Orphan GPCR GPRC5C Facilitates Angiotensin II-Induced Smooth Muscle Contraction.

BACKGROUND: GPCRs (G-protein-coupled receptors) play a central role in the regulation of smooth muscle cell (SMC) contractility, but the function of SMC-expressed orphan GPCR class C group 5 member C (GPRC5C) is unclear. The aim of this project is to define the role of GPRC5C in SMC in vitro and in vivo. METHODS: We studied the role of GPRC5C in the regulation of SMC contractility and differentiation in human and murine SMC in vitro, as well as in tamoxifen-inducible, SMC-specific GPRC5C knockout mice under basal conditions and in vascular disease in vivo. RESULTS: Mesenteric arteries from tamoxifen-inducible, SMC-specific GPRC5C knockout mice showed ex vivo significantly reduced angiotensin II (Ang II)-dependent calcium mobilization and contraction, whereas responses to other relaxant or contractile factors were normal. In vitro, the knockdown of GPRC5C in human aortic SMC resulted in diminished Ang II-dependent inositol phosphate production and lower myosin light chain phosphorylation. In line with this, tamoxifen-inducible, SMC-specific GPRC5C knockout mice showed reduced Ang II-induced arterial hypertension, and acute inactivation of GPRC5C was able to ameliorate established arterial hypertension. Mechanistically, we show that GPRC5C and the Ang II receptor AT1 dimerize, and knockdown of GPRC5C resulted in reduced binding of Ang II to AT1 receptors in HEK293 cells, human and murine SMC, and arteries from tamoxifen-inducible, SMC-specific GPRC5C knockout mice. CONCLUSIONS: Our data show that GPRC5C regulates Ang II-dependent vascular contraction by facilitating AT1 receptor-ligand binding and signaling.

EP4 Receptor Conformation Sensor Suited for Ligand Screening and Imaging of Extracellular Prostaglandins.

Prostaglandins are important lipid mediators with a wide range of functions in the human body. They act mainly via plasma membrane localized prostaglandin receptors, which belong to the G-protein coupled receptor class. Due to their localized formation and short lifetime, it is important to be able to measure the distribution and abundance of prostaglandins in time and/or space. In this study, we present a Foerster resonance energy transfer (FRET)-based conformation sensor of the human prostaglandin E receptor subtype 4 (EP4 receptor), which was capable of detecting prostaglandin E2 (PGE2)-induced receptor activation in the low nanomolar range with a good signal-to-noise ratio. The sensor retained the typical selectivity for PGE2 among arachidonic acid products. Human embryonic kidney cells stably expressing the sensor did not produce detectable amounts of prostaglandins making them suitable for a coculture approach allowing us, over time, to detect prostaglandin formation in Madin-Darby canine kidney cells and primary mouse macrophages. Furthermore, the EP4 receptor sensor proved to be suited to detect experimentally generated PGE2 gradients by means of FRET-microscopy, indicating the potential to measure gradients of PGE2 within tissues. In addition to FRET-based imaging of prostanoid release, the sensor allowed not only for determination of PGE2 concentrations, but also proved to be capable of measuring ligand binding kinetics. The good signal-to-noise ratio at a commercial plate reader and the ability to directly determine ligand efficacy shows the obvious potential of this sensor interest for screening and characterization of novel ligands of the pharmacologically important human EP4 receptor. SIGNIFICANCE STATEMENT: The authors present a biosensor based on the prostaglandin E receptor subtype 4, which is well suited to measure extracellular prostaglandin E2 (PGE2) concentration with high temporal and spatial resolution. It can be used for the imaging of PGE2 levels and gradients by means of Foerster resonance energy transfer microscopy, and for determining PGE2 release of primary cells as well as for screening purposes in a plate reader setting.

Orphan G Protein-Coupled Receptor GPRC5B Controls Smooth Muscle Contractility and Differentiation by Inhibiting Prostacyclin Receptor Signaling.

