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Platelet activation of protease-activated receptor 4 (PAR4) and thrombin are at the top of a chain of events leading to fibrin deposition, microinfarcts, blood-brain barrier disruption, and inflammation. We evaluated mRNA expression of the PAR4 gene F2RL3 in human brain and global cognitive performance in participants with and without cognitive impairment or dementia. Data were acquired from the Religious Orders Study (ROS) and the Rush Memory and Aging Project (MAP). F2RL3 mRNA was elevated in AD cases and was associated with worse retrospective longitudinal cognitive performance. Moreover, F2RL3 expression interacted with clinical AD diagnosis on longitudinal cognition whereas this relationship was attenuated in individuals without cognitive impairment. Additionally, when adjusting for the effects of AD neuropathology, F2RL3 expression remained a significant predictor of cognitive decline. F2RL3 expression correlated positively with transcript levels of proinflammatory markers including TNFα, IL-1β, NFκB, and fibrinogen α/β/γ. Together, these results reveal that F2RL3 mRNA expression is associated with multiple AD-relevant outcomes and its encoded product, PAR4, may play a role in disease pathogenesis., Competing Interests: Conflict of Interest TH serves as a member of the scientific advisory board for Vivid Genomics (outside the work presented herein). No other authors have any relevant conflicts to declare., (Copyright © 2024. Published by Elsevier Inc.)

Here, we demonstrate a structure-based small molecule virtual screening and lead optimization pipeline using a homology model of a difficult-to-drug G-protein-coupled receptor (GPCR) target. Protease-activated receptor 4 (PAR4) is activated by thrombin cleavage, revealing a tethered ligand that activates the receptor, making PAR4 a challenging target. A virtual screen of a make-on-demand chemical library yielded a one-hit compound. From the single-hit compound, we developed a novel series of PAR4 antagonists. Subsequent lead optimization via simultaneous virtual library searches and structure-based rational design efforts led to potent antagonists of thrombin-induced activation. Interestingly, this series of antagonists was active against PAR4 activation by the native protease thrombin cleavage but not the synthetic PAR4 agonist peptide AYPGKF., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Protease-activated receptor 4 (PAR4) is a G protein-coupled receptor activated by thrombin. In the platelet, response to thrombin PAR4 contributes to the predominant procoagulant microparticle formation, increased fibrin deposition, and initiation of platelet-stimulated inflammation. In addition, PAR4 is expressed in other cell types, including endothelial cells. Under inflammatory conditions, PAR4 is overexpressed via epigenetic demethylation of the PAR4 gene, F2RL3 . PAR4 knockout (KO) studies have determined a role for PAR4 in ischemia-reperfusion injury in the brain, and PAR4 KO mice display normal cardiac function but present less myocyte death and cardiac dysfunction in response to acute myocardial infarction. Although PAR4 has been reported to be expressed within the kidney, the contribution of PAR4 to acute kidney injury (AKI) and chronic kidney disease (CKD) is not well understood. Here we report that PAR4 KO mice are protected against kidney injury in two mouse models. First, PAR4 KO mice are protected against induction of markers of both fibrosis and inflammation in two different models of kidney injury: 1 ) 7 days following unilateral ureter obstruction (UUO) and 2 ) an AKI-CKD model of ischemia-reperfusion followed by 8 days of contralateral nephrectomy. We further show that PAR4 expression in the kidney is low in the control mouse kidney but induced over time following UUO. PAR4 KO mice are protected against blood urea nitrogen (BUN) and glomerular filtration rate (GFR) kidney function pathologies in the AKI-CKD model. Following the AKI-CKD model, PAR4 is expressed in the collecting duct colocalizing with Dolichos biflorus agglutinin (DBA), but not in the proximal tubule with Lotus tetragonolobus lectin (LTL). Collectively, the results reported in this study implicate PAR4 as contributing to the pathology in mouse models of acute and chronic kidney injury. NEW & NOTEWORTHY The contribution of the thrombin receptor protease-activated receptor 4 (PAR4) to acute kidney injury (AKI) and chronic kidney disease (CKD) is not well understood. Here we report that PAR4 expression is upregulated after kidney injury and PAR4 knockout (KO) mice are protected against fibrosis following kidney injury in two mouse models. First, PAR4 KO mice are protected against unilateral ureter obstruction. Second, PAR4 KO mice are protected against an AKI-CKD model of ischemia-reperfusion followed by contralateral nephrectomy.

Negative regulation of exocytosis from secretory cells is accomplished through inhibitory signals from Gi/o GPCRs by Gβγ subunit inhibition of 2 mechanisms: decreased calcium entry and direct interaction of Gβγ with soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) plasma membrane fusion machinery. Previously, we disabled the second mechanism with a SNAP25 truncation (SNAP25Δ3) that decreased Gβγ affinity for the SNARE complex, leaving exocytotic fusion and modulation of calcium entry intact and removing GPCR-Gβγ inhibition of SNARE-mediated exocytosis. Here, we report substantial metabolic benefit in mice carrying this mutation. Snap25Δ3/Δ3 mice exhibited enhanced insulin sensitivity and beiging of white fat. Metabolic protection was amplified in Snap25Δ3/Δ3 mice challenged with a high-fat diet. Glucose homeostasis, whole-body insulin action, and insulin-mediated glucose uptake into white adipose tissue were improved along with resistance to diet-induced obesity. Metabolic protection in Snap25Δ3/Δ3 mice occurred without compromising the physiological response to fasting or cold. All metabolic phenotypes were reversed at thermoneutrality, suggesting that basal autonomic activity was required. Direct electrode stimulation of sympathetic neuron exocytosis from Snap25Δ3/Δ3 inguinal adipose depots resulted in enhanced and prolonged norepinephrine release. Thus, the Gβγ-SNARE interaction represents a cellular mechanism that deserves further exploration as an additional avenue for combating metabolic disease.

The nucleus accumbens (NAc) guides reward-related motivated behavior implicated in pathological behavioral states, including addiction and depression. These behaviors depend on the precise neuromodulatory actions of G i/o -coupled G-protein-coupled receptors (GPCRs) at glutamatergic synapses onto medium spiny projection neurons (MSNs). Previous work has shown that discrete classes of G i/o -coupled GPCR mobilize Gβγ to inhibit vesicular neurotransmitter release via t-SNARE protein, SNAP25. However, it remains unknown which Gαi/o systems in the NAc utilize Gβγ-SNARE signaling to dampen glutamatergic transmission. Utilizing patch-clamp electrophysiology and pharmacology in a transgenic mouse line with a C-terminal three-residue deletion of SNAP25 (SNAP25Δ3) weaking the Gβγ-SNARE interaction, we surveyed a broad cohort of G i/o -coupled GPCRs with robust inhibitory actions at glutamatergic synapses in the NAc. We find that basal presynaptic glutamate release probability is reduced in SNAP25Δ3 mice. While κ opioid, CB1, adenosine A1, group II metabotropic glutamate receptors, and histamine H3 receptors inhibit glutamatergic transmission onto MSNs independent of SNAP25, we report that SNAP25 contributes significantly to the actions of GABA B , 5-HT1 B/D , and μ opioid receptors. These findings demonstrate that presynaptic G i/o -coupled GPCRs recruit heterogenous effector mechanisms at glutamatergic synapses in the NAc, with a subset requiring SNA25-dependent Gβγ signaling., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Manz, Zepeda, Zurawski, Hamm and Grueter.)

