Gi Proteins Regulate Adenylyl Cyclase Activity Independent of Receptor Activation

Background and purpose

Despite the view that only β₂- as opposed to β₁-adrenoceptors (βARs) couple to G_i, some data indicate that the β₁AR-evoked inotropic response is also influenced by the inhibition of G_i. Therefore, we wanted to determine if G_i exerts tonic receptor-independent inhibition upon basal adenylyl cyclase (AC) activity in cardiomyocytes.

Experimental approach

We used the G_s-selective (R,R)- and the G_s- and G_i-activating (R,S)-fenoterol to selectively activate β₂ARs (β₁AR blockade present) in combination with G_i inactivation with pertussis toxin (PTX). We also determined the effect of PTX upon basal and forskolin-mediated responses. Contractility was measured ex vivo in left ventricular strips and cAMP accumulation was measured in isolated ventricular cardiomyocytes from adult Wistar rats.

Key results

PTX amplified both the (R,R)- and (R,S)-fenoterol-evoked maximal inotropic response and concentration-dependent increases in cAMP accumulation. The EC₅₀ values of fenoterol matched published binding affinities. The PTX enhancement of the G_s-selective (R,R)-fenoterol-mediated responses suggests that G_i regulates AC activity independent of receptor coupling to G_i protein. Consistent with this hypothesis, forskolin-evoked cAMP accumulation was increased and inotropic responses to forskolin were potentiated by PTX treatment. In non-PTX-treated tissue, phosphodiesterase (PDE) 3 and 4 inhibition or removal of either constitutive muscarinic receptor activation of G_i with atropine or removal of constitutive adenosine receptor activation with CGS 15943 had no effect upon contractility. However, in PTX-treated tissue, PDE3 and 4 inhibition alone increased basal levels of cAMP and accordingly evoked a large inotropic response.

Conclusions and implications

Together, these data indicate that G_i exerts intrinsic receptor-independent inhibitory activity upon AC. We propose that PTX treatment shifts the balance of intrinsic G_i and G_s activity upon AC towards G_s, enhancing the effect of all cAMP-mediated inotropic agents.     

Gαo/i-stimulated proteosomal degradation of RGS20: A mechanism for temporal integration of Gs and Gi pathways

The G_s and G_i pathways interact to control the levels of intracellular cAMP. Although coincident signaling through G_s and G_i-coupled receptors can attenuate G_s-stimulated cAMP levels, it is not known if prior activation of the G_i pathway can affect signaling by G_s-coupled receptors. We have found that activated Gα_o/i interact with RGS20, a GTPase activating protein for members of the Gα_ο/i family. Interaction between Gα_o/i and RGS20 results in decreased cellular levels of RGS20. This decrease was induced by activated Gα_o and Gα_i2 but not by Gα_q, Gα_i1 or Gα_i3. The Gα_o/i-induced decrease in RGS20 can be blocked by proteasomal inhibitors lactacystin or MG132. Activated Gα_o stimulates the ubiquitination of RGS20. The serotonin-1A receptor that couples to G_o/i reduces the levels of RGS20 and this effect is blocked by lactacystin, suggesting that G_o/i promotes the degradation of RGS20. Expression of RGS20 attenuates the inhibition of β-adrenergic receptor-induced cAMP levels mediated by the serotonin-1A receptor. Prior activation of the serotonin-1A receptor results in loss of the RGS20-mediated attenuation, and the loss of attenuation is blocked when lactacystin is included during the prior treatment. These observations suggest that G_o/i-coupled receptors, by stimulating the degradation of RGS20, can regulate how subsequent activation of the G_s and G_i pathways controls cellular cAMP levels, thus allowing for signal integration.     