BACKGROUND: G protein-coupled receptors are important regulators of contractility and differentiation in vascular smooth muscle cells (SMCs), but the specific function of SMC-expressed orphan G protein-coupled receptor class C group 5 member B (GPRC5B) is unclear. METHODS: We studied the role of GPRC5B in the regulation of contractility and dedifferentiation in human and murine SMCs in vitro and in iSM-Gprc5b-KO (tamoxifen-inducible, SMC-specific knockout) mice under conditions of arterial hypertension and atherosclerosis in vivo. RESULTS: Mesenteric arteries from SMC-specific Gprc5b-KOs showed ex vivo significantly enhanced prostacyclin receptor (IP)-dependent relaxation, whereas responses to other relaxant or contractile factors were normal. In vitro, knockdown of GPRC5B in human aortic SMCs resulted in increased IP-dependent cAMP production and consecutive facilitation of SMC relaxation. In line with this facilitation of IP-mediated relaxation, iSM-Gprc5b-KO mice were protected from arterial hypertension, and this protective effect was abrogated by IP antagonists. Mechanistically, we show that knockdown of GPRC5B increased the membrane localization of IP both in vitro and in vivo and that GPRC5B, but not other G protein-coupled receptors, physically interacts with IP. Last, we show that enhanced IP signaling in GPRC5B-deficient SMCs not only facilitates relaxation but also prevents dedifferentiation during atherosclerosis development, resulting in reduced plaque load and increased differentiation of SMCs in the fibrous cap. CONCLUSIONS: Taken together, our data show that GPRC5B regulates vascular SMC tone and differentiation by negatively regulating IP signaling.

Lifetime of muscarinic receptor-G-protein complexes determines coupling efficiency and G-protein subtype selectivity.

G-protein-coupled receptors (GPCRs) are essential for the detection of extracellular stimuli by cells and transfer the encoded information via the activation of functionally distinct subsets of heterotrimeric G proteins into intracellular signals. Despite enormous achievements toward understanding GPCR structures, major aspects of the GPCR-G-protein selectivity mechanism remain unresolved. As this can be attributed to the lack of suitable and broadly applicable assays, we set out to develop a quantitative FRET-based assay to study kinetics and affinities of G protein binding to activated GPCRs in membranes of permeabilized cells in the absence of nucleotides. We measured the association and dissociation kinetics of agonist-induced binding of Gi/o, Gq/11, Gs, and G12/13 proteins to muscarinic M1, M2, and M3 receptors in the absence of nucleotides between fluorescently labeled G proteins and receptors expressed in mammalian cells. Our results show a strong quantitative correlation between not the on-rates of G-protein-M3-R interactions but rather the affinities of Gq and Go proteins to M3-Rs, their GPCR-G-protein lifetime and their coupling efficiencies determined in intact cells, suggesting that the G-protein subtype-specific affinity to the activated receptor in the absence of nucleotides is, in fact, a major determinant of the coupling efficiency. Our broadly applicable FRET-based assay represents a fast and reliable method to quantify the intrinsic affinity and relative coupling selectivity of GPCRs toward all G-protein subtypes.

Voltage Dependence of Prostanoid Receptors.

G protein-coupled receptors (GPCRs) are the largest class of transmembrane receptors and serve as signal mediators to transduce information from extracellular signals such as neurotransmitters, hormones, or drugs to cellular responses. They are exposed to the strong electrical field of the plasma membrane. In the last decade voltage modulation of ligand-induced GPCR activity has been reported for several GPCRs. Using Foerster resonance energy transfer-based biosensors in patch clamp experiments, we discovered a robust voltage dependence of the thromboxane receptor (TP receptor) on the receptor level as well as on downstream signaling. TP receptor activity doubled upon depolarization from -90 to +60 mV in the presence of U46619, a stable analog of prostaglandin H2 Half-maximal effective potential (V0.5) determined for TP receptor was -46 mV, which is within the physiologic range. We identified that depolarization affected the agonist affinity for the TP receptor. Depolarization enhanced responses of several structural analogs of U46619 with modifications to a similar extent all around the molecule, indicating that voltage modulates the general conformation of TP receptor. By means of site direct mutagenesis, we identified TP receptor R2957.40, which showed alteration of voltage sensitivity of TP receptor upon mutation. Voltage sensitivity was not limited to TP receptor because prostaglandin F receptor activated with U46619 and prostaglandin E2 receptor subtype 3 activated with iloprost showed a similar reaction to depolarization as TP receptor. However, prostacyclin receptor activated with iloprost showed no detectable voltage dependence. SIGNIFICANCE STATEMENT: Prostanoids mediate many of their physiological effects via transmembrane receptors expressed in the plasma membrane of excitable cells. We found that agonist-mediated activation of prostaglandin F receptors and prostaglandin E2 receptors as well as thromboxane receptors are activated upon depolarization, whereas prostacyclin receptors are not. The voltage-induced modulation of thromboxane receptor activity was observed on the level of receptor conformation and downstream signaling. The range of voltage dependence was restricted by R2957.40 in the agonist-binding pocket.