Accurate photon counting requires that rods generate highly amplified, reproducible single photon responses (SPRs). The SPR is generated within the rod outer segment (ROS), a multilayered structure built from membranous disks that house rhodopsin. Photoisomerization of rhodopsin at the disk rim causes a local depletion of cGMP that closes ion channels in the plasmalemma located nearby with relative rapidity. In contrast, a photoisomerization at the disk center, distant from the plasmalemma, has a delayed impact on the ion channels due to the time required for cGMP redistribution. Radial differences should be greatest in large diameter rods. By affecting membrane guanylate cyclase activity, bicarbonate could impact spatial inhomogeneity in cGMP content. It was previously known that in the absence of bicarbonate, SPRs are larger and faster at the base of a toad ROS (where the ROS attaches to the rest of the cell) than at the distal tip. Given that bicarbonate enters the ROS at the base and diffuses to the tip and that it expedites flash response recovery, there should be an axial concentration gradient for bicarbonate that would accentuate the base-to-tip SPR differences. Seeking to understand how ROS geometry and bicarbonate affect SPR variability, we used mathematical modeling and made electrophysiological recordings of single rods. Modeling predicted and our experiments confirmed minor radial SPR variability in large diameter, salamander rods that was essentially unchanged by bicarbonate. SPRs elicited at the base and tip of salamander rods were similar in the absence of bicarbonate, but when treated with 30 mM bicarbonate, SPRs at the base became slightly faster than those at the tip, verifying the existence of an axial gradient for bicarbonate. The differences were small and unlikely to undermine visual signaling. However, in toad rods with longer ROSs, bicarbonate somehow suppressed the substantial, axial SPR variability that is naturally present in the absence of bicarbonate. Modeling suggested that the axial gradient of bicarbonate might dampen the primary phototransduction cascade at the base of the ROS. This novel effect of bicarbonate solves a mystery as to how toad vision is able to function effectively in extremely dim light., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Geva, Caruso, Klaus, Hamm, Gurevich, DiBenedetto and Makino.)

Inhibition of neurotransmitter release by neurotransmitter substances constitutes a fundamental means of neuromodulation. In contrast to well-delineated mechanisms that underlie inhibition of evoked release via suppression of voltage-gated Ca 2+ channels, processes that underlie neuromodulatory inhibition of spontaneous release remain unclear. Here, we interrogated inhibition of spontaneous glutamate and GABA release by presynaptic metabotropic GABA B receptors. Our findings show that this inhibition relies on Gβγ subunit action at the membrane, and it is largely independent of presynaptic Ca 2+ signaling for both forms of release. In the case of spontaneous glutamate release, inhibition requires Gβγ interaction with the C terminus of the key fusion machinery component SNAP25, and it is modulated by synaptotagmin-1. Inhibition of spontaneous GABA release, on the other hand, is independent of these pathways and likely requires alternative Gβγ targets at the presynaptic terminal., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Ligand binding to G protein–coupled receptors (GPCRs), such as the α 2a -adrenergic receptor (α 2a AR), results in the activation of heterotrimeric G proteins, which consist of functionally distinct Gα subunits and Gβγ dimers. α 2a AR-dependent inhibition of synaptic transmission regulates functions such as spontaneous locomotor activity, anesthetic sparing, and working memory enhancement and requires the soluble NSF attachment protein receptor (SNARE) complex, a Gβγ effector. To understand how the Gβγ-SNARE complex underlies the α 2a AR-dependent inhibition of synaptic transmission, we examined the specificity of Gβγ subunits for the SNARE complex in adrenergic neurons, in which auto-α 2a ARs respond to epinephrine released from these neurons, and nonadrenergic neurons, in which hetero-α 2a ARs respond to epinephrine released from other neurons. We performed a quantitative, targeted multiple reaction monitoring proteomic analysis of Gβ and Gγ subunits bound to the SNARE complex in synaptosomes from mouse brains. In the absence of stimulation of auto-α 2a ARs, Gβ 1 and Gγ 3 interacted with the SNARE complex. However, Gβ 1 , Gβ 2 , and Gγ 3 were found in the complex when auto-α 2a ARs were activated by epinephrine. Further understanding of the specific usage of distinct Gβγ subunits in vivo may provide insights into the homeostatic regulation of synaptic transmission and the mechanisms of dysfunction that occur in neurological diseases.

The detailed pharmacology and therapeutic potential of the central PAR4 receptors are poorly understood due to a lack of potent, selective, and brain-penetrant tool compounds. Despite this, robust data with biochemical and genetic tools show the therapeutic potential of PAR4 antagonists in traumatic brain injury, Alzheimer's disease, Parkinson's disease, and other neurodegenerative disorders with a neuroinflammatory component. Thus, we performed a functional HTS campaign, identified a fundamentally new PAR4 competitive inhibitor chemotype, optimized this new series (increased potency >45-fold), discovered enantiospecific activity (though opposing preference for human versus mouse PAR4), and engendered high central nervous system penetration (rat K p 's of 0.52 to 4.2 and K p,uu 's of 0.52 to 1.2).

In daylight, cone photoreceptors in the retina are responsible for the bulk of visual perception, yet compared to rods, far less is known quantitatively about their biochemistry. This is partly because it is hard to isolate and purify cone proteins. The issue is also complicated by the synergistic interaction of these parameters in producing systems biology outputs, such as photoresponse. Using a 3-D resolved, finite element model of cone outer segments, here we conducted a study of parameter significance using global sensitivity analysis, by Sobol indices, which was contextualized within the uncertainty surrounding these parameters in the available literature. The analysis showed that a subset of the parameters influencing the circulating dark current, such as the turnover rate of cGMP in the dark, may be most influential for variance with experimental flash response, while the shut-off rates of photoexcited rhodopsin and phosphodiesterase also exerted sizable effect. The activation rate of transducin by rhodopsin and the light-induced hydrolysis rate of cGMP exerted measurable effects as well but were estimated as relatively less significant. The results of this study depend on experimental ranges currently described in the literature and should be revised as these become better established. To that end, these findings may be used to prioritize parameters for measurement in future investigations., Competing Interests: The authors have declared that no competing interests exist.

Retinal rods function as accurate photon counters to provide for vision under very dim light. To do so, rods must generate highly amplified, reproducible responses to single photons, yet outer segment architecture and randomness in the location of rhodopsin photoisomerization on the surface of an internal disk introduce variability to the rising phase of the photon response. Soon after a photoisomerization at a disk rim, depletion of cGMP near the plasma membrane closes ion channels and hyperpolarizes the rod. But with a photoisomerization in the center of a disk, local depletion of cGMP is distant from the channels in the plasma membrane. Thus, channel closure is delayed by the time required for the reduction of cGMP concentration to reach the plasma membrane. Moreover, the local fall in cGMP dissipates over a larger volume before affecting the channels, so response amplitude is reduced. This source of variability increases with disk radius. Using a fully space-resolved biophysical model of rod phototransduction, we quantified the variability attributable to randomness in the location of photoisomerization as a function of disk structure. In mouse rods that have small disks bearing a single incisure, this variability was negligible in the absence of the incisure. Variability was increased slightly by the incisure, but randomness in the shutoff of rhodopsin emerged as the main source of single photon response variability at all but the earliest times. Variability arising from randomness in the transverse location of photoisomerization increased in magnitude and persisted over a longer period in the photon response of large salamander rods. A symmetric arrangement of multiple incisures in the disks of salamander rods greatly reduced this variability during the rising phase, but the incisures had the opposite effect on variability arising from randomness in rhodopsin shutoff at later times., Competing Interests: The authors have declared that no competing interests exist.

