PAR1 Agonist SFLLRN Research Articles

Lipid Receptor GPR31 (G-Protein-Coupled Receptor 31) Regulates Platelet Reactivity and Thrombosis Without Affecting Hemostasis.

12-LOX (12-lipoxygenase) produces a number of bioactive lipids including 12(S)-HETE that are involved in inflammation and platelet reactivity. The GPR31 (G-protein-coupled receptor 31) is the proposed receptor of 12(S)-HETE; however, it is not known whether the 12(S)-HETE-GPR31 signaling axis serves to enhance or inhibit platelet activity. Approach and Results: Using pepducin technology and biochemical approaches, we provide evidence that 12(S)-HETE-GPR31 signals through Gi to enhance PAR (protease-activated receptor)-4-mediated platelet activation and arterial thrombosis using both human platelets and mouse carotid artery injury models. 12(S)-HETE suppressed AC (adenylyl cyclase) activity through GPR31 and resulted in Rap1 (Ras-related protein 1) and p38 activation and low but detectable calcium flux but did not induce platelet aggregation. A GPR31 third intracellular (i3) loop-derived pepducin, GPR310 (G-protein-coupled receptor 310), significantly inhibited platelet aggregation in response to thrombin, collagen, and PAR4 agonist, AYPGKF, in human and mouse platelets but relative sparing of PAR1 agonist SFLLRN in human platelets. GPR310 treatment gave a highly significant 80% protection (P=0.0018) against ferric chloride-induced carotid artery injury in mice by extending occlusion time, without any effect on tail bleeding. PAR4-mediated dense granule secretion and calcium flux were both attenuated by GPR310. Consistent with these results, GPR310 inhibited 12(S)-HETE-mediated and PAR4-mediated Rap1-GTP and RASA3 translocation to the plasma membrane and attenuated PAR4-Akt and ERK activation. GPR310 caused a right shift in thrombin-mediated human platelet aggregation, comparable to the effects of inhibition of the Gi-coupled P2Y12 receptor. Co-immunoprecipitation studies revealed that GPR31 and PAR4 form a heterodimeric complex in recombinant systems. The 12-LOX product 12(S)-HETE stimulates GPR31-Gi-signaling pathways, which enhance thrombin-PAR4 platelet activation and arterial thrombosis in human platelets and mouse models. Suppression of this bioactive lipid pathway, as exemplified by a GPR31 pepducin antagonist, may provide beneficial protective effects against platelet aggregation and arterial thrombosis with minimal effect on hemostasis.

Abstract 6: Continuous Plasma S1p-dependent Signaling by Apically Polarized S1Pr1 Supports Endothelial Barrier Function

Sphingosine-1-phosphate, generated by sphingosine kinases (Sphk1 and 2), acts at its receptor S1Pr1 to maintain vascular homeostasis. The gradient of S1P concentration (low micromolar in plasma vs. low nanomolar in tissue) suggests two non-mutually exclusive models via which S1P maintains the endothelial barrier: 1) plasma S1P communicates continuously with the endothelium to maintain vascular integrity and regulate vascular leak, or 2) compartmentalization of S1P and S1Pr1 enables sensing of plasma extravasation and a dynamic response to leak. To distinguish between these models, we examined endothelial responses to apical vs. basolateral application of S1P in vitro and S1Pr1 subcellular localization in vivo. Addition of S1P to the upper (apical) chamber of transwells containing EA.hy926 endothelial monolayers triggered ERK activation. Surprisingly, addition of these agonists to the lower (basolateral) chamber was without effect. Similar results were obtained with the S1Pr1 agonist SEW2871, but the PAR1 agonist SFLLRN triggered ERK activation from either chamber. By immunofluorescence staining and surface biotinylation of EA.hy926 cells, S1Pr1 protein was present predominantly on the apical surface. Similarly, staining of mouse trachea microvessels revealed S1Pr1 on CD31-positive membranes apical, but not basal, to the endothelial cell nucleus. S1Pr1 staining co-localized with the apical membrane marker podocalyxin but not the basal marker β1-integrin. The level of S1Pr1 protein in endothelial cells in mice lacking plasma S1P (Sphk1 flox/-; Sphk2 -/-; Mx-1 cre) was twice that in littermate controls. Deletion of S1Pr1 from the endothelium (S1Pr1 flox/-; Cdh5 Cre) resulted in a 6-fold increase in vascular permeability compared to littermate controls. Preliminary data suggests that S1Pr1 inactivation alters the phosphorylation state of VE-cadherin. These results suggest that S1Pr1 is polarized to the luminal membrane of endothelial cells where it is continuously activated but only partially downregulated by plasma S1P and that ongoing activation of S1Pr1 by S1P in plasma is required to maintain endothelial barrier function.

