Replacement of the alpha5 helix of Galpha16 with Galphas-specific sequences enhances promiscuity of Galpha16 toward Gs-coupled receptors

G(16) can couple indiscriminately to a large number of G protein-coupled receptors (GPCRs), making it a prime candidate as a universal adaptor for GPCRs. In order to increase the promiscuity of Galpha(16), three chimeras incorporating increasing lengths of G(s)-specific residues (25, 44 or 81 residues) into the C-terminus of Galpha(16) were constructed and named 16s25, 16s44 and 16s81, respectively. The chimeras were examined for their ability to mediate receptor-induced stimulation of phospholipase C (PLC) and Ca(2+) mobilization. 16s25 was more effective than 16s44 and 16s81 at coupling to G(s)-linked receptors. 16s25 coupled productively to 10 different G(s)-coupled receptors examined and, for 50% of these receptors, 16s25-mediated PLC activities were higher than those mediated via Galpha(16). Similar results were observed for agonist-induced Ca(2+) mobilizations. These results show that incorporating the alpha5 helix of Galpha(s) into Galpha(16) can increase the promiscuity of 16s25 towards G(s)-coupled receptors.     

Chimeric G proteins extend the range of insect cell-based functional assays for human G protein-coupled receptors

We previously described a functional assay for G protein-coupled receptors (GPCRs) based on stably transformed insect cells and using the promiscuous G protein Galpha16. We now show that, compared with Galpha16, the use of chimeric Galphaq subunits with C-terminal modifications (qi5-HA, qo5-HA, or qz5-HA) significantly enhances the ability of insect cells to redirect Gi-coupled GPCRs into a Gq-type signal transduction pathway. We coexpressed human Gi-coupled GPCRs, G protein alpha subunits (either a chimeric Galphaq or Galpha16), and the calcium-sensitive reporter protein aequorin in Sf9 cells using a nonlytic protein expression system, and measured agonist-induced intracellular calcium flux using a luminometer. Three of the GPCRs (serotonin 1A, 1D, and dopamine D2) were functionally redirected into a Gq-type pathway when coexpressed with the chimeric G proteins, compared with only one (serotonin 1A) with Galpha16. We determined agonist concentration-response relationships for all three receptors, which yielded EC50 values comparable with those achieved in mammalian cell-based assay systems. However, three other Gi-coupled GPCRs (the opioid kappa1 and delta1 receptors, and serotonin 1E) were not coupled to calcium flux by either the G protein chimeras or Galpha16. Possible reasons and solutions for this result are discussed.     

Development of a Ca(2+)-activated photoprotein, Photina, and its application to high-throughput screening

The present work describes the engineering and characterization of a new Ca(2+)-activated photoprotein (Photina) and its use in mammalian cell lines for implementation of flash luminescence cell-based assays for high-throughput screening (HTS). When used to measure the activation of 2 G protein-coupled receptors (GPCRs), targeting Photina to the mitochondria increased the signal strength as compared to the normal cytoplasmic expression of Photina. The mitochondrial-targeted Photina also produced a higher signal-to-noise ratio than conventional calcium dyes and a consistently stronger signal than aequorin when tested under equivalent conditions. MitoPhotina provided strong and reliable results when used to measure the activity of purinergic receptors endogenously expressed in the Chinese Hamster Ovary cells and heterologously expressed GPCRs in response to their cognate ligands. Several different types of flash luminescence plate readers (FLIPR(3), FLIPR(TETRA), CyBi-Lumax flash HT, Lumilux, Lumibox) in different plate formats (96, 384, 1536 wells) were used to validate the use of Photina in HTS. The cell number had to be adjusted to correspond to the qualities of the different readers, but once so adjusted, it provided equivalent results on each device. The results obtained show robust and reproducible light signals that offer new possibilities for application of photoproteins to the generation of cell-based assays for HTS.     

