Pepducin-based intervention of thrombin-receptor signaling and systemic platelet activation

Transmembrane signaling through G protein-coupled receptors (GPCRs) controls a diverse array of cellular processes including metabolism, growth, motility, adhesion, neuronal signaling and blood coagulation. The numerous GPCRs and their key roles in both normal physiology and disease have made them the target for more than 50% of all prescribed drugs. GPCR agonists and antagonists act on the extracellular side of the receptors, whereas the intracellular surface has not yet been exploited for development of new therapeutic agents. Here, we demonstrate the utility of novel cell-penetrating peptides, termed 'pepducins', that act as intracellular inhibitors of signal transference from receptors to G proteins. Attachment of a palmitate lipid to peptides based on the third intracellular loop of protease-activated receptor 1 (PAR1) or PAR4 (refs. 3-5) yielded potent inhibitors of thrombin-mediated aggregation of human platelets. Infusion of the anti-PAR4 pepducin into mice extended bleeding time and protected against systemic platelet activation, consistent with the phenotype of PAR4-deficient mice. We show that pepducins might be used to ascertain the physiological roles of GPCRs and rapidly determine the potential therapeutic value of blockade of a particular signaling pathway.     

Allosteric Activation of a G Protein-coupled Receptor with Cell-penetrating Receptor Mimetics

G protein-coupled receptors (GPCRs) are remarkably versatile signaling systems that are activated by a large number of different agonists on the outside of the cell. However, the inside surface of the receptors that couple to G proteins has not yet been effectively modulated for activity or treatment of diseases. Pepducins are cell-penetrating lipopeptides that have enabled chemical and physical access to the intracellular face of GPCRs. The structure of a third intracellular (i3) loop agonist, pepducin, based on protease-activated receptor-1 (PAR1) was solved by NMR and found to closely resemble the i3 loop structure predicted for the intact receptor in the on-state. Mechanistic studies revealed that the pepducin directly interacts with the intracellular H8 helix region of PAR1 and allosterically activates the receptor through the adjacent (D/N)PXXYYY motif through a dimer-like mechanism. The i3 pepducin enhances PAR1/Gα subunit interactions and induces a conformational change in fluorescently labeled PAR1 in a very similar manner to that induced by thrombin. As pepducins can potentially be made to target any GPCR, these data provide insight into the identification of allosteric modulators to this major drug target class.     

Turning receptors on and off with intracellular pepducins: new insights into G-protein-coupled receptor drug development

G-protein-coupled receptors (GPCRs) are a large family of remarkably versatile membrane proteins that are attractive therapeutic targets because of their involvement in a vast range of normal physiological processes and pathological diseases. Upon activation, intracellular domains of GPCRs mediate signaling to G-proteins, but these domains have yet to be effectively exploited as drug targets. Cell-penetrating lipidated peptides called pepducins target specific intracellular loops of GPCRs and have recently emerged as effective allosteric modulators of GPCR activity. The lipid moiety facilitates translocation across the plasma membrane, where pepducins then specifically modulate signaling of their cognate receptor. To date, pepducins and related lipopeptides have been shown to specifically modulate the activity of diverse GPCRs and other membrane proteins, including protease-activated receptors (PAR1, PAR2, and PAR4), chemokine receptors (CXCR1, CXCR2, and CXCR4), sphingosine 1-phosphate receptor-3 (S1P3), the melanocortin-4 receptor, the Smoothened receptor, formyl peptide receptor-2 (FPR2), the relaxin receptor (LGR7), G-proteins (Gα(q/11/o/13)), muscarinic acetylcholine receptor and vanilloid (TRPV1) channels, and the GPIIb integrin. This minireview describes recent advances made using pepducin technology in targeting diverse GPCRs and the use of pepducins in identifying potential novel drug targets.     

Two cytoplasmic loops of the glucagon receptor are required to elevate cAMP or intracellular calcium.

