Protein-mediated Signaling Pathways Research Articles

Reduced expression of G protein-coupled receptor kinases in schizophrenia but not in schizoaffective disorder

Alterations of multiple G protein-mediated signaling pathways are detected in schizophrenia. G protein-coupled receptor kinases (GRKs) and arrestins terminate signaling by G protein-coupled receptors exerting a powerful influence on receptor functions. Modifications of arrestin and/or GRKs expression may contribute to schizophrenia pathology. Cortical expression of arrestins and GRKs was measured postmortem in control and subjects with schizophrenia or schizoaffective disorder. Additionally, arrestin/GRK expression was determined in elderly patients with schizophrenia and age-matched control. Patients with schizophrenia, but not schizoaffective disorder, displayed a reduced concentration of arrestin and GRK mRNAs and GRK3 protein. Arrestins and GRK significantly decreased with age. In elderly patients, GRK6 was reduced, with other GRKs and arrestins unchanged. A reduced cortical concentration of GRKs in schizophrenia (resembling that in aging) may result in altered G protein-dependent signaling, thus contributing to prefrontal deficits in schizophrenia. The data suggest distinct molecular mechanisms underlying schizophrenia and schizoaffective disorder.

Optogenetic Control of Motor Coordination by Gi/o Protein-coupled Vertebrate Rhodopsin in Cerebellar Purkinje Cells

G protein-coupled receptors are involved in the modulation of complex neuronal networks in the brain. To investigate the impact of a cell-specific G(i/o) protein-mediated signaling pathway on brain function, we created a new optogenetic mouse model in which the G(i/o) protein-coupled receptor vertebrate rhodopsin can be cell-specifically expressed with the aid of Cre recombinase. Here we use this mouse model to study the functional impact of G(i/o) modulation in cerebellar Purkinje cells (PCs). We show that in vivo light activation of vertebrate rhodopsin specifically expressed in PCs reduces simple spike firing that is comparable with the reduction in firing observed for the activation of cerebellar G(i/o)-coupled GABA(B) receptors. Notably, the light exposure of the cerebellar vermis in freely moving mice changes the motor behavior. Thus, our studies directly demonstrate that spike modulation via G(i/o)-mediated signaling in cerebellar PCs affects motor coordination and show a new promising approach for studying the physiological function of G protein-coupled receptor-mediated signaling in a cell type-specific manner.

Functional selectivity of adenosine receptor ligands

Adenosine receptors are plasma membrane proteins that transduce an extracellular signal into the interior of the cell. Basically every mammalian cell expresses at least one of the four adenosine receptor subtypes. Recent insight in signal transduction cascades teaches us that the current classification of receptor ligands into agonists, antagonists, and inverse agonists relies very much on the experimental setup that was used. Upon activation of the receptors by the ubiquitous endogenous ligand adenosine they engage classical G protein-mediated pathways, resulting in production of second messengers and activation of kinases. Besides this well-described G protein-mediated signaling pathway, adenosine receptors activate scaffold proteins such as β-arrestins. Using innovative and sensitive experimental tools, it has been possible to detect ligands that preferentially stimulate the β-arrestin pathway over the G protein-mediated signal transduction route, or vice versa. This phenomenon is referred to as functional selectivity or biased signaling and implies that an antagonist for one pathway may be a full agonist for the other signaling route. Functional selectivity makes it necessary to redefine the functional properties of currently used adenosine receptor ligands and opens possibilities for new and more selective ligands. This review focuses on the current knowledge of functionally selective adenosine receptor ligands and on G protein-independent signaling of adenosine receptors through scaffold proteins.

Cytosolic RNA Recognition Pathway Activates 14-3-3 Protein Mediated Signaling and Caspase-Dependent Disruption of Cytokeratin Network in Human Keratinocytes