Comparative Docking to Distinct G Protein-Coupled Receptor Conformations Exclusively Yields Ligands with Agonist Efficacy.

G protein-coupled receptors exist in a whole spectrum of conformations that are stabilized by the binding of ligands with different efficacy or intracellular effector proteins. Here, we investigate whether three-dimensional structures of receptor conformations in different states of activation can be used to enrich ligands with agonist behavior in prospective docking calculations. We focused on the ß 2-adrenergic receptor, as it is currently the receptor with the highest number of active-state crystal structures. Comparative docking calculations to distinct conformations of the receptor were used for the in silico prediction of ligands with agonist efficacy. The pharmacology of molecules selected based on these predictions was characterized experimentally, resulting in a hit rate of 37% ligands, all of which were agonists. The ligands furthermore contain a pyrazole moiety that has previously not been described for ß 2-adrenergic receptor ligands, and one of them shows an intrinsic efficacy comparable to salbutamol. SIGNIFICANCE STATEMENT: Structure-based ligand design for G protein-coupled receptors crucially depends on receptor conformation and, hence, their activation state. We explored the influence of using multiple active-conformation X-ray structures on the hit rate of docking calculations to find novel agonists, and how to predict the most fruitful strategy to apply. The results suggest that aggregating the ranks of molecules across docking calculations to more than one active-state structure exclusively yields agonists.

The N-termini of GRK2 and GRK3 simulate the stimulating effects of RKIP on ß-adrenoceptors.

The Raf kinase inhibitor protein (RKIP) activates ß-adrenoceptors (ß-AR) and thereby induces a well-tolerated cardiac contractility and prevents heart failure in mice. Different to RKIP-mediated ß-AR activation, chronic activation of ß-AR by catecholamines was shown to be detrimental for the heart. RKIP is an endogenous inhibitor of G protein coupled receptor kinase 2 (GRK2); it binds GRK2 and thereby inhibits GRK2 mediated ß-AR phosphorylation and desensitization. Here, we evaluate RKIP-mediated effects on ß-AR to explore new strategies for ß-AR modulation. Co-immunoprecipitation assays and pull-down assays revealed subtype specificity of RKIP for the cardiac GRK isoforms GRK2 and GRK3 - not GRK5 - as well as several RKIP binding sites within their N-termini (GRK21-185 and GRK31-185). Overexpression of these N-termini prevented ß2-AR phosphorylation and internalization, subsequently increased receptor signaling in HEK293 cells and cardiomyocyte contractility. Co-immunoprecipitation assays of ß2-AR with these N-terminal GRK fragments revealed a direct interaction suggesting a steric interference of the fragments with the functional GRK-receptor interaction. Altogether, N-termini of GRK2 and GRK3 efficiently simulate RKIP effects on ß-AR signaling in HEK293 cells and in cardiomyocytes by their binding to ß2-AR and, thus, provide important insights for the development of new strategies to modulate ß2-AR signaling.

Related GPCRs couple differently to Gs: preassociation between G protein and 5-HT7 serotonin receptor reveals movement of Gαs upon receptor activation.

How GPCRs and G proteins interact is important for their biologic functions and their functions as pharmacologic targets. It is still an open question whether receptors and G proteins are preassembled in a complex or interact only after receptor activation. We compared the propensity of the two Gs-coupled serotonin (5-HT) receptors 5-HT4 and 5-HT7 to associate with G protein prior to agonist activation. Combining receptor-immobilized fluorescence recovery after photobleaching and fluorescence resonance energy transfer methodologies, we observed that 5-HT7 receptors markedly reduced the diffusion of both Gα and Gßγ at the cell surface, which indicated 5-HT7 receptor preassociation with Gs. This is in sharp contrast to the 5-HT4 receptor for which the diffusion of Gαßγ was not modified, and agonist activation brought together the receptor and Gγ, which is consistent with interaction by collision coupling. Agonist activation of 5-HT7 dissociated Gγ from the receptor, whereas Gαs underwent a rapid conformational change with respect to both Gγ and the receptor, followed by a slower dissociation of Gγ from both Gαs and the receptor. Taken together, these data demonstrate a different propensity among receptors to preassociate with G protein in the absence of ligand and reveals a rapid conformational change in Gαs upon activation by the receptor.-Andressen, K. W., Ulsund, A. H., Krobert, K. A., Lohse, M. J., Bünemann, M., Levy, F. O. Related GPCRs couple differently to Gs: preassociation between G protein and 5-HT7 serotonin receptor reveals movement of Gαs upon receptor activation.