β1‐adrenergic receptor antagonists signal via PDE4 translocation

It is generally assumed that antagonists of G_s‐coupled receptors do not activate cAMP signalling, because they do not stimulate cAMP production via G_s‐protein/adenylyl cyclase activation. Here, we report a new signalling pathway whereby antagonists of β₁‐adrenergic receptors (β₁ARs) increase cAMP levels locally without stimulating cAMP production directly. Binding of antagonists causes dissociation of a preformed complex between β₁ARs and Type‐4 cyclic nucleotide phosphodiesterases (PDE4s). This reduces the local concentration of cAMP‐hydrolytic activity, thereby increasing submembrane cAMP and PKA activity. Our study identifies receptor/PDE4 complex dissociation as a novel mechanism of antagonist action that contributes to the pharmacological properties of β₁AR antagonists and might be shared by other receptor subtypes.     

Multiple G proteins compete for binding with the human gonadotropin releasing hormone receptor

The GnRH receptor is coupled to G proteins of the families G_q and G₁₁. G_q and G₁₁ coupling leads to intracellular signaling through the phospholipase C pathway. GnRHR coupling to other G proteins is controversial. This study provides evidence that G protein families G_s, G_i, G_q and G₁₁ complete for binding with the GnRHR. We quantified interactions of over-expressed G proteins with GnRHR by a competitive binding approach, using measurements of second messengers, IP and cAMP. Transient co-transfection of HEK293 cells with human WT GnRHR and with stimulatory and inhibitory G proteins (G_q, G₁₁ and G_s, G_i) led to either production or inhibition of total inositol phosphate (IP) production, depending on the G protein that was over-expressed. Studies were conducted in different human (COS7, HeLa) and rodent-derived (CHO-K1, GH₃) cell lines in order to confirm that G protein promiscuity observed with the GnRHR was not limited to a particular cell type.     

Single amino acid of an octopamine receptor as a molecular switch for distinct G protein couplings

Octopamine (OA) is thought to be the invertebrate counterpart of noradrenaline and regulates various behavioral patterns of invertebrates by activating OA receptors. As a typical G protein-coupled receptor, BmOAR1, a Bombyx mori α-adrenergic-like OA receptor, is coupled to both G_s and G_q proteins to induce the release of the intracellular second messengers cAMP and Ca²⁺. In this study, we examined the pharmacological and functional properties of the cloned OA receptor, using OA enantiomers. The wild-type OA receptor exhibited significant stereoselectivity for OA enantiomers in cAMP production and binding affinity, but not in calcium signaling response. On the contrary, the Y412F mutant abolished the discrimination between OA enantiomers in the binding affinity and did not evoke any cAMP signaling response. This mutant exhibited levels of potency and efficacy similar to those of the wild-type receptor in the calcium assays. Taken together, these results suggest that Tyr412 might act as a molecular switch to regulate distinct G protein couplings, and a sequential activation model is proposed for such specific-residue-dependent, selective activation in receptors that are coupled to multiple G proteins.     

Innovative functional cAMP assay for studying G protein-coupled receptors: application to the pharmacological characterization of GPR17

In this work, an innovative and non-radioactive functional cAMP assay was validated at the GPR17 receptor. This assay provides a simple and powerful new system to monitor G protein-coupled receptor activity through change in the intracellular cAMP concentration by using a mutant form of Photinus pyralis luciferase into which a cAMP-binding protein moiety has been inserted. Results, expressed as EC₅₀ or IC₅₀ values for agonists and antagonists, respectively, showed a strong correlation with those obtained with [³⁵S]GTPγS binding assay, thus confirming the validity of this approach in the study of new ligands for GPR17. Moreover, this method allowed confirming that GPR17 is coupled with a G_αi.     