A Novel F2RL3 Variant Predicting a Tyr157Cys Substitution in Protease-Activated Receptor 4 Is Associated with Reduced Receptor Function in Platelets

IntroductionProtease-activated receptor 4 (PAR4) is expressed widely in haemopoietic and vascular tissues and mediates thrombin-induced platelet activation primarily through Gq, protein kinase C and Ca2+ dependent pathways. Platelet PAR4 function may be modified by common non-coding variants near F2RL3 which encodes PAR4, and by epigenetic regulators which affect F2RL3 expression. However, coding sequence variants which affect PAR4 expression or function have not been reported previously. Here we characterise a novel missense variant in F2RL3associated with loss of PAR4 function in platelets from heterozygous subjects and reduced surface expression in platelets and a cell model.MethodsThe index case (P1) was identified by re-sequencing platelet G-protein coupled receptor genes in sub-groups of a collection of 2,400 adult cardiac surgery patients (GRAPHICS study; UK REC 12/SW/0286). Platelets were studied by light transmission aggregation (LTA) and by measuring P-selectin exposure, PAC-1 binding and intracellular Ca2+mobilisation in response to activating agonists. Platelet PAR4 expression was measured in platelets by flow cytometry and immunoblot and in transiently transfected HEK293 cells by flow cytometry and fluorescence microscopy.ResultsP1 was a 66 year old male with lifelong mild mucocutaneous bleeding and two gastrointestinal bleeds during aspirin treatment who harboured a heterozygous c.471A>G transition in F2RL3predicting a p.Tyr157Cys (Y157C) substitution in PAR4.Platelet phenotypeCompared to healthy controls, platelets from P1 showed reduced aggregation responses to the PAR4 agonist AYPGKF (60-600 μM; EC50 P1 266 μM vs controls 153 μM (n=11)) and to α-thrombin (5-100 mU.ml-1; EC50 P1 51 mU.ml-1 vs controls 15 mU.ml-1 (n=8)) with the greatest reduction in responses at 150 μM AYPGKF (maximum amplitude (MA) P1 1% vs controls 56%; p<0.0001) and 10 mU.ml-1 α-thrombin (MA P1 3% vs controls 67%; p<0.0001). Platelets from P1 also showed reduced P-selectin exposure, PAC-1 binding and intracellular Ca2+mobilisation in response to AYPGKF (25-1000 μM) compared to healthy controls. Functional responses of platelets from P1 to the PAR1 agonist SFLLRN (0.25-100μM) were similar to controls suggesting a selective defect in PAR4 function. Platelets from P1 showed a mean number of PAR4 receptors/platelet of 13 by flow cytometry, compared to 295 in controls (SEM 98; n=9) indicating markedly reduced surface expression. However, PAR1 surface expression in platelets from P1 was similar to controls. Immunoblots of platelet protein extracts for PAR4 from P1 and controls were similar, suggesting comparable total PAR4 expression.The c.471A>G F2RL3 variant was identified in two sons of P1, who also showed reduced platelet functional responses to AYPGKF and PAR4 surface expression that were similar to P1, indicating co-segregation of platelet phenotype and the variant F2LR3 genotype. However, these subjects did not display abnormal bleeding.Y157C PAR4 in HEK293 cellsVariant PAR4 with a C-terminal CFP tag (Y157C PAR4-CFP) expressed in HEK293 cells showed reduced surface expression by flow cytometry (mean of median fluorescence intensities 28% that of wild-type (WT) PAR4-CFP with SEM 5%; n=3). Consistent with this, fluorescence microscopy revealed predominantly intracellular expression of Y157C PAR4-CFP in the HEK293 cells with limited cell surface expression when compared to WT PAR4-CFP.ConclusionThe Y157 residue is highly conserved within transmembrane domain 3 of PAR4 that, in other G protein coupled receptors, is crucial for maintaining normal receptor structure. Therefore, the Y157C substitution identified in the index case in PAR4 is likely to adversely affect receptor function. Consistent with this, our data from multiple functional assays show reduced platelet responses to α-thrombin and to a PAR4 specific agonist, but not to a PAR1 specific agonist. This platelet phenotype co-segregated with the heterozygous Y157C substitution in the study kindred suggesting a causal association. However, this was not associated with bleeding in all kindred members. In both platelets and HEK293 cells we demonstrated the Y157C substitution was associated with loss of PAR4 function and reduced surface receptor expression.This is the first description of a missense PAR4 variant causing loss of receptor function as a heritable trait. DisclosuresNo relevant conflicts of interest to declare.