Potentiation of receptor responses induced by prolonged binding of Gα13 and leukemia-associated RhoGEF.

Diverse cellular functions are controlled by RhoA-GTPases, which are activated by trimeric G proteins via RhoGEFs, among others. In this study, we focused on the signaling from GPCRs to RhoA via Gα13 and leukemia-associated RhoGEF (LARG). The activation of Gα13 was elucidated in living cells with high temporal and spatial resolution by means of FRET. The inactivation after agonist withdrawal occurred in the same range (t1/2 = 25.3 ± 2.2 s; mean ± sem; n = 22) as described for other Gα proteins. The interaction of Gα13 and LARG and the thereby-induced LARG translocation to the plasma membrane were at least 1 order of magnitude more stable after agonist withdrawal, exceeding Gα13 deactivation in the absence of LARG several fold. Consequently, we observed an almost 100-fold higher agonist sensitivity of the Gα13 LARG interaction compared to the Gα13 activation in the absence of LARG.-Bodmann, E.-L., Krett, A.-L., Bünemann, M. Potentiation of receptor responses induced by prolonged binding of Gα13 and leukemia-associated RhoGEF.

Identification of key phosphorylation sites in PTH1R that determine arrestin3 binding and fine-tune receptor signaling.

The parathyroid hormone receptor 1 (PTH1R) is a member of family B of G-protein-coupled receptors (GPCRs), predominantly expressed in bone and kidney where it modulates extracellular Ca2+ homeostasis and bone turnover. It is well established that phosphorylation of GPCRs constitutes a key event in regulating receptor function by promoting arrestin recruitment and coupling to G-protein-independent signaling pathways. Mapping phosphorylation sites on PTH1R would provide insights into how phosphorylation at specific sites regulates cell signaling responses and also open the possibility of developing therapeutic agents that could target specific receptor functions. Here, we have used mass spectrometry to identify nine sites of phosphorylation in the C-terminal tail of PTH1R. Mutational analysis revealed identified two clusters of serine and threonine residues (Ser489-Ser495 and Ser501-Thr506) specifically responsible for the majority of PTH(1-34)-induced receptor phosphorylation. Mutation of these residues to alanine did not affect negatively on the ability of the receptor to couple to G-proteins or activate extracellular-signal-regulated kinase 1/2. Using fluorescence resonance energy transfer and bioluminescence resonance energy transfer to monitor PTH(1-34)-induced interaction of PTH1R with arrestin3, we show that the first cluster Ser489-Ser495 and the second cluster Ser501-Thr506 operated in concert to mediate both the efficacy and potency of ligand-induced arrestin3 recruitment. We further demonstrate that Ser503 and Thr504 in the second cluster are responsible for 70% of arrestin3 recruitment and are key determinants for interaction of arrestin with the receptor. Our data are consistent with the hypothesis that the pattern of C-terminal tail phosphorylation on PTH1R may determine the signaling outcome following receptor activation.

Membrane Potential Controls the Efficacy of Catecholamine-induced ß1-Adrenoceptor Activity.

G protein-coupled receptors (GPCRs) are membrane-located proteins and, therefore, are exposed to changes in membrane potential (V(M)) in excitable tissues. These changes have been shown to alter receptor activation of certain Gi-and Gq-coupled GPCRs. By means of a combination of whole-cell patch-clamp and Förster resonance energy transfer (FRET) in single cells, we demonstrate that the activation of the Gs-coupled ß1-adrenoreceptor (ß1-AR) by the catecholamines isoprenaline (Iso) and adrenaline (Adr) is regulated by V(M). This voltage-dependence is also transmitted to G protein and arrestin 3 signaling. Voltage-dependence of ß2-AR activation, however, was weak compared with ß1-AR voltage-dependence. Drug efficacy is a major target of ß1-AR voltage-dependence as depolarization attenuated receptor activation, even under saturating concentrations of agonists, with significantly faster kinetics than the deactivation upon agonist withdrawal. Also the efficacy of the endogenous full agonist adrenaline was reduced by depolarization. This is a unique finding since reports of natural full agonists at other voltage-dependent GPCRs only show alterations in affinity during depolarization. Based on a Boltzmann function fit to the relationship of V(M) and receptor-arrestin 3 interaction we determined the voltage-dependence with highest sensitivity in the physiological range of membrane potential. Our data suggest that under physiological conditions voltage regulates the activity of agonist-occupied ß1-adrenoceptors on a very fast time scale.