Comparative analysis of functional assays for characterization of agonist ligands at G protein-coupled receptors

A variety of functional assays are available for agonist or antagonist screening of G protein-coupled receptors (GPCRs), but it is a priori not predictable which assay is the most suitable to identify agonists or antagonists of GPCRs with therapeutic value in humans. More specifically, it is not known how a given set of GPCR agonists compares in different functional assays with respect to potency and efficacy and whether the level of the signaling cascade that is analyzed has any impact on the detection of agonistic responses. To address this question, the authors used the recently cloned human S1P(5) receptor as a model and compared a set of 3 lipid ligands (sphingosine 1-phosphate [S1P], dihydro sphingosine 1-phosphate [dhS1P], and sphingosine) in 5 different functional assays: GTPgammaS binding, inhibition of adenylyl cyclase activity, mobilization of intracellular Ca(2+) via the FLIPR and aequorin technology, and MAP kinase (ERK1/2) activation. S1P induced agonistic responses in all except the ERK1/2 assays with EC(50) values varying by a factor of 10. Whereas dhS1P was identified as a partial agonist in the GTPgammaS assay, it behaved as a full agonist in all other settings. Sphingosine displayed partial agonistic activity exclusively in GTPgammaS binding assays. The findings suggest that assays in a given cellular background may vary significantly with respect to suitability for agonist finding and that ligands producing a response may not readily be detectable in all agonist assays.     

Use of Caenorhabditis elegans G{alpha}q chimeras to detect G-protein-coupled receptor signals

G-protein-coupled receptors (GPCRs) activate heterotrimeric G-proteins (G(i)-, G(s)-, G(q)-, or G(12)-like) to generate specific intracellular responses, depending on the receptor/G-protein coupling. The aim was to enable a majority of GPCRs to generate a predetermined output by signaling through a single G-protein-supported pathway. The authors focused on calcium responses as the output, then engineered Galpha(q) to promote promiscuous receptor interactions. Starting with a human Galpha(q) containing 5 Galpha(z) residues in the C-terminal receptor recognition domain (hGalpha(q/z5)), they evaluated agonist-stimulated calcium responses for 33 diverse GPCRs (G(i)-, G(s)-, and G(q)-coupled) and found 20 of 33 responders. In parallel, they tested Caenorhabditis elegans Galpha(q) containing 5 or 9 C-terminal Galpha(z) residues (cGalpha(q/z5), cGalpha(q/z9)). Signal detection was enhanced with cGalpha(q/z5) and cGalpha(q/z9) (yielding 25/33 and 26/33 responders, respectively). In a separate study of Galpha(s)-coupled receptors, the authors compared hGalpha(q/s5) versus hGalpha(q/s9), cGalpha(q/s9), andcGalphaq/s21 and observed optimal function with cGalpha(q/s9). Cotransfection of an engineered Galpha(q) "cocktail" (cGalpha(q/z5) plus cGalpha(q/s9)) provided a powerful and efficient screening platform. When the chimeras included N-terminal myristoylation sites (to promote membrane localization), calcium responses were sustained or improved, depending on the receptor. This approach toward a "universal functional assay" is particularly useful for orphan GPCRs whose signaling pathways are unknown.     

Selective inhibition of Cav3.3 T-type calcium channels by Galphaq/11-coupled muscarinic acetylcholine receptors

T-type calcium channels play critical roles in controlling neuronal excitability, including the generation of complex spiking patterns and the modulation of synaptic plasticity, although the mechanisms and extent to which T-type Ca(2+) channels are modulated by G-protein-coupled receptors (GPCRs) remain largely unexplored. To examine specific interactions between T-type Ca(2+) channel subtypes and muscarinic acetylcholine receptors (mAChRS), the Cav3.1 (alpha(1G)), Cav3.2 (alpha(1H)), and Cav3.3 (alpha) T-type Ca(2+)(1I)channels were co-expressed with the M1 Galpha(q/11)-coupled mAChR. Perforated patch recordings demonstrate that activation of M1 receptors has a strong inhibitory effect on Cav3.3 T-type Ca(2+) currents but either no effect or a moderate stimulating effect on Cav3.1 and Cav3.2 peak current amplitudes. This differential modulation was observed for both rat and human T-type Ca(2+) channel variants. The inhibition of Cav3.3 channels by M1 receptors is reversible, use-independent, and associated with a concomitant increase in inactivation kinetics. Loss-of-function experiments with genetically encoded antagonists of Galpha and Gbetagamma proteins and gain-of-function experiments with genetically encoded Galpha subtypes indicate that M1 receptor-mediated inhibition of Cav3.3 occurs through Galpha(q/11). This is supported by experiments showing that activation of the M3 and M5 Galpha(q/11)-coupled mAChRs also causes inhibition of Cav3.3 currents, although Galpha(i)-coupled mAChRs (M2 and M4) have no effect. Examining Cav3.1-Cav3.3 chimeric channels demonstrates that two distinct regions of the Cav3.3 channel are necessary and sufficient for complete M1 receptor-mediated channel inhibition and represent novel sites not previously implicated in T-type channel modulation.     

