be changed by different species intercalation (26, 27).As one of the most interesting layered materials, MoS₂ has been extensively studied in a variety of areas such as electrocatalysis (20–22, 30–36). It is known that there is a strong correlation between the electronic structure and catalytic activity of the catalysts (20, 37–41). It is intriguing to continuously tune the morphology and electronic structure of MoS₂ and explore the effects on MoS₂ hydrogen evolution reaction (HER) activity. Very recent studies demonstrated that the monolayered MoS₂ and WS₂ nanosheets with 1T metallic phase synthesized by chemical exfoliation exhibited superior HER catalytic activity to those with 2H semiconducting phase (35, 42), with a possible explanation that the strained 1T phase facilitates the hydrogen binding process during HER (42). However, it only offers two end states of materials and does not offer a continuous tuning. A systematic investigation to correlate the gradually tuned electronic structure, including oxidation state shift and semiconducting–metallic phase transition, and the corresponding HER activity is important but unexplored. We believe that the Li electrochemical intercalation method offers a unique way to tune the catalysts for optimization.In this paper, we demonstrate that the layer spacing, oxidation state, and the ratio of 2H semiconducting to 1T metallic phase of MoS₂ HER catalysts were continuously tuned by Li intercalation to different voltages vs. Li⁺/Li in nanofilms with molecular layers perpendicular to the substrates. Correspondingly, the catalytic activity for HER was observed to be continuously tuned. The lower oxidation state of Mo and 1T metallic phase of MoS₂ turn out to have better HER catalytic activities. The performance of MoS₂ catalyst on both flat and 3D electrodes was dramatically improved when it was discharged to low potentials vs. Li⁺/Li.     

From the Cover: Why the proportion of transmission during early-stage HIV infection does not predict the long-term impact of treatment on HIV incidence

Antiretroviral therapy (ART) reduces the infectiousness of HIV-infected persons, but only after testing, linkage to care, and successful viral suppression. Thus, a large proportion of HIV transmission during a period of high infectiousness in the first few months after infection (“early transmission”) is perceived as a threat to the impact of HIV “treatment-as-prevention” strategies. We created a mathematical model of a heterosexual HIV epidemic to investigate how the proportion of early transmission affects the impact of ART on reducing HIV incidence. The model includes stages of HIV infection, flexible sexual mixing, and changes in risk behavior over the epidemic. The model was calibrated to HIV prevalence data from South Africa using a Bayesian framework. Immediately after ART was introduced, more early transmission was associated with a smaller reduction in HIV incidence rate—consistent with the concern that a large amount of early transmission reduces the impact of treatment on incidence. However, the proportion of early transmission was not strongly related to the long-term reduction in incidence. This was because more early transmission resulted in a shorter generation time, in which case lower values for the basic reproductive number (R₀) are consistent with observed epidemic growth, and R₀ was negatively correlated with long-term intervention impact. The fraction of early transmission depends on biological factors, behavioral patterns, and epidemic stage and alone does not predict long-term intervention impacts. However, early transmission may be an important determinant in the outcome of short-term trials and evaluation of programs.Recent studies have confirmed that effective antiretroviral therapy (ART) reduces the transmission of HIV among stable heterosexual couples (1–3). This finding has generated interest in understanding the population-level impact of HIV treatment on reducing the rate of new HIV infections in generalized epidemic settings (4). Research, including mathematical modeling (5–10), implementation research (11), and major randomized controlled trials (12–14), are focused on how ART provision might be expanded strategically to maximize its public health benefits (15, 16).One concern is that if a large fraction of HIV transmission occurs shortly after a person becomes infected, before the person can be diagnosed and initiated on ART, this will limit the potential impact of HIV treatment on reducing HIV incidence (9, 17, 18). Data suggest that persons are more infectious during a short period of “early infection” after becoming infected with HIV (19–22), although there is debate about the extent, duration, and determinants of elevated infectiousness (18, 23). The amount of transmission that occurs also will depend on patterns of sexual behavior and sexual networks (17, 24–27). There have been estimates for the contribution of early infection to transmission from mathematical models (7, 17, 21, 24–26) and phylogenetic analyses (28–31), but these vary widely, from 5% to above 50% (23).In this study, we use a mathematical model to quantify how the proportion of transmission that comes from persons who have been infected recently affects the impact of treatment scale-up on HIV incidence. The model is calibrated to longitudinal HIV prevalence data from South Africa using a Bayesian framework. Thus, the model accounts for not only the early epidemic growth rate highlighted in previous research (5, 9, 18), but also the heterogeneity and sexual behavior change to explain the peak and decline in HIV incidence observed in sub-Saharan African HIV epidemics (32, 33).The model calibration allows uncertainty about factors that determine the amount of early transmission, including the relative infectiousness during early infection, heterogeneity in propensity for sexual risk behavior, assortativity in sexual partner selection, reduction in risk propensity over the life course, and population-wide reductions in risk behavior in response to the epidemic (32, 33). This results in multiple combinations of parameter values that are consistent with the observed epidemic and variation in the amount of early transmission. We simulated the impact of a treatment intervention and report how the proportion of early transmission correlates with the reduction in HIV incidence from the intervention over the short- and long-term.