Interhelical interaction and receptor phosphorylation regulate the activation kinetics of different human ß1-adrenoceptor variants.

G protein-coupled receptors represent the largest class of drug targets, but genetic variation within G protein-coupled receptors leads to variable drug responses and, thereby, compromises their therapeutic application. One of the most intensely studied examples is a hyperfunctional variant of the human ß1-adrenoceptor that carries an arginine at position 389 in helix 8 (Arg-389-ADRB1). However, the mechanism underlying the higher efficacy of the Arg-389 variant remained unclear to date. Despite its hyperfunctionality, we found the Arg-389 variant of ADRB1 to be hyperphosphorylated upon continuous stimulation with norepinephrine compared with the Gly-389 variant. Using ADRB1 sensors to monitor activation kinetics by fluorescence resonance energy transfer, Arg-389-ADRB1 exerted faster activation speed and arrestin recruitment than the Gly-389 variant. Both activation speed and arrestin recruitment depended on phosphorylation of the receptor, as shown by knockdown of G protein-coupled receptor kinases and phosphorylation-deficient ADRB1 mutants. Structural modeling of the human ß1-adrenoceptor suggested interaction of the side chain of Arg-389 with opposing amino acid residues in helix 1. Site-directed mutagenesis of Lys-85 and Thr-86 in helix 1 revealed that this interaction indeed determined ADRB1 activation kinetics. Taken together, these findings indicate that differences in interhelical interaction regulate the different activation speed and efficacy of ADRB1 variants.

Adenylyl cyclases 5 and 6 underlie PIP3-dependent regulation.

Many different neurotransmitters and hormones control intracellular signaling by regulating the production of the second messenger cAMP. The function of the broadly expressed adenylyl cyclases (ACs) 5 and 6 is regulated by either stimulatory or inhibitory G proteins. By analyzing a well-known rebound stimulation phenomenon after withdrawal of Gi protein in atrial myocytes, we discovered that AC5 and -6 are tightly regulated by the second messenger PIP3. By monitoring cAMP levels in real time by means of Förster resonance energy transfer (FRET)-based biosensors, we reproduced the rebound stimulation in a heterologous expression system specifically for AC5 or -6. Strikingly, this cAMP rebound stimulation was completely blocked by the PI3K inhibitor wortmannin, both in atrial myocytes and in transfected human embryonic kidney cells. Similar effects were observed by heterologous expression of the PIP3 phosphatase and tensin homolog (PTEN). However, general kinase inhibitors or inhibitors of Akt had no effect, suggesting a PIP3-dependent mechanism. These findings demonstrate the existence of a novel general pathway for regulation of AC5 and -6 activity via PIP3 that leads to pronounced alterations of cytosolic cAMP levels.

Voltage regulates adrenergic receptor function.

The present study demonstrates that agonist-mediated activation of α2A adrenergic receptors (α(2A)AR) is voltage-dependent. By resolving the kinetics of conformational changes of α(2A)AR at defined membrane potentials, we show that negative membrane potentials in the physiological range promote agonist-mediated activation of α(2A)AR. We discovered that the conformational change of α(2A)AR by voltage is independent from receptor-G protein docking and regulates receptor signaling, including ß-arrestin binding, activation of G proteins, and G protein-activated inwardly rectifying K(+) currents. Comparison of the dynamics of voltage-dependence of clonidine- vs. norepinephrine-activated receptors uncovers interesting mechanistic insights. For norepinephrine, the time course of voltage-dependent deactivation reflected the deactivation kinetics of the receptor after agonist withdrawal and was strongly attenuated at saturating concentrations. In contrast, clonidine-activated α(2A)AR were switched by voltage even under fully saturating concentrations, and the kinetics of this switch was notably faster than dissociation of clonidine from α(2A)AR, indicating voltage-dependent regulation of the efficacy. We conclude that adrenergic receptors exhibit a unique, agonist-dependent mechanism of voltage-sensitivity that modulates downstream receptor signaling.

