Matthew T. Duvernay

Anterograde Trafficking of G Protein-Coupled Receptors: Function of the C-Terminal F(X)6LL Motif in Export from the Endoplasmic Reticulum

We have reported previously that the F(X)6LL motif in the C termini is essential for export of α2B-adrenergic (α2B-AR) and angiotensin II type 1 receptors (AT1Rs) from the endoplasmic reticulum (ER). Here, we further demonstrate that mutation of the F(X)6LL motif similarly abolished the cell-surface expression of α2B-AR, AT1R, α1B-AR, and β2-AR, suggesting that the F(X)6LL motif plays a general role in ER export of G protein-coupled receptors (GPCRs). Mutation of Phe to Val, Leu, Trp, and Tyr, and mutation of LL to FF and VV, markedly inhibited α2B-AR transport, indicating that the F(X)6LL function cannot be fully substituted by other hydrophobic residues. The structural analysis revealed that the Phe residue in the F(X)6LL motif is buried in the transmembrane domains and possibly interacts with Ile58 in β2-AR and Val42 in α2B-AR, whereas the LL motif is exposed to the cytosolic space. Indeed, mutation of Ile58 in β2-AR and Val42 in α2B-AR markedly disrupted cell surface transport of the receptors. It is noteworthy that the Val and Ile residues are highly conserved among the GPCRs carrying the F(X)6LL motif. Furthermore, the Phe mutant exhibited a stronger interaction with ER chaperones and was more potently rescued by physical and chemical treatments than the LL mutant. These data suggest that the Phe residue is probably involved in folding of α2B-AR and β2-AR, possibly through interaction with other hydrophobic residues in neighboring domains. These data also provide the first evidence implying crucial roles of the C termini possibly through modulating multiple events in anterograde trafficking of GPCRs.

A Single Conserved Leucine Residue on the First Intracellular Loop Regulates ER Export of G Protein‐Coupled Receptors

The intrinsic structural determinants for export trafficking of G protein‐coupled receptors (GPCRs) have been mainly identified in the termini of the receptors. In this report, we determined the role of the first intracellular loop (ICL1) in the transport from the endoplasmic reticulum (ER) to the cell surface of GPCRs. The α2B‐adrenergic receptor (AR) mutant lacking the ICL1 is unable to traffic to the cell surface and to initiate signaling measured as ERK1/2 activation. Mutagenesis studies identify a single Leu48 residue in the ICL1 modulates α2B‐AR export from the ER. The ER export function of the Leu48 residue can be substituted by Phe, but not Ile, Val, Tyr and Trp, and is unlikely involved in correct folding or dimerization of α2B‐AR in the ER. Importantly, the isolated Leu residue is remarkably conserved in the center of the ICL1s among the family A GPCRs and is also required for the export to the cell surface of β2‐AR, α1B‐AR and angiotensin II type 1 receptor. These data indicate a crucial role for a single Leu residue within the ICL1 in ER export of GPCRs.

Protease-activated Receptor (PAR) 1 and PAR4 Differentially Regulate Factor V Expression from Human Platelets

With the recent interest of protease-activated receptors (PAR) 1 and PAR4 as possible targets for the treatment of thrombotic disorders, we compared the efficacy of protease-activated receptor (PAR)1 and PAR4 in the generation of procoagulant phenotypes on platelet membranes. PAR4-activating peptide (AP)–stimulated platelets promoted thrombin generation in plasma up to 5 minutes earlier than PAR1-AP–stimulated platelets. PAR4-AP–mediated factor V (FV) association with the platelet surface was 1.6-fold greater than for PAR1-AP. Moreover, PAR4 stimulation resulted in a 3-fold greater release of microparticles, compared with PAR1 stimulation. More robust FV secretion and microparticle generation with PAR4-AP was attributable to stronger and more sustained phosphorylation of myosin light chain at serine 19 and threonine 18. Inhibition of Rho-kinase reduced PAR4-AP–mediated FV secretion and microparticle generation to PAR1-AP–mediated levels. Thrombin generation assays measuring prothrombinase complex activity demonstrated 1.5-fold higher peak thrombin levels on PAR4-AP–stimulated platelets, compared with PAR1-AP–stimulated platelets. Rho-kinase inhibition reduced PAR4-AP–mediated peak thrombin generation by 25% but had no significant effect on PAR1-AP–mediated thrombin generation. In conclusion, stimulation of PAR4 on platelets leads to faster and more robust thrombin generation, compared with PAR1 stimulation. The greater procoagulant potential is related to more efficient FV release from intracellular stores and microparticle production driven by stronger and more sustained myosin light chain phosphorylation. These data have implications about the role of PAR4 during hemostasis and are clinically relevant in light of recent efforts to develop PAR antagonists to treat thrombotic disorders.