Molecular dissection of G protein preference using Gsalpha chimeras reveals novel ligand signaling of GPCRs

Although only 16 genes have been identified in mammals, several Galpha subunits can be simultaneously activated by G protein-coupled receptors (GPCRs) to modulate their complicated functions. Current GPCR assays are limited in the evaluation of selective Galpha activation, thus not allowing a comprehensive pathway screening. Because adenylyl cyclases are directly activated by G(s)alpha and the carboxyl termini of the various Galpha proteins determine their receptor coupling specificity, we proposed a set of chimeric G(s)alpha where the COOH-terminal five amino acids are replaced by those of other Galpha proteins and used these to dissect the potential Galpha linked to a given GPCR. Unlike G(q)alpha, G(12)alpha, and G(i)alpha outputs, compounding the signals from several Galpha members, the chimeric G(s)alpha proteins provide a superior molecular approach that reflects the previously uncharacterized pathways of GPCRs under the same cAMP platform. This is, to our knowledge, the first time allowing verification of the whole spectrum of Galpha coupling preference of adenosine A1 receptor, reported to couple to multiple G proteins and modulate many physiological processes. Furthermore, we were able to distinguish the uncharacterized pathways between the two neuromedin U receptors (NMURs), which distribute differently but are stimulated by a common agonist. In contrast to the G(q) signals mainly conducted by NMUR1, NMUR2 routed preferentially to the G(i) pathways. Dissecting the potential Galpha coupling to these GPCRs will promote an understanding of their physiological roles and benefit the pharmaceutical development of agonists/antagonists by exploiting the selective affinity toward a certain Galpha subclass.     

A functional assay for G-protein-coupled receptors using stably transformed insect tissue culture cell lines

Insect cells are an underexplored resource for functional G-protein-coupled receptor (GPCR) assays, despite a strong record in biochemical (binding) assays. Here we describe the use of vectors capable of creating stably transformed insect cell lines to generate a cell-based functional GPCR assay. This assay employs the luminescent photoprotein aequorin and the promiscuous G-protein subunit Galpha16 and is broadly applicable to human GPCRs. We demonstrate that the assay can quantitate ligand concentration-activity relationships for seven different human GPCRs, can differentiate between partial and full agonists, and can determine rank order potencies for both agonists and antagonists that match those seen with other assay systems. Human Galpha16 improves signal strength but is not required for activity with some receptors. The coexpression of human and bovine betagamma subunits and/or phospholipase Cbeta makes no difference to agonist efficacy or potency. Two different receptors expressed in the same cell line respond to their specific agonists, and two different cell lines (Sf9 and High 5) are able to functionally detect the same expressed GPCR. Sf9 cells have the capability to produce fully functional human receptors, allied to a low background of endogenous receptors, and so are a valuable system for investigating orphan GPCRs and receptor dimerization.     

GPCR screening via ERK 1/2: a novel platform for screening G protein-coupled receptors

Discovery of novel agonists and antagonists for G protein-coupled receptors (GPCRs) relies heavily on cell-based assays because determination of functional consequences of receptor engagement is often desirable. Currently, there are several key parameters measured to achieve this, including mobilization of intracellular Ca2+ and formation of cyclic adenosine monophosphate or inositol triphosphate. However, no single assay platform is suitable for all situations, and all of the assays have limitations. The authors have developed a new high-throughput homogeneous assay platform for GPCR discovery as an alternative to current assays, which employs detection of phosphorylation of the key signaling molecule p42/44 MAP kinase (ERK 1/2). The authors show that ERK 1/2 is consistently activated in cells stimulated by Gq-coupled GPCRs and provides a new high-throughput platform for screening GPCR drug candidates. The activation of ERK 1/2 in Gq-coupled GPCR systems generates comparable pharmacological data for receptor agonist and antagonist data obtained by other GPCR activation measurement techniques.     