Influence of gαq on the dynamics of m3-acetylcholine receptor-g-protein-coupled receptor kinase 2 interaction.

G-protein-coupled receptor kinase 2 (GRK2) is a serine/threonine kinase with an important function in the desensitization of G-protein-coupled receptors. Based on its ability to bind G-protein ßγ subunits as well as activated Gαq subunits, it can be considered as an effector for G-proteins. The recruitment of GRK2 to activated receptors is well known to be mediated by Gßγ together with negatively charged membrane phospholipids. In the current study, we address the role of Gαq on the interaction of GRK2 with activated Gq-protein-coupled receptors. Therefore, we established new Förster resonance energy transfer (FRET)-based assays to study the interaction of GRK2 with the M3-acetylcholine (M3-ACh) receptor as well as Gq-protein subunits with high spatiotemporal resolution in single living human embryonic kidney 293T cells. M3-ACh receptor stimulation with 10 µM acetylcholine resulted in distinct changes in FRET, which reflects interaction of the respective proteins. GRK2 mutants with reduced binding affinity toward Gαq [GRK2(D110A)] and Gßγ [GRK2(R587Q)] were used to determine the specific role of Gq-protein-binding by GRK2. Comparison of absolute FRET amplitudes demonstrated that Gαq enhances the extent and stability of the GRK2-M3-ACh receptor interaction, and that not only Gßγ but also Gαq can target GRK2 to the membrane. This reveals an important role of Gαq in efficient recruitment of GRK2 to M3-ACh receptors. Furthermore, interactions between Gαq and GRK2 were associated with a prolongation of the interaction between GRK2 and the M3-ACh receptor and enhanced arrestin recruitment by these receptors, indicating that Gαq influences signaling and desensitization.

Engineered hyperphosphorylation of the ß2-adrenoceptor prolongs arrestin-3 binding and induces arrestin internalization.

G protein-coupled receptor phosphorylation plays a major role in receptor desensitization and arrestin binding. It is, however, unclear how distinct receptor phosphorylation patterns may influence arrestin binding and subsequent trafficking. Here we engineer phosphorylation sites into the C-terminal tail of the ß2-adrenoceptor (ß2AR) and demonstrate that this mutant, termed ß2AR(SSS), showed increased isoprenaline-stimulated phosphorylation and differences in arrestin-3 affinity and trafficking. By measuring arrestin-3 recruitment and the stability of arrestin-3 receptor complexes in real time using fluorescence resonance energy transfer and fluorescence recovery after photobleaching, we demonstrate that arrestin-3 dissociated quickly and almost completely from the ß2AR, whereas the interaction with ß2AR(SSS) was 2- to 4-fold prolonged. In contrast, arrestin-3 interaction with a ß2-adrenoceptor fused to the carboxyl-terminal tail of the vasopressin type 2 receptor was nearly irreversible. Further analysis of arrestin-3 localization revealed that by engineering phosphorylation sites into the ß2-adrenoceptor the receptor showed prolonged interaction with arrestin-3 and colocalization with arrestin in endosomes after internalization. This is in contrast to the wild-type receptor that interacts transiently with arrestin-3 at the plasma membrane. Furthermore, ß2AR(SSS) internalized more efficiently than the wild-type receptor, whereas recycling was very similar for both receptors. Thus, we show how the interaction between arrestins and receptors can be increased with minimal receptor modification and that relatively modest increases in receptor-arrestin affinity are sufficient to alter arrestin trafficking.

Dynamics of Gαq-protein-p63RhoGEF interaction and its regulation by RGS2.

Some G-protein-coupled receptors regulate biological processes via Gα12/13- or Gαq/11-mediated stimulation of RhoGEFs (guanine-nucleotide-exchange factors). p63RhoGEF is known to be specifically activated by Gαq/11 and mediates a major part of the acute response of vascular smooth muscle cells to angiotensin II treatment. In order to gain information about the dynamics of receptor-mediated activation of p63RhoGEF, we developed a FRET-based assay to study interactions between Gαq-CFP and Venus-p63RhoGEF in single living cells. Upon activation of histaminergic H1 or muscarinic M3 receptors, a robust FRET signal occurred that allowed for the first time the analysis of the kinetics of this interaction in detail. On- and off-set kinetics of Gαq-p63RhoGEF interactions closely resembled the kinetics of Gαq activity. Analysis of the effect of RGS2 (regulator of G-protein signalling 2) on the dynamics of Gαq activity and their interaction with p63RhoGEF showed that RGS2 is able to accelerate both deactivation of Gαq proteins and dissociation of Gαq and p63RhoGEF to a similar extent. Furthermore, we were able to detect activation-dependent FRET between RGS2 and p63RhoGEF and observed a reduced p63RhoGEF-mediated downstream signalling in the presence of RGS2. In summary, these observations support the concept of a functional activation-dependent p63RhoGEF-Gαq-RGS2 complex.