Abstract 56: S14161 Inhibits Platelet Activation and Thrombus Formation via PI3K Signaling Pathway

The phosphatidylinositol 3-kinase (PI3K) signaling pathway is critical in modulating platelet functions. In the present study, we evaluated the effect of S14161, a recently identified pan-class I PI3K inhibitor, on platelet activation and thrombus formation. Results showed that S14161 inhibited human platelet aggregation induced by collagen, thrombin, U46619, and ADP in a dose-dependent manner. For example, the maximal aggregation induced by collagen were decreased by 32.7% (P&lt;0.05), 59.4% (P&lt;0.001) and 89.2% (P&lt;0.001) (IC50=3.79±1.17 μM) when preincubated with 2.5, 5 and 10 μM of S14161, respectively. Interestingly, platelet aggregates were disaggregated after the peak of aggregation induced by ADP when platelets were pretreated with S14161 (10 μM). S14161 also inhibited platelet aggregation induced by GPVI selective ligand convulxin and CRP, PAR1 agonist SFLLRN, and calcium ionophore A23187. Flow cytometric studies showed that S14161 inhibited convulxin-induced P-selectin expression and fibrinogen binding of single platelet. S14161 also inhibited platelet spreading on fibrinogen and clot retraction, processes mediated by outside-in signaling. Using a microfluidic chamber we demonstrated that S14161 decreased platelet adhesion on collagen-coated surface by about 80%. Western blot showed that S14161 inhibited phosphorylation of Akt at both Ser473 and Thr308 sites, and GSK3β at Ser9 in response to collagen, thrombin, or U46619. Additionally, S14161 inhibited ERK1/2 activation. Finally, the effects of S14161 on thrombus formation in vivo were measured using a ferric chloride-induced carotid artery injury model in mice. The intraperitoneal injection of S14161 (2 mg/kg) to male C57BL/6 mice significantly extended the first occlusion time (5.05±0.99 min, n=9) compared to the vehicle controls (3.72±0.95 min, n=8) (P&lt;0.05), but did not prolong the bleeding time (P&gt;0.05). Taken together, our data showed that S14161 inhibits platelet activation and thrombus formation, and may be developed as a novel therapeutic agent for the prevention of thrombotic disorders.

Exploring the Phe-Gly dipeptide-derived piperazinone scaffold in the search for antagonists of the thrombin receptor PAR1.

A series of Phe-Gly dipeptide-derived piperazinones containing an aromatic urea moiety and a basic amino acid has been synthesized and evaluated as inhibitors of human platelet aggregation induced by the PAR1 agonist SFLLRN and as cytotoxic agents in human cancer cells. The synthetic strategy involves coupling of a protected basic amino acid benzyl amide to 1,2- and 1,2,4-substituted-piperazinone derivatives, through a carbonylmethyl group at the N1-position, followed by formation of an aromatic urea at the exocyclic moiety linked at the C2 position of the piperazine ring and removal of protecting groups. None of the compounds showed activity in the biological evaluation.

Highly functionalized 2-oxopiperazine-based peptidomimetics: An approach to PAR1 antagonists

A series of pseudodipeptide-based chiral 1,3,4,5-tetrasubstituted-2-oxopiperazines has been designed and synthesized as potential PAR1 antagonists. These highly functionalized piperazines were synthesized from aromatic and basic amino acid derived Ψ[CH(CN)NH]pseudodipeptides through a four step pathway that involves reduction of the cyano group to build the 2-oxopiperazine ring, followed by selective functionalization at the N4-, N1-positions, and at the exocyclic moiety at position C5. This regioselective functionalization required the fine tuning of reaction conditions. All new compounds were screened as inhibitors of human platelet aggregation induced by the PAR1 agonist SFLLRN and as cytotoxic agents in human cancer cell lines. Some of the compounds displayed moderate PAR1 antagonist activity, while, others were cytotoxic at μM concentration. No correlation was observed between both types of activities.