Behavioral assays in the mouse can show marked differences between male and female animals of a given genotype. These differences identified in such preclinical studies may have important clinical implications. We recently made a mouse model with impaired presynaptic inhibition through Gβγ-SNARE signaling. Here, we examine the role of sexual dimorphism in the severity of the phenotypes of this model, the SNAP25Δ3 mouse. In males, we already reported that SNAP25Δ3 homozygotes demonstrated phenotypes in motor coordination, nociception, spatial memory and stress processing. We now report that while minimal sexually dimorphic effects were observed for the nociceptive, motor or memory phenotypes, large differences were observed in the stress-induced hyperthermia paradigm, with male SNAP25Δ3 homozygotes exhibiting an increase in body temperature subsequent to handling relative to wild-type littermates, while no such genotype-dependent effect was observed in females. This suggests sexually dimorphic mechanisms of Gβγ-SNARE signaling for stress processing or thermoregulation within the mouse. Second, we examined the effects of heterozygosity with respect to the SNAP25Δ3 mutation. Heterozygote SNAP25Δ3 animals were tested alongside homozygote and wild-type littermates in all of the aforementioned paradigms and displayed phenotypes similar to wild-type animals or an intermediate state. From this, we conclude that the SNAP25Δ3 mutation does not behave in an autosomal dominant manner, but rather displays incomplete dominance for many phenotypes., (© 2020 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Complex circuit interactions within the nucleus accumbens (NAc) facilitate goal-directed behavior. Medium spiny neurons (MSNs) mediate NAc output by projecting to functionally divergent brain regions, a property conferred, in part, by the differential projection patterns of D1- and D2 dopamine receptor-expressing MSNs. Glutamatergic afferents to the NAc direct MSN output by recruiting feedforward inhibitory microcircuits comprised of parvalbumin (PV)-expressing interneurons (INs). Furthermore, the GABA B heteroreceptor (GABA B R), a G i/o -coupled G-protein-coupled receptor, is expressed at glutamatergic synapses throughout the mesolimbic network, yet its physiological context and synaptic mechanism within the NAc remains unknown. Here, we explored GABA B R function at glutamatergic synapses within PV-IN-embedded microcircuits in the NAc core of male mice. We found that GABA B R is expressed presynaptically and recruits a noncanonical signaling mechanism to reduce glutamatergic synaptic efficacy at D1(+) and D1(-) (putative D2) MSN subtypes. Furthermore, PV-INs, a robust source of neuronal GABA in the NAc, heterosynaptically target GABA B R to selectively modulate glutamatergic transmission onto D1(+) MSNs. These findings elucidate a new mechanism of feedforward inhibition and refine mechanisms by which GABA B heteroreceptors modulate mesolimbic circuit function. SIGNIFICANCE STATEMENT Glutamatergic transmission in the nucleus accumbens (NAc) critically contributes to goal-directed behaviors. However, intrinsic microcircuit mechanisms governing the integration of these synapses remain largely unknown. Here, we show that parvalbumin-expressing interneurons within feedforward microcircuits heterosynaptically target GABA B heteroreceptors (GABA B R) on glutamate terminals. Activation of presynaptically-expressed GABA B R decreases glutamatergic synaptic strength by engaging a non-canonical signaling pathway that interferes with vesicular exocytotic release machinery. These findings offer mechanistic insight into the role of GABA B heteroreceptors within reward circuitry, elucidate a novel arm to feedforward inhibitory networks, and inform the growing use of GABA B R-selective pharmacotherapy for various motivational disorders, including addiction, major depressive disorder, and autism (Cousins et al., 2002; Kahn et al., 2009; Jacobson et al., 2018; Stoppel et al., 2018; Pisansky et al., 2019)., (Copyright © 2019 the authors.)

Three SNARE proteins, SNAP-25, syntaxin 1A, and VAMP2 or synaptobrevin 2, constitute the minimal functional machinery needed for the regulated secretion of neurotransmitters. Dynamic changes in the regulated release of neurotransmitters are associated with the induction of long-term plasticity at central synapses. In-vitro studies have validated the C-terminus of SNAP-25 as a target for inhibitory Gi/o-coupled G-protein coupled receptors at a number of synapses. The physiological consequences of the interaction between Gi/o proteins and SNAP-25 in the context of activity-dependent long-term synaptic plasticity are not well understood. Here, we report direct ex-vivo evidence of the involvement of the C-terminus of SNAP-25 in inducing long-term potentiation of synaptic strength at Schaffer collateral-CA1 synapses using a gene-targeted mouse model with truncated C-terminus (carboxyl terminus) of SNAP-25. It has been shown previously that truncation of the three extreme C-terminal residues in SNAP-25[INCREMENT]3 homozygote mice reduces its interaction with the inhibitory Gβγ subunits two-fold. In in-vitro hippocampal slices, we show that these SNAP-25[INCREMENT]3 mice express significantly larger magnitude of long-term potentiation at hippocampal Schaffer collateral-CA1 synapses.

G protein-coupled receptors (GPCRs) that couple to G i/o proteins modulate neurotransmission presynaptically by inhibiting exocytosis. Release of Gβγ subunits from activated G proteins decreases the activity of voltage-gated Ca 2+ channels (VGCCs), decreasing excitability. A less understood Gβγ-mediated mechanism downstream of Ca 2+ entry is the binding of Gβγ to SNARE complexes, which facilitate the fusion of vesicles with the cell plasma membrane in exocytosis. Here, we generated mice expressing a form of the SNARE protein SNAP25 with premature truncation of the C terminus and that were therefore partially deficient in this interaction. SNAP25Δ3 homozygote mice exhibited normal presynaptic inhibition by GABA B receptors, which inhibit VGCCs, but defective presynaptic inhibition by receptors that work directly on the SNARE complex, such as 5-hydroxytryptamine (serotonin) 5-HT 1b receptors and adrenergic α 2a receptors. Simultaneously stimulating receptors that act through both mechanisms showed synergistic inhibitory effects. SNAP25Δ3 homozygote mice had various behavioral phenotypes, including increased stress-induced hyperthermia, defective spatial learning, impaired gait, and supraspinal nociception. These data suggest that the inhibition of exocytosis by G i/o -coupled GPCRs through the Gβγ-SNARE interaction is a crucial component of numerous physiological and behavioral processes., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

G proteins are major transducers of signals from G-protein coupled receptors (GPCRs). They are made up of α, β, and γ subunits, with 16 Gα, 5 Gβ and 12 Gγ subunits. Though much is known about the specificity of Gα subunits, the specificity of Gβγs activated by a given GPCR and that activate each effector in vivo is not known. Here, we examined the in vivo Gβγ specificity of presynaptic α 2a -adrenergic receptors (α 2a ARs) in both adrenergic (auto-α 2a ARs) and non-adrenergic neurons (hetero-α 2a ARs) for the first time. With a quantitative MRM proteomic analysis of neuronal Gβ and Gγ subunits, and co-immunoprecipitation of tagged α 2a ARs from mouse models including transgenic FLAG-α 2a ARs and knock-in HA-α 2a ARs, we investigated the in vivo specificity of Gβ and Gγ subunits to auto-α 2a ARs and hetero-α 2a ARs activated with epinephrine to understand the role of Gβγ specificity in diverse physiological functions such as anesthetic sparing, and working memory enhancement. We detected Gβ 2 , Gγ 2 , Gγ 3 , and Gγ 4 with activated auto α 2a ARs, whereas we found Gβ 4 and Gγ 12 preferentially interacted with activated hetero-α 2a ARs. Further understanding of in vivo Gβγ specificity to various GPCRs offers new insights into the multiplicity of genes for Gβ and Gγ, and the mechanisms underlying GPCR signaling through Gβγ subunits.