The glucagon receptor is a member of a distinct class of G protein-coupled receptors (GPCRs) sharing little amino acid sequence homology with the larger rhodopsin-like GPCR family. To identify the components of the glucagon receptor necessary for G-protein coupling, we replaced sequentially all or part of each intracellular loop (i1, i2, and i3) and the C-terminal tail of the glucagon receptor with the 11 amino acids comprising the first intracellular loop of the D4 dopamine receptor. When expressed in transiently transfected COS-1 cells, the mutant receptors fell into two different groups with respect to hormone-mediated signaling. The first group included the loop i1 mutants, which bound glucagon and signaled normally. The second group comprised the loop i2 and i3 chimeras, which caused no detectable adenylyl cyclase activation in COS-1 cells. However, when expressed in HEK 293T cells, the loop i2 or i3 chimeras caused very small glucagon-mediated increases in cAMP levels and intracellular calcium concentrations, with EC50 values nearly 100-fold higher than those measured for wild-type receptor. Replacement of both loops i2 and i3 simultaneously was required to completely abolish G protein signaling as measured by both cAMP accumulation and calcium flux assays. These results show that the i2 and i3 loops play a role in glucagon receptor signaling, consistent with recent models for the mechanism of activation of G proteins by rhodopsin-like GPCRs.     

Modifying ligand-induced and constitutive signaling of the human 5-HT4 receptor

G protein-coupled receptors (GPCRs) signal through a limited number of G-protein pathways and play crucial roles in many biological processes. Studies of their in vivo functions have been hampered by the molecular and functional diversity of GPCRs and the paucity of ligands with specific signaling effects. To better compare the effects of activating different G-protein signaling pathways through ligand-induced or constitutive signaling, we developed a new series of RASSLs (receptors activated solely by synthetic ligands) that activate different G-protein signaling pathways. These RASSLs are based on the human 5-HT(4b) receptor, a GPCR with high constitutive G(s) signaling and strong ligand-induced G-protein activation of the G(s) and G(s/q) pathways. The first receptor in this series, 5-HT(4)-D(100)A or Rs1 (RASSL serotonin 1), is not activated by its endogenous agonist, serotonin, but is selectively activated by the small synthetic molecules GR113808, GR125487, and RO110-0235. All agonists potently induced G(s) signaling, but only a few (e.g., zacopride) also induced signaling via the G(q) pathway. Zacopride-induced G(q) signaling was enhanced by replacing the C-terminus of Rs1 with the C-terminus of the human 5-HT(2C) receptor. Additional point mutations (D(66)A and D(66)N) blocked constitutive G(s) signaling and lowered ligand-induced G(q) signaling. Replacing the third intracellular loop of Rs1 with that of human 5-HT(1A) conferred ligand-mediated G(i) signaling. This G(i)-coupled RASSL, Rs1.3, exhibited no measurable signaling to the G(s) or G(q) pathway. These findings show that the signaling repertoire of Rs1 can be expanded and controlled by receptor engineering and drug selection.     

Aspartic acid 564 in the third cytoplasmic loop of the luteinizing hormone/choriogonadotropin receptor is crucial for phosphorylation-independent interaction with arrestin2