ADP-ribosylation factors modulate the cell surface transport of G protein-coupled receptors

ADP-ribosylation factors (ARFs) regulate vesicular traffic through recruiting coat proteins. However, their functions in the anterograde transport of nascent G protein-coupled receptors (GPCRs) from the endoplasmic reticulum to the plasma membrane remain poorly explored. Here we show that treatment with brefeldin A, an inhibitor of guanine nucleotide exchange on ARFs, markedly attenuated the cell surface numbers of α2B-adrenergic receptor (AR), β2-AR, angiotensin II type 1 receptor, and chemokine (CXC motif) receptor 4. Functional inhibition of individual ARF GTPases by transient expression of the GDP-bound, GTP-bound, and guanine nucleotide-deficient mutants showed that the five human ARFs differentially modulated receptor cell surface expression and that the ARF1 mutants produced the most profound inhibitory effect. Furthermore, expression of the ARF1 GTPase-activating protein (GAP) ARFGAP1 significantly blocked receptor transport. Interestingly, the GDP- and GTP-bound ARF1 mutants arrested the receptors in distinct intracellular compartments. Consistent with the reduced receptor cell surface expression, extracellular signal-regulated kinase 1 and 2 activation by receptor agonists was significantly attenuated by the GDP-bound mutant ARF1T31N. Moreover, coimmunoprecipitation showed that α2B-AR associated with ARF1 and glutathione transferase pull-down assay indicated that the α2B-AR C terminus directly interacted with ARF1. These data show that ARF1 GTPase is involved in the regulation of cell surface expression of GPCRs at multiple transport steps.

α2B-Adrenergic Receptor Interaction with Tubulin Controls Its Transport from the Endoplasmic Reticulum to the Cell Surface

It is well recognized that the C terminus (CT) plays a crucial role in modulating G protein-coupled receptor (GPCR) transport from the endoplasmic reticulum (ER) to the cell surface. However the molecular mechanisms that govern CT-dependent ER export remain elusive. To address this issue, we used α2B-adrenergic receptor (α2B-AR) as a model GPCR to search for proteins interacting with the CT. By using peptide-conjugated affinity matrix combined with proteomics and glutathione S-transferase fusion protein pull-down assays, we identified tubulin directly interacting with the α2B-AR CT. The interaction domains were mapped to the acidic CT of tubulin and the basic Arg residues in the α2B-AR CT, particularly Arg-437, Arg-441, and Arg-446. More importantly, mutation of these Arg residues to disrupt tubulin interaction markedly inhibited α2B-AR transport to the cell surface and strongly arrested the receptor in the ER. These data provide the first evidence indicating that the α2B-AR C-terminal Arg cluster mediates its association with tubulin to coordinate its ER-to-cell surface traffic and suggest a novel mechanism of GPCR export through physical contact with microtubules.

Synthesis of Indole Derived Protease-Activated Receptor 4 Antagonists and Characterization in Human Platelets

Protease activated receptor-4 (PAR4) is one of the thrombin receptors on human platelets and is a potential target for the management of thrombotic disorders. We sought to develop potent, selective, and novel PAR4 antagonists to test the role of PAR4 in thrombosis and hemostasis. Development of an expedient three-step synthetic route to access a novel series of indole-based PAR4 antagonists also necessitated the development of a platelet based high-throughput screening assay. Screening and subsequent structure activity relationship analysis yielded several selective PAR4 antagonists as well as possible new scaffolds for future antagonist development.

Mathematical model of PAR1-mediated activation of human platelets

Thrombin, one of the major proteases in the coagulation cascade, activates protease activated receptors 1 and 4 (PAR 1 and PAR4) to generate a network of intracellular signals that lead to stable platelet aggregation. Abnormal platelet activation could lead to either thrombosis or bleeding disorders, thus a predictive model of platelet activation would be an invaluable tool for the study of platelet function. In this work, we developed a computational model of PAR1-stimulated human platelet activation fully based on experimental observations. The model is represented by a system of ordinary differential equations (ODEs) describing the kinetics of the interacting components. The model is able to reproduce experimental dose responses and time-courses of cytosolic calcium (Ca(2+)), phosphatidylinositol 4,5-bisphosphate (PIP2), diacylglycerol (DAG), GTP-bound Ras-proximate-1 (Rap1GTP), secretion of dense-granules, and activation of integrin α2bβ3 (GPIIbIIIa). Because of the inherent complexity of such a model, we also provide a simple way to identify and divide the system into interlinked functional modules to reduce the number of unknown parameters. Both the full and the reduced kinetic models are shown to predict platelet behavior in response to PAR1 activation.

Substituted indoles as selective protease activated receptor 4 (PAR-4) antagonists: Discovery and SAR of ML354

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).

Supraclavicular nerve entrapment and clavicular fracture.

Because the supraclavicular nerve lies in close proximity to the clavicle, it is particularly vulnerable to injury in cases of clavicle fracture and in the surgical treatment of these fractures. The development of painful neuromas after iatrogenic transsection and symptomatic nerve entrapment in fracture callus after healing have previously been described. Reported here is a case of acute supraclavicular nerve entrapment and tension after fracture of the clavicle with significant pain relief after fracture fixation and nerve decompression.

Platelet Lipidomic Profiling: Novel Insight into Cytosolic Phospholipase A2α Activity and Its Role in Human Platelet Activation.

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.