Throughout the past five decades, tremendous advancements have been made in our understanding of G protein signaling and presynaptic inhibition, many of which were published in the Journal of Biological Chemistry under the tenure of Herb Tabor as Editor-in-Chief. Here, we identify these critical advances, including the formulation of the ternary complex model of G protein-coupled receptor signaling and the discovery of Gβγ as a critical signaling component of the heterotrimeric G protein, along with the nature of presynaptic inhibition and its physiological role. We provide an overview for the discovery and physiological relevance of the two known Gβγ-mediated mechanisms for presynaptic inhibition: first, the action of Gβγ on voltage-gated calcium channels to inhibit calcium influx to the presynaptic active zone and, second, the direct binding of Gβγ to the SNARE complex to displace synaptotagmin downstream of calcium entry, which has been demonstrated to be important in neurons and secretory cells. These two mechanisms act in tandem with each other in a synergistic manner to provide more complete spatiotemporal control over neurotransmitter release., (© 2019 Zurawski et al.)

Cancer-associated thrombosis is a common first presenting sign of malignancy and is currently the second leading cause of death in cancer patients after their malignancy. However, the molecular mechanisms underlying cancer-associated thrombosis remain undefined. In this study, we aimed to develop a better understanding of how cancer cells affect the coagulation cascade and platelet activation to induce a prothrombotic phenotype. Our results show that colon cancer cells trigger platelet activation in a manner dependent on cancer cell tissue factor (TF) expression, thrombin generation, activation of the protease-activated receptor 4 (PAR4) on platelets and consequent release of ADP and thromboxane A2. Platelet-colon cancer cell interactions potentiated the release of platelet-derived extracellular vesicles (EVs) rather than cancer cell-derived EVs. Our data show that single colon cancer cells were capable of recruiting and activating platelets and generating fibrin in plasma under shear flow. Finally, in a retrospective analysis of colon cancer patients, we found that the number of venous thromboembolism events was 4.5 times higher in colon cancer patients than in a control population. In conclusion, our data suggest that platelet-cancer cell interactions and perhaps platelet procoagulant EVs may contribute to the prothrombotic phenotype of colon cancer patients. Our work may provide rationale for targeting platelet-cancer cell interactions with PAR4 antagonists together with aspirin and/or ADP receptor antagonists as a potential intervention to limit cancer-associated thrombosis, balancing safety with efficacy.

Human platelets express two protease-activated receptors (PARs), PAR1 (F2R) and PAR4 (F2RL3), which are activated by a number of serine proteases that are generated during pathological events and cause platelet activation. Recent interest has focused on PAR4 as a therapeutic target, given PAR4 seems to promote experimental thrombosis and procoagulant microparticle formation, without a broadly apparent role in hemostasis. However, it is not yet known whether PAR4 activity plays a role in platelet-leukocyte interactions, which are thought to contribute to both thrombosis and acute or chronic thrombo-inflammatory processes. We sought to determine whether PAR4 activity contributes to granule secretion from activated platelets and platelet-leukocyte interactions. We performed in vitro and ex vivo studies of platelet granule release and platelet-leukocyte interactions in the presence of PAR4 agonists including PAR4 activating peptide, thrombin, cathepsin G, and plasmin in combination with small-molecule PAR4 antagonists. Activation of human platelets with thrombin, cathepsin G, or plasmin potentiated platelet dense granule secretion that was specifically impaired by PAR4 inhibitors. Platelet-leukocyte interactions and platelet P-selectin exposure the following stimulation with PAR4 agonists were also impaired by activated PAR4 inhibition in either a purified system or in whole blood. These results indicate PAR4-specific promotion of platelet granule release and platelet-leukocyte aggregate formation and suggest that pharmacological control of PAR4 activity could potentially attenuate platelet granule release or platelet-leukocyte interaction-mediated pathological processes.

The growth hormone secretagogue receptor (GHSR) and dopamine receptor (D2R) have been shown to oligomerize in hypothalamic neurons with a significant effect on dopamine signaling, but the molecular processes underlying this effect are still obscure. We used here the purified GHSR and D2R to establish that these two receptors assemble in a lipid environment as a tetrameric complex composed of two each of the receptors. This complex further recruits G proteins to give rise to an assembly with only two G protein trimers bound to a receptor tetramer. We further demonstrate that receptor heteromerization directly impacts on dopamine-mediated Gi protein activation by modulating the conformation of its α-subunit. Indeed, association to the purified GHSR:D2R heteromer triggers a different active conformation of Gαi that is linked to a higher rate of GTP binding and a faster dissociation from the heteromeric receptor. This is an additional mechanism to expand the repertoire of GPCR signaling modulation that could have implications for the control of dopamine signaling in normal and physiopathological conditions., Competing Interests: The authors declare no conflict of interest.

Background: Heterotrimeric G proteins are fundamental signaling proteins composed of three subunits, Gα and a Gβγ dimer. The role of Gα as a molecular switch is critical for transmitting and amplifying intracellular signaling cascades initiated by an activated G protein Coupled Receptor (GPCR). Despite their biochemical and therapeutic importance, the study of G protein evolution has been limited to the scope of a few model organisms. Furthermore, of the five primary Gα subfamilies, the underlying gene structure of only two families has been thoroughly investigated outside of Mammalia evolution. Therefore our understanding of Gα emergence and evolution across phylogeny remains incomplete., Results: We have computationally identified the presence and absence of every Gα gene (GNA-) across all major branches of Deuterostomia and evaluated the conservation of the underlying exon-intron structures across these phylogenetic groups. We provide evidence of mutually exclusive exon inclusion through alternative splicing in specific lineages. Variations of splice site conservation and isoforms were found for several paralogs which coincide with conserved, putative motifs of DNA-/RNA-binding proteins. In addition to our curated gene annotations, within Primates, we identified 15 retrotranspositions, many of which have undergone pseudogenization. Most importantly, we find numerous deviations from previous findings regarding the presence and absence of individual GNA- genes, nuanced differences in phyla-specific gene copy numbers, novel paralog duplications and subsequent intron gain and loss events., Conclusions: Our curated annotations allow us to draw more accurate inferences regarding the emergence of all Gα family members across Metazoa and to present a new, updated theory of Gα evolution. Leveraging this, our results are critical for gaining new insights into the co-evolution of the Gα subunit and its many protein binding partners, especially therapeutically relevant G protein - GPCR signaling pathways which radiated in Vertebrata evolution.

Ligand binding and pathway-specific activation of G protein-coupled receptors is currently being studied with great effort. Individual answers may depend on the nature of the ligands and the effector pathway. Recently, we have presented a detailed model of neuropeptide Y bound to the Y 2 R. Accordingly, the C-terminal part of the peptide binds deeply in the transmembrane bundle and brings the side chain of the most essential Y 36 in close proximity to W 6.48 Here, we investigate the role of this interaction for ligand binding and activation of this receptor. BRET sensors were used for detailed investigation of effector coupling and led to the identification of preassembly of the Y 2 R-G i complex. It further confirmed ligand-dependent recruitment of arrestin3. Using equally sensitive readouts for G i activation and arrestin recruitment as well as quantification with operational models of agonism allowed us to identify a strong inherent bias for G i activation over arrestin3 recruitment for the wild-type receptor. By systematic mutagenesis, we found that W 6.48 does not contribute to the binding affinity, but acts as an allosteric connector to couple ligand binding to G i activation and arrestin3 recruitment. However, even mutagenesis to a small threonine did not lead to a complete loss of signaling. Interestingly, signaling was restored to wild-type levels by ligands that contain a naphthylalanine as the C-terminal residue instead of Y 36 Steric and polar contributions of W 6.48 for the activation of the receptor are discussed in the context of different mechanisms of G protein coupling and arrestin recruitment., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

Prerequisite for structural studies on G protein-coupled receptors is the preparation of highly concentrated, stable, and biologically active receptor samples in milligram amounts of protein. Here, we present an improved protocol for Escherichia coli expression, functional refolding, and reconstitution into bicelles of the human neuropeptide Y receptor type 2 (Y 2 R) for solution and solid-state NMR experiments. The isotopically labeled receptor is expressed in inclusion bodies and purified using SDS. We studied the details of an improved preparation protocol including the in vitro folding of the receptor, e.g., the native disulfide bridge formation, the exchange of the denaturating detergent SDS, and the functional reconstitution into bicelle environments of varying size. Full pharmacological functionality of the Y 2 R preparation was shown by a ligand affinity of 4 nM and G-protein activation. Further, simple NMR experiments are used to test sample quality in high micromolar concentration.