Dynamics of adenylate cyclase regulation via heterotrimeric G-proteins.

A wide variety of G-protein-coupled receptors either activate or inhibit ACs (adenylate cyclases), thereby regulating cellular cAMP levels and consequently inducing proper physiological responses. Stimulatory and inhibitory G-proteins interact directly with ACs, whereas G(q)-coupled receptors exert their effects primarily via Ca2+. Using the FRET-based cAMP sensor Epac1 (exchange protein directly activated by cAMP 1)-cAMPS (adenosine 3',5'-cyclic monophosphorothioate), we studied cAMP levels in single living VSMCs (vascular smooth muscle cells) or HUVECs (human umbilical vein endothelial cells) with subsecond temporal resolution. Stimulation of purinergic (VSMCs) or thrombin (HUVECs) receptors rapidly decreased cAMP levels in the presence of the ß-adrenergic agonist isoprenaline via a rise in Ca2+ and subsequent inhibition of AC5 and AC6. Specifically in HUVECs, we observed that, in the continuous presence of thrombin, cAMP levels climbed slowly after the initial decline with a delay of a little less than 1 min. The underlying mechanism includes phospholipase A2 activity and cyclo-oxygenase-mediated synthesis of prostaglandins. We studied further the dynamics of the inhibition of ACs via G(i)-proteins utilizing FRET imaging to resolve interactions between fluorescently labelled G(i)-proteins and AC5. FRET between Gα(i1) and AC5 developed at much lower concentration of agonist compared with the overall G(i)-protein activity. We found the dissociation of Gα(i1) subunits and AC5 to occur slower than the G(i)-protein deactivation. This led us to the conclusion that AC5, by binding active Gα(i1), interferes with G-protein deactivation and reassembly and thereby might sensitize its own regulation.

Dynamics of Gαi1 interaction with type 5 adenylate cyclase reveal the molecular basis for high sensitivity of Gi-mediated inhibition of cAMP production.

Many physiological and pathophysiological processes are regulated by cAMP. Different therapies directly or indirectly influence the cellular concentration of this second messenger. A wide variety of receptors either activates or inhibits adenylate cyclases in order to induce proper physiological responses. A key event in this signalling system is the direct and dynamic interaction of Gαi1 subunits with adenylate cyclases. We established a FRET-based assay between G-protein subunits and AC5 (type 5 adenylate cyclase) and monitored receptor-stimulated interactions between Gαi1 and AC5 in single intact cells with high temporal resolution. We observed that FRET between Gαi1 and AC5 developed at much lower concentration of agonist compared with the overall Gi-protein activity resulting in a left-shift of the concentration-response curve by approximately one order of magnitude. Furthermore, Gi1-protein-mediated attenuation of AC5-dependent increases in cAMP occurred at comparable low concentrations of agonist. On analysing the dynamics we found the dissociation of the Gαi1 subunits and AC5 to occur significantly slower than the G-protein deactivation and to be insensitive to RGS4 (regulator of G-protein signalling type 4) expression. This led us to the conclusion that AC5, by binding active Gαi1, interferes with G-protein deactivation and reassembly and thereby might sensitize its own regulation.

DP2 receptor activity sensor suited for antagonist screening and measurement of receptor dynamics in real-time.

The DP2 receptor is a G-protein coupled receptor involved in allergic inflammation and is the target of recently developed antagonists already being tested in clinics. To get insights into DP2 receptor dynamics and to study its pharmacology on the level of the receptor, we constructed a fluorescence resonance energy transfer-based conformation sensor. The sensor reflects the selectivity profile of the DP2 receptor-wt and is suited for screening of agonists and antagonists due to its robust response. Furthermore, the sensor enables the direct measurement of DP2 receptor dynamics in real-time and revealed markedly distinct on- and off-rates of prostaglandin D2 between DP2 and DP1 receptors, suggesting a different mechanism of ligand receptor interaction.