Regulation of β-adrenergic responses during in vitro differentiation of mouse erythroleukemia cells

Dimethyl sulfoxide (DMSO)-induced erythroid differentiation of Friend mouse erythroleukemia (MEL) cells is associated with a marked transient modulation of catecholamine sensitivity. Within 24 h after induction and well before the onset of hemoglobin synthesis, we observed a 3-fold increase in β-receptor density and a more than 10-fold increase in receptor-coupled cAMP formation. During the following 4 days, in parallel with the development of normoblast-like cells, receptor numbers returned to preinduction levels while catecholamine-dependent cAMP formation remained significantly elevated. Simultaneously, the apparent potency of the β-adrenoceptor agonist isoprenaline increased 10-fold. Improved receptor—cyclase coupling is probably due to a major shift in the expression of G_i and G_s regulatory proteins. Bacterial toxin-mediated ADP-ribosylation of membrane proteins suggests that the dominating species in native cells is G_i (G_sα:G_iα = 1:7). By contrast, G_s predominates in differentiated cells (G_sα:G_iα = 1.8:1). Receptor-independent forskolin-stimulated cAMP formation showed a pronounced, albeit transient, decrease during differentiation. We suggest that these changes in cellular cAMP responses may be important for transient positive or negative cooperative interactions between hormones and growth factors in the course of erythroid cell development.     

A2A R mediated modulation in IP3 levels altering the [Ca2+]i through cAMP-dependent PKA signalling pathway

Stimulation of A_2A receptors (A_2A R) coupled to G_s/olf protein activates Adenylyl cyclase (AC) leading to the release of cAMP which activates the cAMP-dependent PKA phosphorylation. The possible role of A_2A R in the modulation of free cytosolic Ca²⁺ concentration ([Ca²⁺]_i) involving IP₃, cAMP and PKA was investigated in HEK 293-A_2A R. The levels of IP₃ and cAMP were observed by enzyme immunoassay detection method and [Ca²⁺]_i using Fluo-4 AM. Moreover, cAMP-dependent PKA was determined using the PKA Colorimetric Activity Kit. We observed that the cells pre-treated with A_2A R agonist NECA showed increased levels of cAMP, PKA, IP₃ and [Ca²⁺]_i levels. However, the reverse effect was observed with A_2A R antagonists (ZM241385 and caffeine). Blocking the G_αq/PLC/DAG/IP₃ pathway with neomycin, a PLC inhibitor did not affect the modulation of IP₃ and [Ca²⁺]_i levels in HEK 293-A_2A R cells. To investigate the G_αi/AC/cAMP/PKA, HEK 293-A_2A R cells pre-treated with pertussis toxin followed by forskolin in the presence of A_2A R agonist (NECA) showed no effect on cAMP levels. Further, G_αs/AC/cAMP/PKA pathway was investigated to elucidate the role of cAMP-dependent PKA in IP₃ mediated [Ca²⁺]_i modulation. In the HEK 293-A_2A R cells pre-treated with PKA inhibitor KT5720 and treated with NECA led to inhibit the IP₃ and [Ca²⁺]_i levels. The study distinctly demonstrated that A_2A R modulates IP₃ levels to release the [Ca²⁺]_i via cAMP-dependent PKA. The role of A_2A R mediated G_αs pathway inducing IP₃ mediated [Ca²⁺]_i release may open new avenues in the therapy of neurodegenerative disorder.     

G protein-coupled Receptor 30 (GPR30) Forms a Plasma Membrane Complex with Membrane-associated Guanylate Kinases (MAGUKs) and Protein Kinase A-anchoring Protein 5 (AKAP5) That Constitutively Inhibits cAMP Production