The skin is the primary boundary between the body and the environment. In addition to its properties as a physical barrier, skin keratinocytes actively participate in many defense mechanisms. Viral double-stranded RNA (dsRNA) is the most important viral structure involved in activation of immune response. Intracellular detection of dsRNA by cytoplasmic receptors activates well-characterized antiviral response, as well as pro-inflammatory response and apoptosis of virus-infected cells. Here, we have used quantitative subcellular proteomics to characterize the signaling pathways activated by cytosolic dsRNA recognition pathway in human keratinocytes. Cytoplasmic and mitochondrial proteomes were analyzed using 2-DE in combination with MS, immunoblotting and confocal microscopy. We have identified 239 reproducibly differentially expressed proteins upon dsRNA stimulation. The identified proteins include several key proteins involved in cytoskeletal dynamics, cell signaling, cell death, and stress response. Our analysis provides novel information how the cytokeratin network is disrupted in a caspase-dependent manner upon dsRNA stimulation as well as Encephalomyocarditis virus or Vesicular stomatitis virus infection. We show that this caspase-dependent disruption of cytokeratin is activated by cytoplasmic RNA recognition pathway. In addition, we show that viral infection activates 14-3-3 protein mediated signaling pathways in human keratinocytes which suggest an important role of 14-3-3 proteins in antiviral innate immune response.

Systems-level analysis of a G protein mediated signaling pathway

Systems-level analysis of a G protein mediated signaling pathway

Inhibition of Heterotrimeric G Protein Signaling by a Small Molecule Acting on Gα Subunit

The simultaneous activation of many distinct G protein-coupled receptors (GPCRs) and heterotrimeric G proteins play a major role in various pathological conditions. Pan-inhibition of GPCR signaling by small molecules thus represents a novel strategy to treat various diseases. To better understand such therapeutic approach, we have characterized the biomolecular target of BIM-46187, a small molecule pan-inhibitor of GPCR signaling. Combining bioluminescence and fluorescence resonance energy transfer techniques in living cells as well as in reconstituted receptor-G protein complexes, we observed that, by direct binding to the Galpha subunit, BIM-46187 prevents the conformational changes of the receptor-G protein complex associated with GPCR activation. Such a binding prevents the proper interaction of receptors with the G protein heterotrimer and inhibits the agonist-promoted GDP/GTP exchange. These observations bring further evidence that inhibiting G protein activation through direct binding to the Galpha subunit is feasible and should constitute a new strategy for therapeutic intervention.

Lanthanum suppresses osteoblastic differentiation via pertussis toxin‐sensitive G protein signaling in rat vascular smooth muscle cells

A major cellular event in vascular calcification is the phenotypic transformation of vascular smooth muscle cells (VSMCs) into osteoblast-like cells. After demonstrating that lanthanum chloride (LaCl(3)) suppresses hydrogen peroxide-enhanced calcification in rat calcifying vascular cells (CVCs), here we report its effect on the osteoblastic differentiation of rat VSMCs, a process leading to the formation of CVCs. Cells were isolated from aortic media of male SD rats, and passages between three and eight were cultured in Dulbeccol's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS) and 10 mM beta-glycerophosphate (beta-GP) in the presence or absence of LaCl(3). Exposure of cells to LaCl(3) suppressed the beta-GP-induced elevations in calcium deposition, alkaline phosphatase (ALP) activity, and Cbfa1/Runx2 expression, as well as the concomitant loss of SM alpha-actin. Furthermore, LaCl(3) activated the phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), and the blockage of either pathway with a specific inhibitor abolished the effects of LaCl(3). In addition, pretreatment of the cells with pertussis toxin (PTx), an inhibitor of G protein-mediated signaling pathway, repealed all the changes induced by LaCl(3). These findings demonstrate that LaCl(3) suppresses the beta-GP-induced osteoblastic differentiation and calcification in rat VSMCs, and its effect is mediated by the activation of both ERK and JNK MAPK pathways via PTx-sensitive G proteins.

Mutant 5-Hydroxytryptamine1AReceptor D116A Is a Receptor Activated Solely by Synthetic Ligands with a Rich Pharmacology

