Elevated protease-activated receptor 4 (PAR4) gene expression in Alzheimer's disease predicts cognitive decline.

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).

Discovery of Protease-Activated Receptor 4 (PAR4)-Tethered Ligand Antagonists Using Ultralarge Virtual Screening.

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.

Role of protease-activated receptor 4 in mouse models of acute and chronic kidney injury.

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.

Gβγ-SNAP25 exocytotic brake removal enhances insulin action, promotes adipocyte browning, and protects against diet-induced obesity.

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.

SNAP25 differentially contributes to G-coupled receptor function at glutamatergic synapses in the nucleus accumbens.

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-coupled G-protein-coupled receptors (GPCRs) at glutamatergic synapses onto medium spiny projection neurons (MSNs). Previous work has shown that discrete classes of G-coupled GPCR mobilize Gβγ to inhibit vesicular neurotransmitter release via t-SNARE protein, SNAP25.

Pepperberg plot: Modeling flash response saturation in retinal rods of mouse.

Retinal rods evolved to be able to detect single photons. Despite their exquisite sensitivity, rods operate over many log units of light intensity. Several processes inside photoreceptor cells make this incredible light adaptation possible.

Effects of cell size and bicarbonate on single photon response variability in retinal rods.

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.

Presynaptic mechanisms underlying GABA-receptor-mediated inhibition of spontaneous neurotransmitter release.

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 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 receptors.

Specificities of Gβγ subunits for the SNARE complex before and after stimulation of α-adrenergic receptors.

Ligand binding to G protein–coupled receptors (GPCRs), such as the α-adrenergic receptor (αAR), results in the activation of heterotrimeric G proteins, which consist of functionally distinct Gα subunits and Gβγ dimers. αAR-dependent inhibition of synaptic transmission regulates functions such as spontaneous locomotor activity, anesthetic sparing, and working memory enhancement and requires the soluble attachment protein receptor (SNARE) complex, a Gβγ effector. To understand how the Gβγ-SNARE complex underlies the αAR-dependent inhibition of synaptic transmission, we examined the specificity of Gβγ subunits for the SNARE complex in adrenergic neurons, in which auto-αARs respond to epinephrine released from these neurons, and nonadrenergic neurons, in which hetero-αARs respond to epinephrine released from other neurons.

Phototransduction in retinal cones: Analysis of parameter importance.

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.

Position of rhodopsin photoisomerization on the disk surface confers variability to the rising phase of the single photon response in vertebrate rod photoreceptors.

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.

Sexual Dimorphism in Stress-induced Hyperthermia in SNAP25Δ3 mice, a mouse model with disabled Gβγ regulation of the exocytotic fusion apparatus.

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.

Author Correction: The in vivo specificity of synaptic Gβ and Gγ subunits to the α adrenergic receptor at CNS synapses.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Heterosynaptic GABA Receptor Function within Feedforward Microcircuits Gates Glutamatergic Transmission in the Nucleus Accumbens Core.

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).

Disabling Gβγ-SNAP-25 interaction in gene-targeted mice results in enhancement of long-term potentiation at Schaffer collateral-CA1 synapses in the hippocampus.

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.

Disabling the Gβγ-SNARE interaction disrupts GPCR-mediated presynaptic inhibition, leading to physiological and behavioral phenotypes.

G protein-coupled receptors (GPCRs) that couple to G proteins modulate neurotransmission presynaptically by inhibiting exocytosis. Release of Gβγ subunits from activated G proteins decreases the activity of voltage-gated Ca channels (VGCCs), decreasing excitability. A less understood Gβγ-mediated mechanism downstream of Ca entry is the binding of Gβγ to SNARE complexes, which facilitate the fusion of vesicles with the cell plasma membrane in exocytosis.

The in vivo specificity of synaptic Gβ and Gγ subunits to the α adrenergic receptor at CNS synapses.

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.

The expanding roles and mechanisms of G protein-mediated presynaptic inhibition.

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 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.

Protease-activated receptor 4 activity promotes platelet granule release and platelet-leukocyte interactions.

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.

The role of coagulation and platelets in colon cancer-associated thrombosis.

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.

GHSR-D2R heteromerization modulates dopamine signaling through an effect on G protein conformation.

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.

G Protein Preassembly Rescues Efficacy of W Toggle Mutations in Neuropeptide Y Receptor.

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 YR.

Improved Folding of the Y G Protein-Coupled Receptor into Bicelles.

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 expression, functional refolding, and reconstitution into bicelles of the human neuropeptide Y receptor type 2 (YR) for solution and solid-state NMR experiments. The isotopically labeled receptor is expressed in inclusion bodies and purified using SDS.