GPR30, or G protein-coupled estrogen receptor, is a G protein-coupled receptor reported to bind 17β-estradiol (E₂), couple to the G proteins G_s and G_i/o, and mediate non-genomic estrogenic responses. However, controversies exist regarding the receptor pharmacological profile, effector coupling, and subcellular localization. We addressed the role of the type I PDZ motif at the receptor C terminus in receptor trafficking and coupling to cAMP production in HEK293 cells and CHO cells ectopically expressing the receptor and in Madin-Darby canine kidney cells expressing the native receptor. GPR30 was localized both intracellularly and in the plasma membrane and subject to limited basal endocytosis. E₂ and G-1, reported GPR30 agonists, neither stimulated nor inhibited cAMP production through GPR30, nor did they influence receptor localization. Instead, GPR30 constitutively inhibited cAMP production stimulated by a heterologous agonist independently of G_i/o. Moreover, siRNA knockdown of native GPR30 increased cAMP production. Deletion of the receptor PDZ motif interfered with inhibition of cAMP production and increased basal receptor endocytosis. GPR30 interacted with membrane-associated guanylate kinases, including SAP97 and PSD-95, and protein kinase A-anchoring protein (AKAP) 5 in the plasma membrane in a PDZ-dependent manner. Knockdown of AKAP5 or St-Ht31 treatment, to disrupt AKAP interaction with the PKA RIIβ regulatory subunit, decreased inhibition of cAMP production, and St-Ht31 increased basal receptor endocytosis. Therefore, GPR30 forms a plasma membrane complex with a membrane-associated guanylate kinase and AKAP5, which constitutively attenuates cAMP production in response to heterologous agonists independently of G_i/o and retains receptors in the plasma membrane.     

Pertussis Toxin-Catalyzed ADP-Ribosylation: Effects on the Coupling of Inhibitory Receptors to the Adenylate Cyclase System

Abstract

The adenylate cyclase system consists of stimulatory and inhibitory hormone and drug receptors coupled through different GTP-binding proteins to a catalytic unit, responsible for the synthesis of cAMP from ATP. Pertussis toxin blocks the effect of inhibitory agonists on the catalytic unit by enzymatically inactivating the inhibitory GTP-binding protein (G_i). Study of the inhibitory arm of the cyclase system has been facilitated by the dissection of the overall process of hormonal inhibition of cAMP formation into a series of reactions characteristic of the individual protein components of this complex system; pertussis toxin has proven to be a useful tool with which to study these individual reactions. Exposure of cells or membranes to pertussis toxin in the presence of NAD results in ADP-ribosylation of a 41,000 Da subunit of G_i. ADP-ribosylation of G_i has a number of effects on the overall and partial reactions of the cyclase system, including a loss of a) hormonal inhibition of cAMP formation, b) hormonal stimulation of GTPase and c) agonist-induced release of membrane-bound guanyl nucleotides. In addition, in toxin-treated membranes, the affinity of inhibitory receptors for agonist but not antagonist is decreased with no significant change in receptor number.     

Label-free optical biosensor for probing integrative role of adenylyl cyclase in G protein-coupled receptor signaling

Adenylyl cyclase is considered as an integrator for receptor signaling. However, its integrative role in receptor signaling is largely studied at the level of point of contacts in complex pathways. Here we used forskolin as a pharmacological probe and the resonant waveguide grating (RWG) biosensor to examine the signal integration of G protein-coupled receptors (GPCRs) at the cyclase-cyclic AMP-PKA module. The biosensor is a refractive index sensitive optical biosensor that is capable of detecting ligand-induced dynamic mass redistribution in cells without labels and cellular manipulations. Stimulation of seven cell lines with forskolin led to distinct optical responses, indicative of distinct expressions and/or organization of cyclase isoforms. The forskolin response in A431 was sensitive to the activities of protein kinase A, Rho kinase, and MAP kinases. Desensitization assays showed that the forskolin pretreatment heterologously desensitized G_s signaling, partially attenuated G_q signaling, but had complicate impacts on G_i signaling. This study documents the integrative role of adenylyl cyclase in GPCR signaling and the power of forskolin as a pharmacological probe to differentiate receptor signaling using the label-free biosensor cellular assays.     