Gβγ dimers are one of the essential signaling units of activated G protein-coupled receptors (GPCRs). There are five Gβ and 12 Gγ subunits in humans; numerous studies have demonstrated that different Gβ and Gγ subunits selectively interact to form unique Gβγ dimers, which in turn may target specific receptors and effectors. Perturbation of Gβγ signaling can lead to impaired physiological responses. Moreover, previous targeted multiple-reaction monitoring (MRM) studies of Gβ and Gγ subunits have shown distinct regional and subcellular localization patterns in four brain regions. Nevertheless, no studies have quantified or compared their individual protein levels. In this study, we have developed a quantitative MRM method not only to quantify but also to compare the protein abundance of neuronal Gβ and Gγ subunits. In whole and fractionated crude synaptosomes, we were able to identify the most abundant neuronal Gβ and Gγ subunits and their subcellular localizations. For example, Gβ 1 was mostly localized at the membrane while Gβ 2 was evenly distributed throughout synaptosomal fractions. The protein expression levels and subcellular localizations of Gβ and Gγ subunits may affect the Gβγ dimerization and Gβγ-effector interactions. This study offers not only a new tool for quantifying and comparing Gβ and Gγ subunits but also new insights into the in vivo distribution of Gβ and Gγ subunits, and Gβγ dimer assembly in normal brain function.

G i/o -coupled G protein-coupled receptors can inhibit neurotransmitter release at synapses via multiple mechanisms. In addition to Gβγ-mediated modulation of voltage-gated calcium channels (VGCC), inhibition can also be mediated through the direct interaction of Gβγ subunits with the soluble N -ethylmaleimide attachment protein receptor (SNARE) complex of the vesicle fusion apparatus. Binding studies with soluble SNARE complexes have shown that Gβγ binds to both ternary SNARE complexes, t-SNARE heterodimers, and monomeric SNAREs, competing with synaptotagmin 1(syt1) for binding sites on t-SNARE. However, in secretory cells, Gβγ, SNAREs, and synaptotagmin interact in the lipid environment of a vesicle at the plasma membrane. To approximate this environment, we show that fluorescently labeled Gβγ interacts specifically with lipid-embedded t-SNAREs consisting of full-length syntaxin 1 and SNAP-25B at the membrane, as measured by fluorescence polarization. Fluorescently labeled syt1 undergoes competition with Gβγ for SNARE-binding sites in lipid environments. Mutant Gβγ subunits that were previously shown to be more efficacious at inhibiting Ca 2+ -triggered exocytotic release than wild-type Gβγ were also shown to bind SNAREs at a higher affinity than wild type in a lipid environment. These mutant Gβγ subunits were unable to inhibit VGCC currents. Specific peptides corresponding to regions on Gβ and Gγ shown to be important for the interaction disrupt the interaction in a concentration-dependent manner. In in vitro fusion assays using full-length t- and v-SNAREs embedded in liposomes, Gβγ inhibited Ca 2+ /synaptotagmin-dependent fusion. Together, these studies demonstrate the importance of these regions for the Gβγ-SNARE interaction and show that the target of Gβγ, downstream of VGCC, is the membrane-embedded SNARE complex., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

G-protein βγ subunits (Gβγ) interact with presynaptic proteins and regulate neurotransmitter release downstream of Ca 2+ influx. To accomplish their roles in sensory signaling, photoreceptor synapses use specialized presynaptic proteins that support neurotransmission at active zone structures known as ribbons. While several G-protein coupled receptors (GPCRs) influence synaptic transmission at ribbon synapses of cones and other retinal neurons, it is unknown whether Gβγ contributes to these effects. We tested whether activation of one particular GPCR, a metabotropic glutamate receptor (mGluR), can reduce cone synaptic transmission via Gβγ in tiger salamander retinas. In recordings from horizontal cells, we found that an mGluR agonist (L-AP4) reduced cone-driven light responses and mEPSC frequency. In paired recordings of cones and horizontal cells, L-AP4 slightly reduced cone I Ca (∼10%) and caused a larger reduction in cone-driven EPSCs (∼30%). Proximity ligation assay revealed direct interactions between SNAP-25 and Gβγ subunits in retinal synaptic layers. Pretreatment with the SNAP-25 cleaving protease BoNT/A inhibited L-AP4 effects on synaptic transmission, as did introduction of a peptide derived from the SNAP-25 C terminus. Introducing Gβγ subunits directly into cones reduced EPSC amplitude. This effect was inhibited by BoNT/A, supporting a role for Gβγ/SNAP-25 interactions. However, the mGluR-dependent reduction in I Ca was not mimicked by Gβγ, indicating that this effect was independent of Gβγ. The finding that synaptic transmission at cone ribbon synapses is regulated by Gβγ/SNAP-25 interactions indicates that these mechanisms are shared by conventional and ribbon-type synapses. Gβγ liberated from other photoreceptor GPCRs is also likely to regulate synaptic transmission. SIGNIFICANCE STATEMENT Dynamic regulation of synaptic transmission by presynaptic G-protein coupled receptors shapes information flow through neural circuits. At the first synapse in the visual system, presynaptic metabotropic glutamate receptors (mGluRs) regulate cone photoreceptor synaptic transmission, although the mechanisms and functional impact of this are unclear. We show that mGluRs regulate light response encoding across the cone synapse, accomplished in part by triggering G-protein βγ subunits (Gβγ) interactions with SNAP-25, a core component of the synaptic vesicle fusion machinery. In addition to revealing a role in visual processing, this provides the first demonstration that Gβγ/SNAP-25 interactions regulate synaptic function at a ribbon-type synapse, contributing to an emerging picture of the ubiquity of Gβγ/SNARE interactions in regulating synaptic transmission throughout the nervous system., (Copyright © 2017 the authors 0270-6474/17/374619-17$15.00/0.)