Arrestin2 binding to the active but unphosphorylated luteinizing hormone/choriogonadotropin receptor (LH/CG R) in ovarian follicles is triggered by activation of ADP-ribosylation factor 6 (ARF6) and leads to uncoupling of this receptor from cAMP signaling. We sought to determine how arrestin2 binds to LH/CG R, if binding is of high affinity, and if the receptor also binds arrestin3. Desensitization of intact LH/CG R was equally sensitive to ectopic constructs of arrestin2 that bind other G protein-coupled receptors (GPCRs) either in a phosphorylation-independent or -dependent manner. Intact LH/CG R was not desensitized by ectopic arrestin3 constructs. Surface plasmon resonance studies showed that arrestin2 bound a synthetic third intracellular (3i) LH/CG R loop peptide with picomolar affinity; arrestin3 bound with millimolar affinity. To determine whether Asp-564 in the 3i loop mimicked the phosphorylated residue of other GPCRs, human embryonic kidney (HEK) cells were transfected with wild-type (WT) and D564G LH/CG R. An agonist-stimulated ARF6-dependent arrestin2 undocking pathway to drive desensitization of WT receptor was recapitulated in HEK cell membranes, and ectopic arrestin2 promoted desensitization of WT LH/CG R. However, D564G LH/CG R in HEK cells was not desensitized, and synthetic 3i D564G peptide did not bind arrestin2. Synthetic 3i loop peptides containing D564E, D564V, or D564N also did not bind arrestin2. We conclude that the ARF6-mediated mechanism to release a pool of membrane-delimited arrestin to bind GPCRs may be a widespread mechanism to deliver arrestin to GPCRs for receptor desensitization. Unlike other GPCRs that additionally require receptor phosphorylation, LH/CG R activation is sufficient to expose a conformation in which Asp-564 in the 3i loop confers high affinity binding selectively to arrestin2.     

Multiple functions of G protein-coupled receptor kinases

Desensitization is a physiological feedback mechanism that blocks detrimental effects of persistent stimulation. G protein-coupled receptor kinase 2 (GRK2) was originally identified as the kinase that mediates G protein-coupled receptor (GPCR) desensitization. Subsequent studies revealed that GRK is a family composed of seven isoforms (GRK1–GRK7). Each GRK shows a differential expression pattern. GRK1, GRK4, and GRK7 are expressed in limited tissues. In contrast, GRK2, GRK3, GRK5, and GRK6 are ubiquitously expressed throughout the body. The roles of GRKs in GPCR desensitization are well established. When GPCRs are activated by their agonists, GRKs phosphorylate serine/threonine residues in the intracellular loops and the carboxyl-termini of GPCRs. Phosphorylation promotes translocation of β-arrestins to the receptors and inhibits further G protein activation by interrupting receptor-G protein coupling. The binding of β-arrestins to the receptors also helps to promote receptor internalization by clathrin-coated pits. Thus, the GRK-catalyzed phosphorylation and subsequent binding of β-arrestin to GPCRs are believed to be the common mechanism of GPCR desensitization and internalization. Recent studies have revealed that GRKs are also involved in the β-arrestin-mediated signaling pathway. The GRK-mediated phosphorylation of the receptors plays opposite roles in conventional G protein- and β-arrestin-mediated signaling. The GRK-catalyzed phosphorylation of the receptors results in decreased G protein-mediated signaling, but it is necessary for β-arrestin-mediated signaling. Agonists that selectively activate GRK/β-arrestin-dependent signaling without affecting G protein signaling are known as β-arrestin-biased agonists. Biased agonists are expected to have potential therapeutic benefits for various diseases due to their selective activation of favorable physiological responses or avoidance of the side effects of drugs. Furthermore, GRKs are recognized as signaling mediators that are independent of either G protein- or β-arrestin-mediated pathways. GRKs can phosphorylate non-GPCR substrates, and this is found to be involved in various physiological responses, such as cell motility, development, and inflammation. In addition to these effects, our group revealed that GRK6 expressed in macrophages mediates the removal of apoptotic cells (engulfment) in a kinase activity-dependent manner. These studies revealed that GRKs block excess stimulus and also induce cellular responses. Here, we summarized the involvement of GRKs in β-arrestin-mediated and G protein-independent signaling pathways.     