Functional Receptor Coupling to Gi Is A Mechanism of Agonist-Promoted Desensitization of the β2-Adrenergic Receptor

Abstract

The β₂-adrenergic receptor (β₂AR) couples to G_s, activating adenylyl cyclase (AC) and increasing cAMP. Such signaling undergoes desensitization with continued agonist exposure. β₂AR also couple to G_i after receptor phosphorylation by the cAMP dependent protein kinase A, but the efficiency of such coupling is not known. Given the PKA dependence of β₂AR-G_i coupling, we explored whether this may be a mechanism of agonist-promoted desensitization. HEK293 cells were transfected to express β₂AR or β₂AR and G_iα2, and then treated with vehicle or the agonist isoproterenol to evoke agonist-promoted β₂AR desensitization. Membrane AC activities showed that G_iα2 overexpression decreased basal levels, but the fold-stimulation of the AC over basal by agonist was not altered. However, with treatment of the cells with isoproterenol prior to membrane preparation, a marked decrease in agonist-stimulated AC was observed with the cells overexpressing G_iα2. in the absence of such overexpression, β₂AR desensitization was 23 ± 7%, while with 5-fold G_iα2 overexpression desensitization was 58 ± 5% (p<0.01, n=4). the effect of G_i on desensitization was receptor-specific, in that forskolin responses were not altered by G_iα2 overexpression. Thus, acquired β₂AR coupling to G_i is an important mechanism of agonist-promoted desensitization, and pathologic conditions that increase G_i levels contribute to β₂AR dysfunction.     

The thrombin receptor in adrenal medullary microvascular endothelial cells is negatively coupled to adenylyl cyclase through a Gi protein

The effects of thrombin on adenylyl cyclase activity were examined in rat adrenal medullary microvascular endothelial cells (RAMEC). Confluent RAMEC monolayers were stimulated for 5 min with cAMP-generating agents in the absence and presence of thrombin, and intracellular cAMP was measured with a radioligand binding assay. Thrombin (0.001–0.25 U/ml) dose-dependently inhibited IBMX-, isoproterenol- and forskolin-stimulated cAMP accumulation. A peptide agonist of the thrombin receptor, γ-thrombin, and the serine proteases trypsin and plasmin, also inhibited agonist-stimulated cAMP levels, while proteolytically inactive PPACK- or DIP-α-thrombins were without effect. Moreover, the thrombin inhibitor hirudin abolished the inhibitory effect of thrombin but not of the peptide agonist. These results suggest that the inhibitory action of thrombin on cAMP accumulation is mediated by a proteolytically-activated thrombin receptor. The inhibitor of G_i-proteins pertussis toxin abolished the inhibitory effect of thrombin on isoproterenol- or IBMX-stimulated cAMP production, while the phorbol ester PMA partly impaired it. The protein kinase C inhibitors staurosporine or H7 and the intracellular Ca²⁺ chelator BAPTA-AM were without effect. Collectively, our data suggest that the thrombin receptor in RAMEC is negatively coupled to adenylyl cyclase through a pertussis toxin-sensitive G_i-protein.     

Tyrosine 308 Is Necessary for Ligand-directed Gs Protein-biased Signaling of β2-Adrenoceptor

Interaction of a given G protein-coupled receptor to multiple different G proteins is a widespread phenomenon. For instance, β₂-adrenoceptor (β₂-AR) couples dually to G_s and G_i proteins. Previous studies have shown that cAMP-dependent protein kinase (PKA)-mediated phosphorylation of β₂-AR causes a switch in receptor coupling from G_s to G_i. More recent studies have demonstrated that phosphorylation of β₂-AR by G protein-coupled receptor kinases, particularly GRK2, markedly enhances the G_i coupling. We have previously shown that although most β₂-AR agonists cause both G_s and G_i activation, (R,R′)-fenoterol preferentially activates β₂-AR-G_s signaling. However, the structural basis for this functional selectivity remains elusive. Here, using docking simulation and site-directed mutagenesis, we defined Tyr-308 as the key amino acid residue on β₂-AR essential for G_s-biased signaling. Following stimulation with a β₂-AR-G_s-biased agonist (R,R′)-4′-aminofenoterol, the G_i disruptor pertussis toxin produced no effects on the receptor-mediated ERK phosphorylation in HEK293 cells nor on the contractile response in cardiomyocytes expressing the wild-type β₂-AR. Interestingly, Y308F substitution on β₂-AR enabled (R,R′)-4′-aminofenoterol to activate G_i and to produce these responses in a pertussis toxin-sensitive manner without altering β₂-AR phosphorylation by PKA or G protein-coupled receptor kinases. These results indicate that, in addition to the phosphorylation status, the intrinsic structural feature of β₂-AR plays a crucial role in the receptor coupling selectivity to G proteins. We conclude that specific interactions between the ligand and the Tyr-308 residue of β₂-AR stabilize receptor conformations favoring the receptor-G_s protein coupling and subsequently result in G_s-biased agonism.     