Like other biogenic amine G protein-coupled receptors, mutation of the conserved aspartatic residue into alanine at position 116 (D116A(3.32)) in the 5-hydroxytryptamine (5-HT)(1A) receptor greatly affects 5-HT binding and signal transduction. [(3)H]8-Hydroxy-2-dipropylaminotetralin (8-OH-DPAT) and [(3)H]-N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl)cyclohexanecarboxamide trihydrochloride (WAY100,635) are capable to bind the 5-HT(1A)-D116A mutant and, using these radioligands, we show here that this mutation dramatically reduces the affinities of the selective 5-HT(1A) agonists N-(3-chloro-4-fluorobenzoyl)-4-fluoro-4-[(5-methylpyridin-2-yl)-methylamino methyl]piperidine (F13640), 3-chloro-4-fluorophenyl-(4-fluorophenyl-4-{[(5-methyl-6 methylamino-pyridin-2-ylmethyl)-amino]-methyl}-piperidin-1-yl-methanone (F13714), and 2-[5-[3-(4-methylsulfonylamino)benzyl-1,4-oxadiazol-5-yl]-1H-indole-3-yl]ethylamine (L694247) and that of 5-carboxamidotryptamine. Although to a lesser extent, the binding of buspirone, (+)-flesinoxan, (-)-pindolol, and (-)-8-OH-DPAT are also highly decreased. In contrast, affinities of the 5-HT(1A) ligands WAY100,635, spiperone, (-)-4-(dipropylamino)-1,3,4,5-tetrahydrobenz {c,d}indole-6-carboxamide (LY228,729), and 1-[2-(4-fluorobenzoylamino)ethyl]-4-(7-methoxynaphtyl) piperazine (S14506) and the prototypical 5-HT(1A) agonist (+)-8-OH-DPAT are only slightly affected by the mutation, suggesting a moderate contribution of Asp116 to the binding pocket for these latter. Furthermore, LY228,729, S14506, and (+)-8-OH-DPAT induce a potent and efficacious coupling of the 5-HT(1A)-D116A receptor to G protein activation as measured by Ca(2+) mobilization and guanosine 5'-O-(3-[(35)S]thio)triphosphate binding in Chinese hamster ovary cells as well as by G protein-coupled inwardly rectifying potassium channel current activation in Xenopus laevis oocytes. It is interesting that the selective 5-HT(1A) antagonist WAY100,635 shows potent partial agonist activity at the 5-HT(1A)-D116A mutant, whereas spiperone maintains its inverse agonist properties. The pharmacological approach reported here re-evaluates the binding and functional properties of the 5-HT(1A)-D116A receptor and describes for the first time this mutant as a receptor activated solely by synthetic ligands (RASSL), with a rich pharmacology. By bioengineering animal models incorporating this RASSL, one may further explore the role of 5-HT(1A) receptor signaling in the central nervous system as well as G(i) protein-mediated signaling pathways in other tissues.

Mammary Tumorigenesis through LPA Receptor Signaling

Lysophosphatidic acid (LPA) is a lipid growth factor that is produced by an extracellular phospholipase, termed autotaxin (ATX), and acts via G protein-coupled receptors. In this issue of Cancer Cell, Liu et al. show that transgenic overexpression of ATX or LPA receptors leads to invasive and metastatic mammary cancer.

Systems analysis of the single photon response in invertebrate photoreceptors

Photoreceptors of Drosophila compound eye employ a G protein-mediated signaling pathway that transduces single photons into transient electrical responses called "quantum bumps" (QB). Although most of the molecular components of this pathway are already known, the system-level understanding of the mechanism of QB generation has remained elusive. Here, we present a quantitative model explaining how QBs emerge from stochastic nonlinear dynamics of the signaling cascade. The model shows that the cascade acts as an "integrate and fire" device and explains how photoreceptors achieve reliable responses to light although keeping low background in the dark. The model predicts the nontrivial behavior of mutants that enhance or suppress signaling and explains the dependence on external calcium, which controls feedback regulation. The results provide insight into physiological questions such as single-photon response efficiency and the adaptation of response to high incident-light level. The system-level analysis enabled by modeling phototransduction provides a foundation for understanding G protein signaling pathways less amenable to quantitative approaches.

G protein β3 subunit C825T polymorphism modifies the presentation of temporal lobe epilepsy