Analysis of proteinase-activated receptor 2 and TLR4 signal transduction: a novel paradigm for receptor cooperativity

Proteinase-activated receptor 2 (PAR(2)), a seven-transmembrane G protein-coupled receptor, is activated at inflammatory sites by proteolytic cleavage of its extracellular N terminus by trypsin-like enzymes, exposing a tethered, receptor-activating ligand. Synthetic agonist peptides (AP) that share the tethered ligand sequence also activate PAR(2), often measured by Ca(2+) release. PAR(2) contributes to inflammation through activation of NF-kappaB-regulated genes; however, the mechanism by which this occurs is unknown. Overexpression of human PAR(2) in HEK293T cells resulted in concentration-dependent, PAR(2) AP-inducible NF-kappaB reporter activation that was protein synthesis-independent, yet blocked by inhibitors that uncouple G(i) proteins or sequester intracellular Ca(2+). Because previous studies described synergistic PAR(2)- and TLR4-mediated cytokine production, we hypothesized that PAR(2) and TLR4 might interact at the level of signaling. In the absence of TLR4, PAR(2)-induced NF-kappaB activity was inhibited by dominant negative (DN)-TRIF or DN-TRAM constructs, but not by DN-MyD88, findings confirmed using cell-permeable, adapter-specific BB loop blocking peptides. Co-expression of TLR4/MD-2/CD14 with PAR(2) in HEK293T cells led to a synergistic increase in AP-induced NF-kappaB signaling that was MyD88-dependent and required a functional TLR4, despite the fact that AP exhibited no TLR4 agonist activity. Co-immunoprecipitation of PAR(2) and TLR4 revealed a physical association that was AP-dependent. The response to AP or lipopolysaccharide was significantly diminished in TLR4(-/-) and PAR (-/-)(2) macrophages, respectively, and SW620 colonic epithelial cells exhibited synergistic responses to co-stimulation with AP and lipopolysaccharide. Our data suggest a unique interaction between two distinct innate immune response receptors and support a novel paradigm of receptor cooperativity in inflammatory responses.     

The carboxyl tail of protease-activated receptor-1 is required for chemotaxis. Correlation of signal termination and directional migration.

The G protein-coupled thrombin receptor, protease-activated receptor 1 (PAR1), mediates many of the actions of thrombin on cells including chemotaxis. In contrast to the reversible agonist binding that regulates signaling by most G protein-coupled receptors (GPCRs), PAR1 is activated by an irreversible proteolytic mechanism. Although activated PAR1 is phosphorylated, uncoupled, and internalized like typical GPCRs, signal termination is additionally dependent on lysosomal degradation of cleaved and activated receptors. In the present study we exploit two PAR1 mutants to examine the link between chemotaxis and receptor shutoff. One, a carboxyl tail deletion mutant (Y397Z), is defective in phosphorylation and internalization. The other, a carboxyl tail chimeric receptor (P/S), is phosphorylated and internalized upon activation but recycles to the plasma membrane like reversibly activated GPCRs. Expression of these receptors in a hematopoietic cell line disrupted cell migration along thrombin gradients. Thrombin activation of cells expressing P/S or Y397Z resulted in persistent signaling independent of the continued presence of thrombin. Signaling in response to the soluble agonist peptide SFLLRN was reversible for P/S but persisted for Y397Z. Strikingly, cells expressing P/S responded chemokinetically to thrombin but chemotactically to SFLLRN. In contrast, Y397Z-mediated migration was largely chemokinetic to both agonists. These studies suggest that termination of PAR1 signaling at the level of the receptor is necessary for gradient detection and directional migration.     

Reversing systemic inflammatory response syndrome with chemokine receptor pepducins

We describe a new therapeutic approach for the treatment of lethal sepsis using cell-penetrating lipopeptides-termed pepducins-that target either individual or multiple chemokine receptors. Interleukin-8 (IL-8), a ligand for the CXCR1 and CXCR2 receptors, is the most potent endogenous proinflammatory chemokine in sepsis. IL-8 levels rise in blood and lung fluids to activate neutrophils and other cells, and correlate with shock, lung injury and high mortality. We show that pepducins derived from either the i1 or i3 intracellular loops of CXCR1 and CXCR2 prevent the IL-8 response of both receptors and reverse the lethal sequelae of sepsis, including disseminated intravascular coagulation and multi-organ failure in mice. Conversely, pepducins selective for CXCR4 cause a massive leukocytosis that does not affect survival. CXCR1 and CXCR2 pepducins conferred nearly 100% survival even when treatment was postponed, suggesting that our approach might be beneficial in the setting of advanced disease.     