Basic Mechanism Leading to Stimulation of Glycogenolysis by Isoproterenol,EGF, Elevated Extracellular K+ Concentrations,or GABA

Glycogenolysis, in brain parenchyma an astrocyte-specific process, has changed from being envisaged as an emergency procedure to playing central roles during brain response to whisker stimulation, memory formation, astrocytic K⁺ uptake and stimulated release of ATP. It is activated by several transmitters and by even very small increases in extracellular K⁺ concentration, and to be critically dependent upon an increase in free cytosolic Ca²⁺ concentration ([Ca²⁺]_i), whereas cAMP plays only a facilitatory role together with increased [Ca²⁺]_i. Detailed knowledge about the signaling pathways eliciting glycogenolysis is therefore of interest and was investigated in the present study in well differentiated cultures of mouse astrocytes. The β-adrenergic agonist isoproterenol stimulated glycogenolysis by a β₁-adrenergic effect, which initiated a pathway in which cAMP/protein kinase A activated a G_i/G_s shift, leading to Ca²⁺-activated glycogenolysis. Inhibition of this pathway downstream of cAMP but upstream of the G_i/G_s shift abolished the glycogenolysis. However, inhibitors operating downstream of the Ca²⁺-sensitive step, but preventing transactivation-mediated epidermal growth factor (EGF) receptor stimulation, a later step in the activated pathway, also caused inhibition of glycogenolysis. For this reason the effect of EGF was investigated and it was found to be glycogenolytic. Large increases in extracellular K⁺ activated glycogenolysis by a nifedipine-inhibited L-channel opening allowing influx of Ca²⁺, known to be glycogenolysis-dependent. Small increases (addition of 5 mM KCl) caused a smaller effect by a similarly glycogenolysis-reliant opening of an IP₃ receptor-dependent ouabain signaling pathway. The same pathway could be activated by GABA (also in brain slices) due to its depolarizing effect in astrocytes.     

A Fluorescent Reporter Assay for the Detection of Ligands Acting Through GI Protein-Coupled Receptors

Abstract

Accompanying the advances in basic biology of G protein-coupled receptors (GPCRs) is the practical need among biopharmaceutical companies for sensitive assays to assess GPCR function, particularly formats that are compatible with high-throughput drug screening. Here we describe a novel cell-based assay format for the high-throughput detection of ligands for G, protein-coupled receptors. Two G_i-GPCRs, μ-opioid receptor (μ-OPR) and 5-hydroxytryptamine receptor la (5HTlaR) are employed as model receptor targets. The key feature of this assay system is the isolation of stable, clonal Chinese hamster ovary (CHO) cell lines that carry three separate expression plasmids: (1) a chimeric G_q/i5 protein (which re-directs a negative G_i-type signal to a positive Gq-type response), (2) a given G_i-GPCR, and (3) a β-lactamase (βla) reporter gene responsive to G_i-GPCR signaling. Cell-based assays built using this format show appropriate rank order of potency among a reference set of receptor agonist and antagonist compounds. Such assays are also robust, reliable, and can be used for industrial-scale applications such as high-throughput screening for drug leads.     

Galanin—A neuropeptide with inhibitory actions

Summary 1. Galanin is a 29 (in humans 30) amino acids long neuropeptide with mostly inhibitory, hyperpolarizing actions.2. Differential structural requirements of truncated forms of galanin and differential agonist/antagonist behaviour of chimeric peptides, high affinity galanin receptor ligands suggest the presence of pharmacologically distinct galanin receptor subtypes.3. The galanin receptor from human Bowes melanoma cell line—a member of G-protein coupled receptor superfamily—has been cloned.4. Galanin acts via G_i/G_o proteins inhibiting cAMP production, inositol phosphate turnover, opening K⁺ channels or closing Ca²⁺ channels.     