Experimental studies suggest a role of G protein-mediated signaling pathways in epileptogenesis. A genetic variation affecting the G protein subunit Gbeta3 denoted the C825T polymorphism has been reported to increase the signaling efficiency through G(i) proteins and to modify responses to certain drugs. The C825T polymorphism has also been associated with several diseases including hypertension, diabetes type II, obesity, and major depressive disorder. In this study, we have explored whether the G protein polymorphism C825T is associated with or influences temporal lobe epilepsy (TLE). The study included 227 TLE patients, 186 controls, and 106 family members of TLE patients. DNA was extracted from blood samples and typing of the polymorphism was performed. Case record forms were analyzed for all the homozygote TLE patients and homozygote controls, i.e., carrying the TT genotype as well as for 28 matched TLE patients (16 females, 12 males) without the polymorphism (CC genotype). Typing of the C825T polymorphism showed that 6.0% of the TLE patients, 7.0% of the controls, and 7.5% of the family members were homozygote for the polymorphism; i.e., carrying the TT genotype. TLE patients carrying the TT genotype had higher severity score on eight out of nine predefined parameters compared with the TLE patients without polymorphism, i.e., carrying CC genotype. TT genotype TLE patients also had increased body mass index, body weight, and waist circumference compared with the TLE patients carrying the CC genotype. There was no increased frequency of hypertension or diabetes. There was no increased frequency of TLE between the carriers of the TT genotype compared with the healthy controls and/or family members without epilepsy. However, the TLE patients with the TT genotype showed tendencies of a more severe disease phenotype.

Crosstalk between integrin and G protein pathways involved in mechanotransduction in mandibular condylar chondrocytes under pressure

To investigate the role of integrin and G protein pathways in the mechanotransduction process within MCCs and explore the possible crosstalk between the two traditional signal pathways, in vitro-cultured rabbit MCCs were treated with pressure. The mRNA level of α5β1 integrin was determined by in situ hybridization and the distributions of vinculin, Gαq/11 protein, F-actin and intracellular calcium were studied with a laser scanning confocal microscope. Increased integrin α5β1 expression, enhanced stress fiber assembly, elevated G protein and vinculin level and up-regulated IP3 channel sensitivity were found in the mechanotransduction process of MCCs under pressure. Furthermore, the vinculin and the Gαq/11 were observed co-localized with each other, and the F-actin reassembly and stress fibers formation could be inhibited by intracellular calcium channel blocking, which gave direct evidence that the traditional integrin-mediated or G protein-mediated signaling pathways coordinately regulate the function of MCCs under mechanical stimulation.

Abstract 939: Galpha-i Signaling in Neural Crest Cells is Required for Normal Migration of Cardiac Neural Crest Cells, Cardiac Development, and Survival

G protein coupled receptors (GPCRs) have long been known to play crucial roles in transducing environmental signals to the adult cardiovascular system. In recent years, the roles of G protein-mediated signaling pathways in orchestrating the interactions of different tissues during cardiovascular development have become increasingly evident. To analyze the role of G protein signaling pathways in vivo we have generated mice where the function of the heterotrimeric G alpha subunit Gai can be ablated in a cell type specific manner utilizing the Cre-loxP system. We have mated these mice to two different neural crest-specific Cre lines in order to probe the effects of loss of Gai mediated signaling on the ability of neural crest cells (NCC) to contribute to the developing outflow tract and aortic arch arteries. METHODS: We have generated mice that express the Gai-inhibiting pertussis toxin S1 subunit (PTX) from the ROSA26 locus in a Cre recombination dependent manner (ROSA-PTX mice). These were mated to mice expressing either the Wnt1 Cre or P0 Cre transgene. Wnt1Cre is active in both premigratory and migratory NCC, whereas P0Cre is active only in migratory NCC and their derivatives. RESULTS: P0Cre-ROSA-PTX mice were normal at birth and demonstrated no structural heart defects. In contrast, Wnt1Cre-ROSA-PTX mice were present in normal numbers at late gestation but died perinatally due in part to cardiac outflow tract defects. Excision reporter and in situ hybridization studies suggest this is secondary to a delay/blockage of cardiac NCC migration into the developing outflow tract. NCC migration into the pharyngeal arches was unaffected in these mice and no craniofacial, thymic, or aortic arch abnormalities were observed. CONCLUSIONS: These results indicate that Gai-mediated signaling is required in premigratory or early migratory cardiac NCC for normal development of the outflow tract. In contrast, endothelin A receptor knockout mice (currently the only GPCR knock out with a neural crest phenotype) are thought to exhibit defects of postmigratory NCC function. RNA profiling of NCC for GPCRs involved in this Gai-dependent pathway has revealed several potential candidate receptors, including orphan receptors. Further analysis of these receptors is underway.

Defining the Role of Astrocytes in Neuromodulation

Astrocytes undergo elevations in intracellular calcium following activation of metabotropic receptors, which may trigger glutamate secretion and excitation of surrounding neurons. In this issue of Neuron, Fiacco et al. use transgenic mice that express a foreign G(q)-coupled receptor in astrocytes to show that selective stimulation of astrocytes is not sufficient to induce the release of glutamate.