Novel roles for arrestins in the post-endocytic trafficking of G protein-coupled receptors

G protein-coupled receptors (GPCRs) represent the largest family of transmembrane signaling molecules in the human genome. As such, they interact with numerous intracellular molecules, which can act either to propagate or curtail signaling from the receptor. Their primary mode of cellular activation occurs through heterotrimeric G proteins, which in turn can activate a wide spectrum of effector molecules, including phosphodiesterases, phospholipases, adenylyl cyclases and ion channels. Active GPCRs are also the target of G protein-coupled receptor kinases, which phosphorylate the receptors culminating in the binding of the protein arrestin. This results in rapid desensitization through inhibition of G protein binding, as well as novel mechanisms of cellular activation that involve the scaffolding of cellular kinases to GPCR-arrestin complexes. Arrestins can also serve to mediate the internalization of certain GPCRs, a process which plays an important role in regulating cellular activity both by mediating long-term desensitization through down regulation (degradation) of receptors and by recycling desensitized receptors back to the cell surface to initiate additional rounds of signaling. The mechanisms that regulate the subsequent intracellular trafficking of GPCRs following internalization are largely unknown. Recently however, it has become clear that the pattern of receptor phosphorylation and subsequent binding of arrestin play a critical role in the intracellular trafficking of internalized receptors, thereby dictating the ultimate fate of the receptor. In addition, arrestins have now been shown to be required for the recycling of GPCRs that are capable of internalizing through arrestin-independent mechanisms. This review will summarize recent advances in our understanding of the roles of arrestins in post-endocytic GPCR trafficking.     

Proteinase-activated receptors (PARs) – focus on receptor-receptor-interactions and their physiological and pathophysiological impact

Proteinase-activated receptors (PARs) are a subfamily of G protein-coupled receptors (GPCRs) with four members, PAR₁, PAR₂, PAR₃ and PAR₄, playing critical functions in hemostasis, thrombosis, embryonic development, wound healing, inflammation and cancer progression. PARs are characterized by a unique activation mechanism involving receptor cleavage by different proteinases at specific sites within the extracellular amino-terminus and the exposure of amino-terminal “tethered ligand“ domains that bind to and activate the cleaved receptors. After activation, the PAR family members are able to stimulate complex intracellular signalling networks via classical G protein-mediated pathways and beta-arrestin signalling. In addition, different receptor crosstalk mechanisms critically contribute to a high diversity of PAR signal transduction and receptor-trafficking processes that result in multiple physiological effects.In this review, we summarize current information about PAR-initiated physical and functional receptor interactions and their physiological and pathological roles. We focus especially on PAR homo- and heterodimerization, transactivation of receptor tyrosine kinases (RTKs) and receptor serine/threonine kinases (RSTKs), communication with other GPCRs, toll-like receptors and NOD-like receptors, ion channel receptors, and on PAR association with cargo receptors. In addition, we discuss the suitability of these receptor interaction mechanisms as targets for modulating PAR signalling in disease.     