Design, synthesis and biological evaluation of new peptide-based ureas and thioureas as potential antagonists of the thrombin receptor PAR1

By applying a diversity oriented synthesis strategy for the search of new antagonists of the thrombin receptor PAR1, a series of peptide-based ureas and thioureas, including analogues of the PAR1 reference antagonist RWJ-58259, has been designed and synthesized. The general synthetic scheme involves reduction of basic amino acid-derived amino nitriles by hydrogen transfer from hydrazine monohydrate in the presence of Raney Ni, followed by reaction with diverse isocyanates and isothiocyanates, and protecting group removal. All new compounds have been evaluated as inhibitors of human platelet aggregation induced by the PAR1 agonist SFLLRN. Some protected peptide-based ureas displayed significant antagonist activity.

Identification of signal transduction pathways involved in the formation of platelet subpopulations upon activation

Platelets are formed elements of blood. Upon activation, they externalize phosphatidylserine, thus accelerating membrane-dependent reactions of blood coagulation. Activated platelets form two subpopulations, only one of which expresses phosphatidylserine. This study aimed to identify signalling pathways responsible for this segregation. Gel-filtered platelets, intact or loaded with calcium-sensitive dyes, were activated and labelled with annexin V and antibodies, followed by flow cytometric analysis. Calcium Green and Fura Red dyes were compared, and only the latter was able to detect calcium level differences in the platelet subpopulations. Phosphatidylserine-positive platelets produced by thrombin had stably high intracellular calcium level; addition of convulxin increased and stabilized calcium level in the phosphatidylserine-negative subpopulation. PAR1 agonist SFLLRN also induced calcium rise and subpopulation formation, but the resulting platelets were not coated with alpha-granule proteins. Adenylatecyclase activator forskolin inhibited phosphatidylserine-positive platelets formation several-fold, while its inhibitor SQ22536 had no effect. This suggests that adenylatecyclase inactivation is necessary, but not rate-limiting, for subpopulation segregation. Inhibition of mitogen-activated protein kinase kinase (U0126) and glycoprotein IIb-IIIa (Monafram(®)) was without effect, whereas inhibitors of phosphatidylinositol 3-kinase (wortmannin) and Src tyrosine kinase (PP2) decreased the procoagulant subpopulation threefold. These data identify the principal signalling pathways controlling platelet heterogeneity.

Protein Kinase C Delta Mediates the Activation of Protein Kinase D In Platelets

AbstractAbstract 2021Protein kinase D (PKD) is a subfamily of serine/threonine specific family of kinases, comprised of PKD1, PKD2 and PKD3 (PKCm, PKD2 and PKCn in humans). They are part of alternate DAG receptors along with RasGRPs, Munc 13s, chimerins and DAG kinases. Members of the novel class of PKC isoforms such as PKCd, PKCh, and PKCe associate with PKD in smooth muscle cells and COS-7 cells. The mechanism of activation of PKD and the specific PKC isoforms required for its activation are not known to date. This study is aimed at investigating the pathways involved in the activation of PKD in platelets. We show that human as well as murine platelets express PKD. PKD could be activated with PAR4 agonist AYPGKF, PAR1 agonist SFLLRN and GPVI agonist convulxin. AYPGKF and SFLLRN induced PKD phosphorylation as early as 30 sec and convulxin induced PKD phosphorylation at 1 minute. PKD phosphorylation induced by AYPGKF and convulxin were sustained for 5 minutes but phosphorylation induced by SFLLRN was attenuated after 2 minutes of stimulation. AYPGKF-induced PKD phosphorylation was reduced with a calcium chelator, dimethyl BAPTA, indicating that calcium-mediated signals might play a role in activation of PKD. PKD phosphorylation in response to AYPGKF was abolished with a Gq inhibitor, YM-254890, but was not affected by Gi-coupled P2Y12 receptor antagonist ARC-69931MX, indicating that PKD phosphorylation is Gq-, but not Gi- or G 12/13-dependent. PKD phosphorylation was abolished with pan-PKC inhibitors, GF109203X and Ro31-8220, indicating that PKCs are required for PKD activation in platelets. PKD phosphorylation was significantly inhibited with a PKC delta inhibitor, rottlerin, but was not affected by the classical PKC inhibitor, Go6976, suggesting that novel PKC isoforms are important for PKD activation. In addition, 2MeSADP that fails to activate PKCd did not induce phosphorylation of PKD in platelets. Furthermore, phosphorylation of PKD induced by AYPGKF was significantly reduced in PKCd-deficient platelets compared to that of wild type platelets. Hence, we conclude that PKD is a common signaling target downstream of various agonist receptors in platelets, and Gq-mediated signals and novel PKC isoforms, in particular PKCd is required for activation of PKD. Disclosures:No relevant conflicts of interest to declare.