Human platelets display a unique dual receptor system for responding to its primary endogenous activator, α-thrombin. Because of the lack of efficacious antagonists, the field has relied on synthetic peptides and pepducins to describe protease-activated receptor PAR1 and PAR4 signaling. The precise contributions of each receptor have not been established in the context of thrombin. We took advantage of newly discovered PAR antagonists to contrast the contribution of PAR1 and PAR4 to thrombin-mediated activation of the platelet fibrin receptor (GPIIbIIIa). PAR1 is required for platelet activation at low but not high concentrations of thrombin, and maximal platelet activation at high concentrations of thrombin requires PAR4. As the concentration of thrombin is increased, PAR1 signaling is quickly overcome by PAR4 signaling, leaving a narrow window of low thrombin concentrations that exclusively engage PAR1. PAR4 antagonism reduces the maximum thrombin response by over 50%. Thus, although the PAR1 response still active at higher concentrations of thrombin, this response is superseded by PAR4. Truncation of a known PAR4 antagonist and identification of the minimum pharmacophore converted the mechanism of inhibition from noncompetitive to competitive, such that the antagonist could be outcompeted by increasing doses of the ligand. Fragments retained efficacy against both soluble and tethered ligands with lower cLogP values and an increased free fraction in plasma. These reversible, competitive compounds represent a route toward potentially safer PAR4 antagonists for clinical utility and the development of tools such as radioligands and positron emission tomography tracers that are not currently available to the field for this target., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

This letter describes the further deconstruction of the known PAR4 inhibitor chemotypes (MWs 490-525 and with high plasma protein binding) to identify a minimum PAR4 pharmacophore devoid of metabolic liabilities and improved properties. This exercise identified a greatly simplified 2-methoxy-6-arylimidazo[2,1-b][1,3,4]thiadiazole scaffold that afforded nanomolar inhibition of both activating peptide and γ-thrombin mediated PAR4 stimulation, while reducing both molecular weight and the number of hydrogen bond donors/acceptors by ∼50%. This minimum PAR4 pharmacophore, with competitive inhibition, versus non-competitive of the larger chemotypes, allows an ideal starting point to incorporate desired functional groups to engender optimal DMPK properties towards a preclinical candidate., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Reduced platelet aggregation and a mild bleeding phenotype have been observed in patients chronically taking selective serotonin reuptake inhibitors (SSRIs). However, it remains unclear how SSRIs, which inhibit the plasma membrane serotonin transporter (SERT), modulate hemostasis. Here, we examine how sustained inhibition of SERT activity alters serotonergic signaling and influences platelet activation and hemostasis. Pharmaceutical blockade (citalopram dosing) or genetic ablation (SERT(-/-)) of SERT function in vivo led to reduced serotonin (5-hydroxytryptamine (5-HT)) blood levels that paralleled a mild bleeding phenotype in mice. Transfusion of wild-type platelets to SERT(-/-) mice normalized bleeding times to wild-type levels, suggesting that loss of SERTs causes a deficiency in platelet activation. Although SERT(-/-) platelets displayed no difference in P-selectin or αIIbβ3 activation upon stimulation with thrombin, ADP-mediated αIIbβ3 activation is reduced in SERT(-/-) platelets. Additionally, synergistic potentiation of αIIbβ3 activation by ADP and 5-HT is lost in SERT(-/-) platelets. Acute treatment of wild-type platelets with 5-HT2A receptor (5-HT2AR) antagonists or SSRIs revealed that functional 5-HT2ARs, not SERTs, are necessary for the synergistic activation of αIIbβ3 by dual 5-HT/ADP stimulation. Pharmacological studies using radiolabeled guanosine 5'-3-O-([(35)S]thio)triphosphate and [(3)H]ketanserin revealed that platelets isolated from SERT(-/-) or citalopram-treated mice have reduced activation of G-proteins coupled to 5-HT2ARs and receptor surface expression. Taken together, these data demonstrate that sustained SERT loss of function reduces 5-HT2AR surface expression that is critical for the synergistic activation of αIIbβ3 by 5-HT and ADP. These results highlight an antiplatelet strategy centered on blocking or desensitizing 5-HT2AR to attenuate ADP-mediated αIIbβ3 activation., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

G protein-coupled receptor-mediated heterotrimeric G protein activation is a major mode of signal transduction in the cell. Previously, we and other groups reported that the α5 helix of Gαi1, especially the hydrophobic interactions in this region, plays a key role during nucleotide release and G protein activation. To further investigate the effect of this hydrophobic core, we disrupted it in Gαi1 by inserting 4 alanine amino acids into the α5 helix between residues Gln(333) and Phe(334) (Ins4A). This extends the length of the α5 helix without disturbing the β6-α5 loop interactions. This mutant has high basal nucleotide exchange activity yet no receptor-mediated activation of nucleotide exchange. By using structural approaches, we show that this mutant loses critical hydrophobic interactions, leading to significant rearrangements of side chain residues His(57), Phe(189), Phe(191), and Phe(336); it also disturbs the rotation of the α5 helix and the π-π interaction between His(57) and Phe(189) In addition, the insertion mutant abolishes G protein release from the activated receptor after nucleotide binding. Our biochemical and computational data indicate that the interactions between α5, α1, and β2-β3 are not only vital for GDP release during G protein activation, but they are also necessary for proper GTP binding (or GDP rebinding). Thus, our studies suggest that this hydrophobic interface is critical for accurate rearrangement of the α5 helix for G protein release from the receptor after GTP binding., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Here, we describe the development of a series of highly selective PAR4 antagonists with nanomolar potency and selectivity versus PAR1, derived from the indole-based 3. Of these, 9j (PAR4 IC50 = 445 nM, PAR1 response IC50 > 30 μM) and 10h (PAR4 IC50 = 179 nM, PAR1 response IC50 > 30 μM) maintained an overall favorable in vitro DMPK profile, encouraging rat/mouse in vivo pharmacokinetics (PK) and activity against γ-thrombin.

Among the opioid receptors, the κ-opioid receptor (κOR) has been gaining considerable attention as a potential therapeutic target for the treatment of complex CNS disorders including depression, visceral pain, and cocaine addiction. With an interest in discovering novel ligands targeting κOR, we searched natural products for unusual scaffolds and identified collybolide (Colly), a nonnitrogenous sesquiterpene from the mushroom Collybia maculata. This compound has a furyl-δ-lactone core similar to that of Salvinorin A (Sal A), another natural product from the plant Salvia divinorum Characterization of the molecular pharmacological properties reveals that Colly, like Sal A, is a highly potent and selective κOR agonist. However, the two compounds differ in certain signaling and behavioral properties. Colly exhibits 10- to 50-fold higher potency in activating the mitogen-activated protein kinase pathway compared with Sal A. Taken with the fact that the two compounds are equipotent for inhibiting adenylyl cyclase activity, these results suggest that Colly behaves as a biased agonist of κOR. Behavioral studies also support the biased agonistic activity of Colly in that it exhibits ∼10-fold higher potency in blocking non-histamine-mediated itch compared with Sal A, and this difference is not seen in pain attenuation by these two compounds. These results represent a rare example of functional selectivity by two natural products that act on the same receptor. The biased agonistic activity, along with an easily modifiable structure compared with Sal A, makes Colly an ideal candidate for the development of novel therapeutics targeting κOR with reduced side effects.

Abnormal platelet reactivity is associated with recurrent ischemia and bleeding following percutaneous coronary intervention (PCI). Protease-activated receptor-1 (PAR1), encoded by F2R, is a high affinity thrombin receptor on platelets and the target of the antiplatelet drug vorapaxar. The intronic single nucleotide polymorphism F2R IVS-14 A/T affects PAR1 receptor density and function. We hypothesized that carriers of the T allele, who have been shown to have decreased platelet reactivity, would be at lower risk for thrombotic events, but higher risk for bleeding following PCI. Using BioVU, the Vanderbilt DNA repository linked to the electronic medical record, we studied 660 patients who underwent PCI for unstable or stable coronary artery disease. Primary outcome measures were major adverse cardiovascular events (MACE, composite of revascularization, MI, stroke, death) and bleeding (assessed by Bleeding Academic Research Consortium scale) over 24 months. The minor allele (T) frequency was 14.8 %. There were no genotypic differences in the frequency of MACE (33.7, 28.8, and 31.6 % for A/A, A/T, and T/T respectively, P = 0.50) or bleeding (15.7, 14.7, and 18.8 % for A/A, A/T, and T/T respectively, P = 0.90). In a Cox regression model, fully adjusted for age, race, sex, BMI, and smoking status, carrying a T allele was not associated with MACE (HR 1.19, 95 % CI 0.89-1.59, P = 0.23) or bleeding (HR 0.73, 95 % CI 0.37-1.4, P = 0.34). In conclusion, in our population, F2R IVS-14 PAR1 variability does not affect risk of MACE or bleeding following PCI.