Vasoactive intestinal peptide (VIP) stimulates [Ca2+]i and cyclic AMPin CHO cells expressing Galpha16

The stimulatory effect of vasoactive intestinal peptide (VIP) and analogues on [Ca2+]i has been investigated in chinese hamster ovary (CHO) cells stably transfected with the reporter gene aequorin, and expressing either the human VPAC1or VPAC2 receptor in absence or in presence of the Galpha16. In cells that were not transfected with Galpha16 and expressed a similar density of receptors, the VIP induced [Ca2+]i ncrease was higher in VPAC1 than in VPAC2 receptor expressing cells. In aequorin/Galpha16 cotransfected cells, the VIP-induced response was higher, reaching 70 to 80% of the maximal calcium response, obtained after digitonin treatment, in response to both VPAC1 and VPAC2 receptor stimulation.The results suggest that in hematopoietic cells, which express both VIP receptors and Galpha16, the signalling pathway of VIP could be mediated through both cyclic AMP and [Ca2+]i increase.     

Potentiation of GPCR-signaling via membrane targeting of G protein alpha subunits

Different assay technologies are available that allow ligand occupancy of G protein coupled receptors to be converted into robust functional assay signals. Of particular interest are universal screening systems such that activation of any GPCR can be detected with a common assay end point. The promiscuous G protein Galpha16 and chimeric G proteins are broadly used tools for setting up almost universal assay systems. Many efforts focused on making G proteins more promiscuous, however no attempts have been made to make promiscuos G proteins more sensitive by interfering with their cellular protein distribution. As a model system, we used a promiscuous G protein alphaq subunit, that lacks the highly conserved six amino acid N-terminal extension and bears four residues of alphai sequence at its C-terminus replacing the corresponding alphaq sequence (referred to as delta6qi4). When expressed in COS7 cells, delta6qi4 undergoes palmitoylation at its N-terminus. Cell fractionation and immunoblotting analysis indicated localization in the particulate and cytosolic fraction. Interestingly, introduction of a consensus site for N-terminal myristoylation (the resulting mutant referred to as delta6qi4myr) created a protein that was dually acylated and exclusively located in the particulate fraction. As a measure of G protein activation delta6qi4 and delta6qi4myr were coexpressed (in CHO cells) with a series of different Gi/o coupled receptors and ligand induced increases in intracellular Ca2+ release were determined with the FLIPR technology (Fluorescence plate imaging reader from Molecular Devices Corp.). All of the receptors interacted more efficiently with delta6qi4myr as compared with delta6qi4. It could be shown that increased functional responses of agonist activated GPCRs are due to the higher content of delta6qi4myr in the plasma membrane. Our results indicate that manipulation of subcellular localization of G protein alpha subunits-moving them from the cytosol to the plasma membrane-potentiates signaling of agonist activated GPCRs. It is concluded that addition of myristoylation sites into otherwise exclusively palmitoylated G proteins is a new and sensitive approach and may be applicable when functional assays are expected to yield weak signals as is the case when screening extracts of tissues for biologically active GPCR ligands.     