Activation of Platelet Function Through G Protein–Coupled Receptors

Because of their ability to become rapidly activated at places of vascular injury, platelets are important players in primary hemostasis as well as in arterial thrombosis. In addition, they are also involved in chronic pathological processes including the atherosclerotic remodeling of the vascular system. Although primary adhesion of platelets to the vessel wall is largely independent of G protein-mediated signaling, the subsequent recruitment of additional platelets into a growing platelet thrombus requires mediators such as ADP, thromboxane A(2), or thrombin, which act through G protein-coupled receptors. Platelet activation via G protein-coupled receptors involves 3 major G protein-mediated signaling pathways that are initiated by the activation of the G proteins G(q), G(13), and G(i). This review summarizes recent progress in understanding the mechanisms underlying platelet activation and thrombus extension via G protein-mediated signaling pathways.

Regulation of cardiomyocyte signaling by RGS proteins: Differential selectivity towards G proteins and susceptibility to regulation

Many signals that regulate cardiomyocyte growth, differentiation and function are mediated via heterotrimeric G proteins, which are under the control of RGS proteins (Regulators of G protein Signaling). Several RGS proteins are expressed in the heart, but so far little is known about their function and regulation. Using adenoviral gene transfer, we conducted the first comprehensive analysis of the capacity and selectivity of the major cardiac RGS proteins (RGS2–RGS5) to regulate central G protein-mediated signaling pathways in adult ventricular myocytes (AVM). All four RGS proteins potently inhibited G q/11-mediated phospholipase C β stimulation and cell growth (assessed in neonatal myocytes). Importantly, RGS2 selectively inhibited G q/11 signaling, whereas RGS3, RGS4 and RGS5 had the capacity to regulate both G q/11 and G i/o signaling (carbachol-induced cAMP inhibition). G s signaling was unaffected, and, contrary to reports in other cell lines, RGS2–RGS5 did not appear to regulate adenylate cyclase directly in AVM. Since RGS proteins can be highly regulated in their expression by many different stimuli, we also tested the hypothesis that RGS expression is subject to G protein-mediated regulation in AVM and determined the specificity with which enhanced G protein signaling alters endogenous RGS expression in AVM. RGS2 mRNA and protein were markedly but transiently up-regulated by enhanced G q/11 signaling (α 1-adrenergic stimulation or Gα q * overexpression), possibly by a negative feedback mechanism. In contrast, the other negative regulators of G q/11 signaling (RGS3–RGS5) were unchanged. Endogenous RGS2 (but not RGS3–RGS5) expression was also up-regulated in cells with enhanced AC signaling (β-adrenergic or forskolin stimulation). Taken together, these findings suggest diverse roles of RGS proteins in regulating myocyte signaling. RGS2 emerged as the only selective and highly regulated inhibitor of G q/11 signaling that could potentially become a promising target for ameliorating G q/11-mediated signaling and growth.

Crosstalk between chemokines and neuronal receptors bridges immune and nervous systems

Chemokine receptors, a family of Gi protein-coupled receptors responsible for cell migration, are widely expressed by cells of immune and nervous systems. Activation of receptors on the surface of leukocytes, such as opioid, vasoactive intestinal peptide, or adenosine receptors, often has inhibitory effects on chemokine receptors by a mechanism termed heterologous desensitization, resulting in suppression of immune responses. Conversely, activation of chemokine receptors also induces heterologous desensitization of mu-opioid receptors (MOR), a class of key analgesic receptors on neurons. Furthermore, prior exposure of neuronal cells to chemokine treatment enhances the sensitivity of transient receptor potential vanilloid 1 (TRPV1), a heat- and ligand-gated calcium channel, which is critical for sensing of pain. Consequently, during inflammation, activation of chemokine receptors on neurons contributes to hyperalgesia by inhibiting MOR and concomitantly sensitizing TRPV1 via Gi protein-mediated signaling pathways. These observations suggest that the crosstalk between chemokine receptors and neuropeptide membrane receptors serves as a bridge between the immune and nervous systems.

Importin alpha transports CaMKIV to the nucleus without utilizing importin beta.