Structure-function analysis of protease-activated receptor 4 tethered ligand peptides. Determinants of specificity and utility in assays of receptor function

Thrombin activates protease-activated receptors (PARs) by specific cleavage of their amino-terminal exodomains to unmask a tethered ligand that binds intramolecularly to the body of the receptor to effect transmembrane signaling. Peptides that mimic such ligands are valuable as agonists for probing PAR function, but the tethered ligand peptide for PAR4, GYPGKF, lacks potency and is of limited utility. In a structure-activity analysis of PAR4 peptides, AYPGKF was approximately 10-fold more potent than GYPGKF and, unlike GYPGKF, elicited PAR4-mediated responses comparable in magnitude to those elicited by thrombin. AYPGKF was relatively specific for PAR4 in part due to the tyrosine at position 2; substitution of phenylalanine or p-fluorophenylalanine at this position produced peptides that activated both PAR1 and PAR4. Because human platelets express both PAR1 and PAR4, it might be desirable to inhibit both receptors. Identifying a single agonist for both receptors raises the possibility that a single antagonist for both receptors might be developed. The AYPGKF peptide is a useful new tool for probing PAR4 function. For example, AYPGKF activated and desensitized PAR4 in platelets and, like thrombin, triggered phosphoinositide hydrolysis but not inhibition of adenylyl cyclase in PAR4-expressing cells. The latter shows that, unlike PAR1, PAR4 couples to G(q) and not G(i).     

C5a receptor oligomerization. I. Disulfide trapping reveals oligomers and potential contact surfaces in a G protein-coupled receptor

G protein-coupled receptors (GPCRs), stimulated by hormones and sensory stimuli, act as molecular switches to relay activation to heterotrimeric G proteins. Recent studies suggest that GPCRs form dimeric or oligomeric structures, a phenomenon that has long been established for growth factor receptors. The elucidation of the domains of GPCRs that mediate receptor association is of critical importance for understanding the function of GPCR oligomers. Using a disulfide-trapping strategy to probe the intermolecular contact surfaces, we demonstrate cross-linking of C5a receptors in membranes prepared from both human neutrophils and stably transfected mammalian cells that is mediated by a cysteine in the second intracellular loop. To explore other surfaces that might be involved in the oligomerization of C5a receptors, we constructed receptors with individual cysteines in other intracellular regions. C5a receptors with a cysteine in the first intracellular loop or the carboxyl terminus displayed the fastest kinetics of dimer formation, whereas an intracellular loop 3 cysteine displayed minimal cross-linking. Since the rate of disulfide trapping reflects the proximity of sulfhydryl groups, assuming similar accessibility and flexibility, these results imply a symmetric dimer interface that may involve either transmembrane helices 1 and 2 or helix 4. However, neither model can account for the ability of the native cysteine in the second intracellular loop to mediate efficient crosslinking. Based on these observations, we propose that C5a receptors form higher order oligomers (i.e. tetramers) or clusters in the membrane.     

Biased agonism of the calcium-sensing receptor

After the discovery of molecules modulating G protein-coupled receptors (GPCRs) that are able to selectively affect one signaling pathway over others for a specific GPCR, thereby "biasing" the signaling, it has become obvious that the original model of GPCRs existing in either an "on" or "off" conformation is too simple. The current explanation for this biased agonism is that GPCRs can adopt multiple active conformations stabilized by different molecules, and that each conformation affects intracellular signaling in a different way. In the present study we sought to investigate biased agonism of the calcium-sensing receptor (CaSR), by looking at 12 well-known orthosteric CaSR agonists in 3 different CaSR signaling pathways: G(q/11) protein, G(i/o) protein, and extracellular signal-regulated kinases 1 and 2 (ERK1/2). Here we show that apart from G(q/11) and G(i/o) signaling, ERK1/2 is activated through recruitment of β-arrestins. Next, by measuring activity of all three signaling pathways we found that barium, spermine, neomycin, and tobramycin act as biased agonist in terms of efficacy and/or potency. Finally, polyamines and aminoglycosides in general were biased in their potencies toward ERK1/2 signaling. In conclusion, the results of this study indicate that several active conformations of CaSR, stabilized by different molecules, exist, which affect intracellular signaling distinctly.     