Protein Kinase C-mediated Phosphorylation and Activation of PDE3A Regulate cAMP Levels in Human Platelets

The elevation of [cAMP](i) is an important mechanism of platelet inhibition and is regulated by the opposing activity of adenylyl cyclase and phosphodiesterase (PDE). In this study, we demonstrate that a variety of platelet agonists, including thrombin, significantly enhance the activity of PDE3A in a phosphorylation-dependent manner. Stimulation of platelets with the PAR-1 agonist SFLLRN resulted in rapid and transient phosphorylation of PDE3A on Ser(312), Ser(428), Ser(438), Ser(465), and Ser(492), in parallel with the PKC (protein kinase C) substrate, pleckstrin. Furthermore, phosphorylation and activation of PDE3A required the activation of PKC, but not of PI3K/PKB, mTOR/p70S6K, or ERK/RSK. Activation of PKC by phorbol esters also resulted in phosphorylation of the same PDE3A sites in a PKC-dependent, PKB-independent manner. This was further supported by the finding that IGF-1, which strongly activates PI3K/PKB, but not PKC, did not regulate PDE3A. Platelet activation also led to a PKC-dependent association between PDE3A and 14-3-3 proteins. In contrast, cAMP-elevating agents such as PGE(1) and forskolin-induced phosphorylation of Ser(312) and increased PDE3A activity, but did not stimulate 14-3-3 binding. Finally, complete antagonism of PGE(1)-evoked cAMP accumulation by thrombin required both G(i) and PKC activation. Together, these results demonstrate that platelet activation stimulates PKC-dependent phosphorylation of PDE3A on Ser(312), Ser(428), Ser(438), Ser(465), and Ser(492) leading to a subsequent increase in cAMP hydrolysis and 14-3-3 binding.

Do heterotrimeric G proteins redistribute upon G protein-coupled receptor stimulation in platelets?

Previous studies have proposed that stimulation of G protein-coupled receptors can cause a redistribution of G proteins to other receptors. The redistribution would cause a greater functional sensitivity of unsensitized ‘secondary’ receptors toward their agonists. Using platelets as a model system, we utilized a proximal signaling event, intracellular calcium mobilization, to determine if agonist stimulation of particular Gq-coupled receptors would result in increased sensitivity for stimulation of other Gq-coupled receptors. Platelets express three Gq-coupled receptors for thrombin, thromboxane A2, and ADP with different potencies. Varying concentrations of a primary agonist (PAR-1 agonist SFLLRN, or the TXA2 agonist U46619) was followed by a constant submaximal concentration of a secondary agonist (U46619, or the P2Y1 agonist ADP). We observed that initial stimulation by SFLLRN was followed by a decrease in the extent of secondary U46619 or ADP-mediated calcium mobilization in comparison to control responses (i.e. without primary stimulation). To extend these studies we examined calcium mobilization in platelets from mice that were either wild-type or homozygous null for the PAR-4 or P2Y1 receptors, hypothesizing that the loss of PAR-4 or P2Y1 receptors would cause redistribution of its Gαq proteins to other receptors, and elicit a greater response when stimulated with other agonists than in platelets from a wild-type mouse. However, our results showed almost identical levels of peak calcium between wild-type or PAR-4 null mice when stimulated with either ADP or U46619. Similar results were obtained for the P2Y1 null mice stimulated with AYPGKF or U46619. We conclude that stimulation of one Gq coupled receptor does not result in redistribution of Gq to other Gq-coupled receptors.