Gi/o-coupled G protein-coupled receptors can exert an inhibitory effect on vesicle release through several G protein-driven mechanisms, more than one of which may be concurrently present in individual presynaptic terminals. The synaptosomal-associated protein of 25 kDa (SNAP25) is a key downstream effector of Gβγ subunits. It has previously been shown that proteolytic cleavage of SNAP25 by botulinum toxin A reduces the ability of Gβγ to compete with the calcium sensor synaptotagmin 1 (Syt1) for binding to SNAP25 in a calcium-dependent manner. These truncated SNAP25 proteins sustain a low level of exocytosis but are unable to support serotonin-mediated inhibition of exocytosis in lamprey spinal neurons. Here, we generate a SNAP25 extreme C-terminal mutant that is deficient in its ability to bind Gβγ while retaining normal calcium-dependent Syt1 binding to soluble N-ethylmaleimide attachment protein receptor (SNARE) and vesicle release. The SNAP25Δ3 mutant, in which residue G204 is replaced by a stop codon, features a partial reduction in Gβ1γ2 binding in vitro as well as a partial reduction in the ability of the lamprey 5-hydroxytryptamine1b-type serotonin receptor to reduce excitatory postsynaptic current amplitudes, an effect previously shown to be mediated through the interaction of Gβγ with SNAP25. Syt1 calcium-dependent binding to SNAP25Δ3 was reduced by a small extent compared with the wild type. We conclude that the extreme C terminus of SNAP25 is a critical region for the Gβγ-SNARE interaction., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

With a newer, more selective and efficacious cytosolic phospholipase A2α (cPLA2α) inhibitor available, we revisited the role of cPLA2α activity in platelet activation and discovered that a component of platelet signaling, even larger than previously appreciated, relies on this enzyme. In a whole blood shear-based flow chamber assay, giripladib, a cPLA2α inhibitor, reduced platelet adhesion and accumulation on collagen. Moreover, giripladib differentially affected P-selectin expression and GPIIbIIIa activation depending on the agonist employed. While protease-activated receptor 1 (PAR1)-mediated platelet activation was unaffected by giripladib, the levels of PAR4- and GPVI-mediated platelet activation were significantly reduced. Meanwhile, the thromboxane A2 receptor antagonist SQ29548 had no effect on PAR-, GPVI-, or puriniergic receptor-mediated platelet activation, suggesting that another eicosanoid produced downstream of arachidonic acid liberation by cPLA2α was responsible for this large component of PAR4- and GPVI-mediated platelet activation. In parallel, we profiled PAR-mediated changes in glycerophospholipid (GPL) mass with and without giripladib to better understand cPLA2α-mediated lipid metabolism. Phosphatidylcholine and phosphatidylethanolamine (PE) demonstrated the largest consumption of mass during thrombin stimulation. Additionally, we confirm phosphatidylinositol as a major substrate of cPLA2α. A comparison of PAR1- and PAR4-induced metabolism revealed the consumption of more putative arachidonyl-PE species downstream of PAR1 activation. Instead of enhanced cPLA2α activity and therefore more arachidonic acid liberation downstream of PAR4, these results indicate the major role that cPLA2α activity plays in platelet function and suggest that a novel eicosanoid is produced in response to platelet activation that represents a large component of PAR4- and GPVI-mediated responses.

Rhodopsin is a prototypical member of the G protein-coupled receptors (GPCRs). This photoreceptor is responsible for initiating the visual signaling transduction cascade upon interaction with its heterotrimeric G protein, transducin (Gt), after light activation. Like all transmembrane proteins, rhodopsin is embedded within a phospholipid bilayer. Many studies have proposed that the membrane composition of this bilayer is an important factor for receptor function during the activation process. Here we describe the methods and assays used to evaluate the function of purified and reconstituted rhodopsin in bicelles.

Introduction: The blood coagulation system is a tightly regulated balance of procoagulant and anticoagulant factors, disruption of which can cause clinical complications. Diabetics are known to have a hypercoagulable phenotype, along with increased circulating levels of methylglyoxal (MGO) and decreased activity of the anticoagulant plasma protein antithrombin III (ATIII). MGO has been shown to inhibit ATIII activity in vitro, however the mechanism of inhibition is incompletely understood. As such, we designed this study to investigate the kinetics and mechanism of MGO-mediated ATIII inhibition., Methods: MGO-mediated ATIII inhibition was confirmed using inverse experiments detecting activity of the ATIII targets thrombin and factor Xa. Fluorogenic assays were performed in both PBS and plasma after incubation of ATIII with MGO, at molar ratios comparable to those observed in the plasma of diabetic patients. LC-coupled tandem mass spectrometry was utilized to investigate the exact mechanism of MGO-mediated ATIII inhibition., Results and Conclusions: MGO concentration-dependently attenuated inhibition of thrombin and factor Xa by ATIII in PBS-based assays, both in the presence and absence of heparin. In addition, MGO concentration-dependently inhibited ATIII activity in a plasma-based system, to the level of plasma completely deficient in ATIII, again both in the presence and absence of heparin. Results from LC-MS/MS experiments revealed that MGO covalently adducts the active site Arg 393 of ATIII through two distinct glyoxalation mechanisms. We posit that active site adduction is the mechanism of MGO-mediated inhibition of ATIII, and thus contributes to the underlying pathophysiology of the diabetic hypercoagulable state and complications thereof., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Although resistance to the P2Y12 antagonist clopidogrel is linked to altered drug metabolism, some studies suggest that these pharmacokinetic abnormalities only partially account for drug resistance. To circumvent pharmacokinetic complications and target P2Y12 receptor function we applied the direct P2Y12 antagonist 2-methylthio-AMP (2-methylthioadenosine 5'-monophosphate triethylammonium salt) to purified platelets ex vivo. Platelets were purified from healthy and type 2 diabetes mellitus (T2DM) patients and stimulated with thrombin or the selective protease-activated receptor agonists, protease-activated receptor 1-activating peptide (PAR1-AP), or PAR4-AP. Platelet activation as measured by αIIbβ3 activation, and P-selectin expression was monitored in 141 subjects. Our results demonstrate that, compared with healthy subjects, platelets from diabetic patients are resistant to inhibition by 2-methylthio-AMP, demonstrating P2Y12 pharmacodynamic defects among diabetic patients. Inhibition of thrombin-mediated αIIbβ3 activation by 2-methylthio-AMP was lower in diabetic platelets versus healthy platelets. Subgroup analysis revealed a racial difference in the resistance to 2-methylthio-AMP. We found no resistance in platelets from diabetic African Americans; they were inhibited by 2-methylthio-AMP equally as well as platelets from healthy African Americans. In contrast, platelets from Caucasian patients with diabetes were resistant to P2Y12 antagonism compared with healthy Caucasians. Multivariable analysis demonstrated that other variables, such as obesity, age, or gender, could not account for the differential resistance to 2-methylthio-AMP among races. These results suggest that in addition to altered drug metabolism, P2Y12 receptor function itself is altered in the Caucasian diabetic population. The racial difference in platelet function in T2DM is a novel finding, which may lead to differences in treatment as well as new targets for antiplatelet therapy., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)