Regulation of melanin-concentrating hormone receptor 1 signaling by RGS8 with the receptor third intracellular loop

Melanin-concentrating hormone (MCH) receptor 1 (MCH1R) belongs to the class A G protein-coupled receptors (GPCRs). The MCH-MCH1R system plays a central role in energy metabolism, and thus the regulation of signaling pathways activated by this receptor is of particular interest. Regulator of G protein signaling (RGS) proteins work by increasing the GTPase activity of G protein alpha subunits and attenuate cellular responses coupled with G proteins. Recent evidence has shown that RGS proteins are not simple G protein regulators but equally inhibit the signaling from various GPCRs. Here, we demonstrate that RGS8, which is highly expressed in the brain, functions as a negative modulator of MCH1R signaling. By using biochemical approaches, RGS8 was found to selectively and directly bind to the third intracellular (i3) loop of MCH1R in vitro. When expressed in HEK293T cells, RGS8 and MCH1R colocalized to the plasma membrane and RGS8 potently inhibited the calcium mobilization induced by MCH. The N-terminal 9 amino acids of RGS8 were required for the optimal capacity to downregulate the receptor signaling. Furthermore, Arg(253) and Arg(256) at the distal end of the i3 loop were found to comprise a structurally important site for the functional interaction with RGS8, since coexpression of RGS8 with R253Q/R256Q mutant receptors resulted in a loss of induction of MCH-stimulated calcium mobilization. This functional association suggests that RGS8 may represent a new therapeutic target for the development of novel pharmaceutical agents.     

Arrestin-2 differentially regulates PAR4 and ADP receptor signaling in platelets

Arrestins can facilitate desensitization or signaling by G protein-coupled receptors (GPCR) in many cells, but their roles in platelets remain uncharacterized. Because of recent reports that arrestins can serve as scaffolds to recruit phosphatidylinositol-3 kinases (PI3K)s to GPCRs, we sought to determine whether arrestins regulate PI3K-dependent Akt signaling in platelets, with consequences for thrombosis. Co-immunoprecipitation experiments demonstrate that arrestin-2 associates with p85 PI3Kα/β subunits in thrombin-stimulated platelets, but not resting cells. The association is inhibited by inhibitors of P2Y12 and Src family kinases (SFKs). The function of arrestin-2 in platelets is agonist-specific, as PAR4-dependent Akt phosphorylation and fibrinogen binding were reduced in arrestin-2 knock-out platelets compared with WT controls, but ADP-stimulated signaling to Akt and fibrinogen binding were unaffected. ADP receptors regulate arrestin recruitment to PAR4, because co-immunoprecipitates of arrestin-2 with PAR4 are disrupted by inhibitors of P2Y1 or P2Y12. P2Y1 may regulate arrestin-2 recruitment to PAR4 through protein kinase C (PKC) activation, whereas P2Y12 directly interacts with PAR4 and therefore, may help to recruit arrestin-2 to PAR4. Finally, arrestin2(-/-) mice are less sensitive to ferric chloride-induced thrombosis than WT mice, suggesting that arrestin-2 can regulate thrombus formation in vivo. In conclusion, arrestin-2 regulates PAR4-dependent signaling pathways, but not responses to ADP alone, and contributes to thrombus formation in vivo.     