Mechanisms of Protease-activated Receptor-4 Actions in Cardiomyocytes: ROLE OF Src TYROSINE KINASE

Protease-activated receptor (PAR)-4 is a low affinity thrombin receptor with slow activation and desensitization kinetics relative to PAR-1. This study provides novel evidence that cardiomyocytes express functional PAR-4 whose signaling phenotype is distinct from PAR-1 in cardiomyocytes. AYPGKF, a modified PAR-4 agonist with increased potency at PAR-4, activates p38-mitogen-activated protein kinase but is a weak activator of phospholipase C, extracellular signal-regulated kinase, and cardiomyocyte hypertrophy; AYPGKF and thrombin, but not the PAR-1 agonist SFLLRN, activate Src. The observation that AYPGKF and thrombin activate Src in cardiomyocytes cultured from PAR-1(-/-) mice establishes that Src activation is via PAR-4 (and not PAR-1) in cardiomyocytes. Further studies implicate Src and epidermal growth factor receptor (EGFR) kinase activity in the PAR-4-dependent p38-mitogen-activated protein kinase signaling pathway. Thrombin phosphorylates EGFRs and ErbB2 via a PP1-sensitive pathway in PAR-1(-/-) cells that stably overexpress PAR-4; the Src-mediated pathway for EGFR/ErbB2 transactivation underlies the protracted phases of thrombin-dependent extracellular signal-regulated kinase activation in PAR-1(-/-) cells that overexpress PAR-4 and in cardiomyocytes. These studies identify a unique signaling phenotype for PAR-4 (relative to other cardiomyocyte G protein-coupled receptors) that is predicted to contribute to cardiac remodeling and influence the functional outcome at sites of cardiac inflammation.

The effects of stimulating protease-activated receptor-1 and -2 in A172 human glioblastoma.

Human glioblastoma cell line A172 expressed protease-activated receptor-1 and -2 (PAR-1 and PAR-2). We investigated the effects of the stimulation of these receptors by receptor-activating agonist peptides on the Ca2+ signaling, protein kinase C translocation, cell morphology and cell proliferation in A172. Both PAR-1 agonist SFLLRN and PAR-2 agonist SLIGKV induced an increase in [Ca2+]i. The prior treatment of A172 with PAR-2 agonist SLIGKV did not influence the [Ca2+]i response to PAR-1 agonist SFLLRN or thrombin, however, the prior treatment with PAR-1 agonist SFLLRN or thrombin completely abolished the second response to PAR-2 agonist SLIGKV. Treatment with each agonist peptide produced thinner and fewer processes in A172. The PAR-2 agonist inhibited the proliferation of A172 significantly while PAR-1 agonist did not. PKC-alpha and gamma were translocated from cytosol to membrane with either PAR-1 or PAR-2 stimulation, however, L was specifically translocated with SFLLRN, and lambda with SLIGKV, respectively. These results indicated that PAR-1 and PAR-2 stimulation produced a similar [Ca2+]i response and morphological changes in A172 glioblastoma while the effects on the cell proliferation and activation of PKC isozymes were distinct, suggesting that different signal transduction pathways were activated by these receptors. The uni-directional cross desensitization implies a functional linkage between PAR-1 and PAR-2 receptors.

A Novel PAR-1-Type Thrombin Receptor Signaling Pathway: Cyclic AMP-Independent Activation of PKA in SNB-19 Glioblastoma Cells

Cellular effects of thrombin are mediated by members of a new subfamily of G protein-coupled receptors designated proteinase-activated receptors (PARs) with the prototype PAR-1. Investigation of PAR-1-induced signaling has been shown to be very important in clarifying thrombin‘s role in cell metabolism, differentiation, and growth. We evaluated connection of PAR-1 with the cAMP/PKA pathway in SNB-19 glioblastoma cells. Alpha-thrombin and the synthetic PAR-1 agonist SFLLRN stimulated PKA as shown by increased PKA activity and translocation of the catalytic PKA α subunits (PKAcatα) into the nucleus. However, no effect on cAMP could be observed. PKAcatα was found to be associated with nuclear factor-kappa B (NF-κB) p65 and its inhibitor protein IκB in SNB-19 cells. After PAR-1 stimulation, this association was markedly diminished. We conclude that PAR-1 mediates PKA activation without altering cAMP levels but includes NF-κB-associated PKAcatα in SNB-19 glioblastoma cells. This is the first evidence for a cAMP-independent PKA signaling by a G protein-coupled receptor.