RGS17/RGSZ2, a novel regulator of Gi/o, Gz, and Gq signaling

To identify novel regulators of Galpha(o), the most abundant G-protein in brain, we used yeast two-hybrid screening with constitutively active Galpha(o) as bait and identified a new regulator of G-protein signaling (RGS) protein, RGS17 (RGSZ2), as a novel human member of the RZ (or A) subfamily of RGS proteins. RGS17 contains an amino-terminal cysteine-rich motif and a carboxyl-terminal RGS domain with highest homology to hRGSZ1- and hRGS-Galpha-interacting protein. RGS17 RNA was strongly expressed as multiple species in cerebellum and other brain regions. The interactions between hRGS17 and active forms of Galpha(i1-3), Galpha(o), Galpha(z), or Galpha(q) but not Galpha(s) were detected by yeast two-hybrid assay, in vitro pull-down assay, and co-immunoprecipitation studies. Recombinant RGS17 acted as a GTPase-activating protein (GAP) on free Galpha(i2) and Galpha(o) under pre-steady-state conditions, and on M2-muscarinic receptor-activated Galpha(i1), Galpha(i2), Galpha(i3), Galpha(z), and Galpha(o) in steady-state GTPase assays in vitro. Unlike RGSZ1, which is highly selective for G(z), RGS17 exhibited limited selectivity for G(o) among G(i)/G(o) proteins. All RZ family members reduced dopamine-D2/Galpha(i)-mediated inhibition of cAMP formation and abolished thyrotropin-releasing hormone receptor/Galpha(q)-mediated calcium mobilization. RGS17 is a new RZ member that preferentially inhibits receptor signaling via G(i/o), G(z), and G(q) over G(s) to enhance cAMP-dependent signaling and inhibit calcium signaling. Differences observed between in vitro GAP assays and whole-cell signaling suggest additional determinants of the G-protein specificity of RGS GAP effects that could include receptors and effectors.     

Spinophilin/neurabin reciprocally regulate signaling intensity by G protein-coupled receptors

Spinophilin (SPL) and neurabin (NRB) are structurally similar scaffolding proteins with several protein binding modules, including actin and PP1 binding motifs and PDZ and coiled-coil domains. SPL also binds regulators of G protein signaling (RGS) proteins and the third intracellular loop (3iL) of G protein-coupled receptors (GPCRs) to reduce the intensity of Ca(2+) signaling by GPCRs. The role of NRB in Ca(2+) signaling is not known. In the present work, we used biochemical and functional assays in model systems and in SPL(-/-) and NRB(-/-) mice to show that SPL and NRB reciprocally regulate Ca(2+) signaling by GPCRs. Thus, SPL and NRB bind all members of the R4 subfamily of RGS proteins tested (RGS1, RGS2, RGS4, RGS16) and GAIP. By contract, SPL, but not NRB, binds the 3iL of the GPCRs alpha(1B)-adrenergic (alpha(1B)AR), dopamine, CCKA, CCKB and the muscarinic M3 receptors. Coexpression of SPL or NRB with the alpha(1B)AR in Xenopus oocytes revealed that SPL reduces, whereas NRB increases, the intensity of Ca(2+) signaling by alpha(1B)AR. Accordingly, deletion of SPL in mice enhanced binding of RGS2 to NRB and Ca(2+) signaling by alphaAR, whereas deletion of NRB enhanced binding of RGS2 to SPL and reduced Ca(2+) signaling by alphaAR. This was due to reciprocal modulation by SPL and NRB of the potency of RGS2 to inhibit Ca(2+) signaling by alphaAR. These findings suggest a novel mechanism of regulation of GPCR-mediated Ca(2+) signaling in which SPL/NRB forms a functional pair of opposing regulators that modulates Ca(2+) signaling intensity by GPCRs by determining the extent of inhibition by the R4 family of RGS proteins.     

Sphingosine 1-phosphate is a ligand of the human gpr3, gpr6 and gpr12 family of constitutively active G protein-coupled receptors