Biased signaling: potential agonist and antagonist of PAR2

Protease activated receptor 2 (PAR2) has emerged as one of the promising therapeutic targets to inhibit rapidly metastasizing breast cancer cells. However, its elusive molecular mechanism of activation and signaling has made it a difficult target for drug development. In this study, in silico methods were used to unfold PAR2 molecular mechanism of signaling based on the concept of GPCR receptor plasticity. Although, there are no conclusive evidences of the presence of specific endogenous ligands for PAR2, the efficacy of synthetic agonist and antagonist in PAR2 signaling has opened up the possibilities of ligand-mediated signaling. Furthermore, it has been proved that ligands specific for one GPCR can induce signaling in GPCRs belonging to other subfamilies. Therefore, the aim of this study was to identify potential agonists and antagonists from the GPCR ligand library (GLL), which may induce biased signaling in PAR2 using the concept of existence of multiple ligand-stabilized receptor conformations. The results of our in silico study suggest that PAR2 may show biased signaling mainly with agonists of serotonin type 1, β-adrenergic type 1,3 and antagonists of substance K (NK1), serotonin type 2, dopamine type 4, and thromboxane receptors. Further, this study also throws light on the putative ligand-specific conformations of PAR2. Thus, the results of this study provide structural insights to putative conformations of PAR2 and also gives initial clues to medicinal chemists for rational drug design targeting this challenging receptor.     

Design and biological assessment of membrane-tethering neuroprotective peptides derived from the pituitary adenylate cyclase-activating polypeptide type 1 receptor

BackgroundThe pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 receptor (PAC1), a class B G protein-coupled receptor (GPCR), has emerged as a promising target for treating neurodegenerative conditions. Unfortunately, despite years of research, no PAC1-specific agonist has been discovered, as activity on two other GPCRs, VPAC1 and VPAC2, is retained with current analogs. Cell signaling is related to structural modifications in the intracellular loops (ICLs) of GPCRs. Thus, we hypothesized that peptides derived from the ICLs (called pepducins) of PAC1 might initiate, as allosteric ligands, signaling cascades after recognition of the parent receptor and modulation of its conformational landscape.MethodsThree pepducins were synthesized and evaluated for their ability to 1) promote cell survival; 2) stimulate various signaling pathways associated with PAC1 activation; 3) modulate selectively PAC1, VPAC1 or VPAC2 activation; and 4) sustain mobility and prevent death of dopaminergic neurons in a zebrafish model of neurodegeneration.ResultsAssays demonstrated that these molecules promote SH-SY5Y cell survival, a human neuroblastoma cell line expressing PAC1, and activate signaling via Gα_s and Gα_q, with distinct potencies and efficacies. Also, PAC1-Pep1 and PAC1-Pep2 activated selectively PAC1-mediated Gα_s stimulation. Finally, experiments, using a zebrafish neurodegeneration model, showed a neuroprotective action with all three pepducins and in particular, revealed the ability of PAC1-Pep1 and PAC1-Pep3 to preserve fish mobility and tyrosine hydroxylase expression in the brain.ConclusionWe have developed the first neuroprotective pepducins derived from PAC1, a class B GPCR.General significancePAC1-derived pepducins represent attractive templates for the development of innovative neuroprotecting molecules.     

Cathepsin G activates protease-activated receptor-4 in human platelets

Of the four known protease-activated receptors (PARs), PAR1 and PAR4 are expressed by human platelets and mediate thrombin signaling. Whether these receptors are redundant, interact, or play at least partially distinct roles is unknown. It is possible that PAR1 and/or PAR4 might confer responsiveness to proteases other than thrombin. The neutrophil granule protease, cathepsin G, is known to cause platelet secretion and aggregation. We now report that this action of cathepsin G is mediated by PAR4. Cathepsin G triggered calcium mobilization in PAR4-transfected fibroblasts, PAR4-expressing Xenopus oocytes, and washed human platelets. An antibody raised against the PAR4 thrombin cleavage site blocked platelet activation by cathepsin G but not other agonists. Desensitization with a PAR4 activating peptide had a similar effect. By contrast, inhibition of PAR1 function had no effect on platelet responses to cathepsin G. When neutrophils were present, the neutrophil agonist fMet-Leu-Phe triggered calcium signaling in Fura-2-loaded platelets. Strikingly, this neutrophil-dependent platelet activation was blocked by the PAR4 antibody. These data show that PAR4 mediates platelet responses to cathepsin G and support the hypothesis that cathepsin G might mediate neutrophil-platelet interactions at sites of vascular injury or inflammation.