Highly efficacious factor Xa inhibitors containing α-substituted phenylcycloalkyl P4 moieties

We previously disclosed a series of highly potent FXa inhibitors bearing α-substituted (CH₂NR¹R²) phenylcyclopropyl P4 moieties in the pyrazolodihydropyridone core system. Herein, we describe our continuous SAR efforts in this series. Effects of the C-3 substitution of the pyrazolodihydropyridone core and the α-substitution (R group) of the cyclopropyl ring on FXa binding affinity (FXa K_i), human plasma anticoagulant activity (PT EC_2×) and permeability are discussed. A set of compounds obtained from optimization of the R group and the C-3 substituent were orally bioavailable in dogs. Furthermore, representative compounds were highly efficacious in the rabbit arterio-venous shunt thrombosis model (EC₅₀s = 29–81 nM).     

Impedance responses reveal β₂-adrenergic receptor signaling pluridimensionality and allow classification of ligands with distinct signaling profiles

The discovery that drugs targeting a single G protein-coupled receptor (GPCR) can differentially modulate distinct subsets of the receptor signaling repertoire has created a challenge for drug discovery at these important therapeutic targets. Here, we demonstrate that a single label-free assay based on cellular impedance provides a real-time integration of multiple signaling events engaged upon GPCR activation. Stimulation of the β₂-adrenergic receptor (β₂AR) in living cells with the prototypical agonist isoproterenol generated a complex, multi-featured impedance response over time. Selective pharmacological inhibition of specific arms of the β₂AR signaling network revealed the differential contribution of G_s-, G_i- and Gβγ-dependent signaling events, including activation of the canonical cAMP and ERK1/2 pathways, to specific components of the impedance response. Further dissection revealed the essential role of intracellular Ca²⁺ in the impedance response and led to the discovery of a novel β₂AR-promoted Ca²⁺ mobilization event. Recognizing that impedance responses provide an integrative assessment of ligand activity, we screened a collection of β-adrenergic ligands to determine if differences in the signaling repertoire engaged by compounds would lead to distinct impedance signatures. An unsupervised clustering analysis of the impedance responses revealed the existence of 5 distinct compound classes, revealing a richer signaling texture than previously recognized for this receptor. Taken together, these data indicate that the pluridimensionality of GPCR signaling can be captured using integrative approaches to provide a comprehensive readout of drug activity.     

Parathyroid hormone 2 receptor is a functional marker of nociceptive myelinated fibers responsible for neuropathic pain

We have previously demonstrated that parathyroid hormone 2 (PTH2) receptors are expressed in dorsal root ganglion (DRG) neurons and that its endogenous agonist tuberoinfundibular peptide of 39 residues (TIP39) causes nociceptive paw flexor responses after intraplantar administration. Here we found that the PTH2 receptor is selectively localized on myelinated A‐, but not unmyelinated C‐fibers using immunohistochemical labeling, based on PTH2 receptor expression on antibody N52‐positive medium/large‐sized DRG neurons, but not on TRPV1, substance P, P2X₃ receptor or isolectin B4‐binding protein‐positive small‐sized DRG neurons. Pharmacological studies showed that TIP39‐induced nociceptive responses were mediated by activation of G_s and cAMP‐dependent protein kinase. We also found that nociceptive responses induced by TIP39‐ or the cAMP analog 8‐bromo‐cAMP were significantly greater following partial sciatic nerve injury induced neuropathic pain, without changes in PTH2 receptor expression. Together these data suggest that activation of PTH2 receptors stimulates nociceptive A‐fiber through G_s‐cAMP‐dependent protein kinase signaling, and this pathway has elevated sensitization following nerve injury.