Ca(2+)/calmodulin-dependent protein kinase type IV (CaMKIV) plays an essential role in the transcriptional activation of cAMP response element-binding protein-mediated signaling pathways. Although CaMKIV is localized predominantly in the nucleus, the molecular mechanism of the nuclear import of CaMKIV has not been elucidated. We report here that importin alpha is able to carry CaMKIV into the nucleus without the need for importin beta or any other soluble proteins in digitonin-permeabilized cells. An importin beta binding-deficient mutant (DeltaIBB) of importin alpha also carried CaMKIV into the nucleus, which strongly suggests that CaMKIV is transported in an importin beta-independent manner. While CaMKIV directly interacted with the C-terminal region of importin alpha, the CaMKIV/importin alpha complex did not form a ternary complex with importin beta, which explains the nonrequirement of importin beta for the nuclear transport of CaMKIV. The cytoplasmic microinjection of importin alpha-DeltaIBB enhanced the rate of nuclear translocation of CaMKIV in vivo. This is the first report to demonstrate definitely that mammalian importin alpha solely carries a cargo protein into the nucleus without utilizing the classical importin beta-dependent transport system.

Unresponsiveness of Platelets Lacking Both Gαq and Gα13: IMPLICATIONS FOR COLLAGEN-INDUCED PLATELET ACTIVATION

The diffusible platelet stimuli ADP and thromboxane A(2) activate multiple G protein-mediated signaling pathways and function as important secondary mediators of platelet activation as they are released from activated platelets. Because they can also increase their own formation and release, their effects are amplified; eventually, all major G protein-mediated signaling pathways are activated. The multiple positive feedback mechanisms operating during platelet activation have obscured the exact analysis of the roles individual G protein-mediated signaling pathways play during the platelet activation process. In this report, we show that platelets lacking G(q) and G(13) are completely unresponsive to diffusible stimuli such as ADP, thromboxane A(2), or thrombin, even when applied at very high concentrations in combination, whereas all stimuli are able to induce platelet aggregation, shape change, and RhoA activation in platelets lacking only one Galpha subunit. This shows that G(q) or G(13) is required to induce some platelet activation, whereas the activation of G(i)-mediated signaling alone is not sufficient to induceactivation of mouse platelets. In addition, platelets lacking Galpha(q) and Galpha(13) adhered normally to collagen under high shearbut did not aggregate any more in response to collagen, indicating that collagen-induced platelet activation but not platelet adhesion requires intact G protein-mediated signaling pathways.

A New Spin on an Old Cycle

During a search for natural ligands of orphan GPCRs [heterotrimeric guanine-nucleotide-binding protein (G protein)-coupled receptors], He et al. uncovered an unexpected link between cell metabolism and cell signaling with intriguing implications for the pathogenesis of metabolic disease. The citric acid cycle, in which fuel molecules are broken down to yield energy and CO 2 , takes place in the mitochondria. However, citric acid cycle intermediates are found in the blood at micromolar concentrations, where their levels are controlled by absorption and extrusion by the kidneys, as well as by respiration and metabolism. He et al. purified a compound that activated cells expressing the orphan GPCR GPR91, which they identified as the citric acid cycle intermediate succinate. When applied to 293 cells stably expressing human GPR91, succinate elicited increases in intracellular calcium and inositol phosphate, activated extracellular signal-regulated kinase, and inhibited adenosine 3′,5′-monophosphate production. These data, together with sensitivity to pertussis toxin, indicated that succinate stimulation of GPR91 activated at least two G protein-mediated signaling pathways (G i or G o and G q ). A second citric acid cycle intermediate, α-ketoglutarate, activated a G q pathway through the closely related orphan GPCR GPR99. The mRNAs for GPR91 and GPR99 were predominantly localized to the kidney, and intravenous injection of succinate elicited increases in plasma renin activity and mean arterial blood pressure in rats; in mice, the effect of succinate on blood pressure depended on GPR91 expression. Thus, succinate and α-ketoglutarate serve as ligands for GPR91 and GPR99, respectively, and succinate may thereby link cell metabolism and blood pressure regulation. W. He, F. J.-P. Miao, D. C.-H. Lin, R. T. Schwandner, Z. Wang, J. Gao, J.-L. Chen, H. Tian, L. Ling, Citric acid cycle intermediates as ligands for orphan G-protein-coupled receptors. Nature 429 , 188-193 (2004). [Online Journal]