Herein we report the discovery and SAR of an indole-based protease activated receptor-4 (PAR-4) antagonist scaffold derived from a similarity search of the Vanderbilt HTS collection, leading to MLPCN probe ML354 (VU0099704). Using a novel PAC-1 fluorescent αIIbβ3 activation assay this probe molecule antagonist was found to have an IC50 of 140nM for PAR-4 with 71-fold selectivity versus PAR-1 (PAR-1IC50=10μM)., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

G protein activation by G protein-coupled receptors is one of the critical steps for many cellular signal transduction pathways. Previously, we and other groups reported that the α5 helix in the G protein α subunit plays a major role during this activation process. However, the precise signaling pathway between the α5 helix and the guanosine diphosphate (GDP) binding pocket remains elusive. Here, using structural, biochemical, and computational techniques, we probed different residues around the α5 helix for their role in signaling. Our data showed that perturbing the Phe-336 residue disturbs hydrophobic interactions with the β2-β3 strands and α1 helix, leading to high basal nucleotide exchange. However, mutations in β strands β5 and β6 do not perturb G protein activation. We have highlighted critical residues that leverage Phe-336 as a relay. Conformational changes are transmitted starting from Phe-336 via β2-β3/α1 to Switch I and the phosphate binding loop, decreasing the stability of the GDP binding pocket and triggering nucleotide release. When the α1 and α5 helices were cross-linked, inhibiting the receptor-mediated displacement of the C-terminal α5 helix, mutation of Phe-336 still leads to high basal exchange rates. This suggests that unlike receptor-mediated activation, helix 5 rotation and translocation are not necessary for GDP release from the α subunit. Rather, destabilization of the backdoor region of the Gα subunit is sufficient for triggering the activation process., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Receptor-mediated activation of the Gα subunit of heterotrimeric G proteins requires allosteric communication between the receptor binding site and the guanine nucleotide binding site, which are separated by >30 Å. Structural changes in the allosteric network connecting these sites are predicted to be transient in the wild-type Gα subunit, making studies of these connections challenging. In the current work, site-directed mutants that alter the energy barriers between the activation states are used as tools to better understand the transient features of allosteric signaling in the Gα subunit. The observed differences in relative receptor affinity for intact Gαi1 subunits versus C-terminal Gαi1 peptides harboring the K345L mutation are consistent with this mutation modulating the allosteric network in the protein subunit. Measurement of nucleotide exchange rates, affinity for metarhodopsin II, and thermostability suggest that the K345L Gαi1 variant has reduced stability in both the GDP-bound and nucleotide-free states as compared with wild type but similar stability in the GTPγS-bound state. High resolution x-ray crystal structures reveal conformational changes accompanying the destabilization of the GDP-bound state. Of these, the conformation for Switch I was stabilized by an ionic interaction with the phosphate binding loop. Further site-directed mutagenesis suggests that this interaction between Switch I and the phosphate binding loop is important for receptor-mediated nucleotide exchange in the wild-type Gαi1 subunit.

G protein βγ subunits play essential roles in regulating cellular signaling cascades, yet little is known about their distribution in tissues or their subcellular localization. While previous studies have suggested specific isoforms may exhibit a wide range of distributions throughout the central nervous system, a thorough investigation of the expression patterns of both Gβ and Gγ isoforms within subcellular fractions has not been conducted. To address this, we applied a targeted proteomics approach known as multiple-reaction monitoring to analyze localization patterns of Gβ and Gγ isoforms in pre- and postsynaptic fractions isolated from cortex, cerebellum, hippocampus, and striatum. Particular Gβ and Gγ subunits were found to exhibit distinct regional and subcellular localization patterns throughout the brain. Significant differences in subcellular localization between pre- and postsynaptic fractions were observed within the striatum for most Gβ and Gγ isoforms, while others exhibited completely unique expression patterns in all four brain regions examined. Such differences are a prerequisite for understanding roles of individual subunits in regulating specific signaling pathways throughout the central nervous system.

G(i/o)-protein-coupled receptors (GPCRs) ubiquitously inhibit neurotransmission, principally via Gβγ, which acts via a number of possible effectors. GPCR effector specificity has traditionally been attributed to Gα, based on Gα's preferential effector targeting in vitro compared with Gβγ's promiscuous targeting of various effectors. In synapses, however, Gβγ clearly targets unique effectors in a receptor-dependent way to modulate synaptic transmission. It remains unknown whether Gβγ specificity in vivo is due to specific Gβγ isoform-receptor associations or to spatial separation of distinct Gβγ pathways through macromolecular interactions. We thus sought to determine how Gβγ signaling pathways within axons remain distinct from one another. In rat hippocampal CA1 axons, GABA(B) receptors (GABA(B)Rs) inhibit presynaptic Ca(2+) entry, and we have now demonstrated that 5-HT(1B) receptors (5-HT(1B)Rs) liberate Gβγ to interact with SNARE complex C terminals with no effect on Ca(2+) entry. Both GABA(B)Rs and 5-HT(1B)Rs inhibit Ca(2+)-evoked neurotransmitter release, but 5-HT(1B)Rs have no effect on Sr(2+)-evoked release. Sr(2+), unlike Ca(2+), does not cause synaptotagmin to compete with Gβγ binding to SNARE complexes. 5-HT(1B)Rs also fail to inhibit release following cleavage of the C terminus of the SNARE complex protein SNAP-25 with botulinum A toxin. Thus, GABA(B)Rs and 5-HT(1B)Rs both localize to presynaptic terminals, but target distinct effectors. We demonstrate that disruption of SNARE complexes and vesicle priming with botulinum C toxin eliminates this selectivity, allowing 5-HT(1B)R inhibition of Ca(2+) entry. We conclude that receptor-effector specificity requires a microarchitecture provided by the SNARE complex during vesicle priming.

We present a model of interaction of Gi protein with the activated receptor (R*) rhodopsin, which pinpoints energetic contributions to activation and reconciles the β2 adrenergic receptor-Gs crystal structure with new and previously published experimental data. In silico analysis demonstrated energetic changes when the Gα C-terminal helix (α5) interacts with the R* cytoplasmic pocket, thus leading to displacement of the helical domain and GDP release. The model features a less dramatic domain opening compared with the crystal structure. The α5 helix undergoes a 63° rotation, accompanied by a 5.7-Å translation, that reorganizes interfaces between α5 and α1 helices and between α5 and β6-α5. Changes in the β6-α5 loop displace αG. All of these movements lead to opening of the GDP-binding pocket. The model creates a roadmap for experimental studies of receptor-mediated G-protein activation.

G-protein coupled receptors catalyze nucleotide exchange on G proteins, which results in subunit dissociation and effector activation. In the recent β2AR-Gs structure, portions of Switch I and II of Gα are not fully elucidated. We paired fluorescence studies of receptor-Gαi interactions with the β2AR-Gs and other Gi structures to investigate changes in Switch I and II during receptor activation and GTP binding. The β2/β3 loop containing Leu194 of Gαi is located between Switches I and II, in close proximity to IC2 of the receptor and the C-terminus of Gα, thus providing an allosteric connection between these Switches and receptor activation. We compared the environment of residues in myristoylated Gαi proteins in the heterotrimer to that upon receptor activation and subsequent GTP binding. Upon receptor activation, residues in both Switch regions are less solvent-exposed, as compared to the heterotrimer. Upon GTPγS binding, the environment of several residues in Switch I resemble the receptor-bound state, while Switch II residues display effects on their environment which are consistent with their role in GTP binding and Gβγ dissociation. The ability to merge available crystal structures with solution studies is a powerful tool to gain insight into conformational changes associated with receptor-mediated Gi protein activation., (Copyright © 2013 Elsevier Inc. All rights reserved.)