Five G protein-coupled receptors (GPCRs) for the lysophospholipid sphingosine 1-phosphate (S1P) have been cloned and characterized so far. We report here about the identification of gpr3, gpr6 and gpr12 as additional members of the S1P-GPCR family. When expressed transiently in HEK293 cells, gpr3, gpr6 and gpr12 confer constitutive activation of adenylate cyclase (AC) similar in amplitude to that seen with fully activated G(alpha)(s)-coupled receptors. Culturing the transfected cells in medium with charcoal-stripped serum (devoid of lipids) significantly reduces cyclic adenosine monophosphate (cAMP) levels, suggesting a lipid-like ligand. A library containing 200 bioactive lipids was applied in functional assays recording intracellular Ca(2+) mobilization. S1P and dihydrosphingosine 1-phosphate (DHS1P) were identified as functional activators exhibiting nanomolar EC(50) values. In the presence of the S1P and LPA receptor antagonist suramin, gpr3-, gpr6- and gpr12-mediated intracellular Ca(2+) mobilization via S1P is enhanced. Besides constitutive activation of G(alpha)(s) type of G proteins, all three receptors are capable of constitutively activating inhibitory G(alpha)(i/o) proteins: (i) in the presence of pertussis toxin, gpr3-, gpr6- and gpr12-mediated stimulation of AC is enhanced; and (ii) overexpression of G(alpha)(i) significantly reduces the stimulatory action on intracellular cAMP levels. Agonist (S1P)-mediated internalization can be visualized in intact HEK293 cells using a gpr6 green fluorescent protein (GFP) fusion protein. In summary, our data suggest that gpr3, gpr6 and gpr12 are a family of constitutively active receptors with dual coupling to G(alpha)(s) and G(alpha)(i) type of G proteins. Constitutive activation of AC and mobilization of [Ca(2+)](i) can be modulated by the sphingophospholipids S1P and DHS1P, adding three additional members to the family of S1P receptors.     

Stimulation of increases in intracellular calcium and prostaglandin E2 generation in Chinese hamster ovary cells expressing receptor-Galpha16 fusion proteins

We examined whether fusion proteins of G protein-coupled receptors with the alpha subunit of G(16) (Galpha(16)) could activate downstream signals. We expressed fusion proteins of G(i)-coupled receptors, i.e. CX(3)C chemokine receptor 1 (CX(3)CR1) and M(2) receptor, in Chinese hamster ovary cells. An agonist for CX(3)CR1 induced greater increases in intracellular Ca(2+) and prostaglandin E(2) generation in cells expressing CX(3)CR1-Galpha(16) fusion protein than in cells expressing CX(3)CR1 alone or both CX(3)CR1 and Galpha(16) separately. Similarly, agonist-induced prostaglandin E(2) generation was greater in cells expressing M(2)-Galpha(16) fusion protein than ones expressing M(2) alone or both M(2) and Galpha(16) separately. In cells expressing fusion proteins with Galpha(16) of G(q)-coupled receptors, i.e. urotensin II receptor and M(1) receptor, the relevant agonists induced similar increases in intracellular Ca(2+) and prostaglandin E(2) generation as in ones expressing the receptor alone. In cells expressing urotensin II receptor-Galpha(16) fusion protein, prostaglandin E(2) generation exhibited a lower EC(50) value than the intracellular Ca(2+) increase. These results indicate that agonist-stimulated receptor-Galpha(16) fusion proteins are coupled to downstream signaling pathways, and suggest that receptor-Galpha(16) fusion proteins may be useful for screening for ligands of orphan G protein-coupled receptors and G(i)-coupled receptors.     

Galpha(12) and galpha(13) inhibit Ca(2+)-dependent exocytosis through Rho/Rho-associated kinase-dependent pathway

The release of neurotransmitters is known to be regulated by activation of heterotrimeric G protein-coupled receptors, although precise mechanisms have not yet been elucidated. To assess the role of the G(12) family of heterotrimeric G proteins in the regulation of neurotransmitter release, we established PC12 cell lines that expressed constitutively active Galpha(12) or Galpha(13) using an isopropyl-beta-D-thiogalactoside-inducible expression system. In the cells, expression of constitutively active Galpha(12) or Galpha(13) inhibited the high K(+)-evoked [(3)H]dopamine release without any effect on the high K(+)-induced increase in intracellular Ca(2+) concentration. A Ca(2+) ionophore ionomycin-induced [(3)H]dopamine release was also inhibited by the expression of active Galpha(12) or Galpha(13). These inhibitory effects of Galpha(12) and Galpha(13) on [(3)H]dopamine release were mimicked by the expression of constitutively active RhoA. In addition, Y-27632, and inhibitor of Rho-associated kinase, a downstream Rho effector, completely abolished the inhibition of [(3)H]dopamine release by Galpha(12), Galpha(13), and RhoA. These results indicate that Ca(2+)-dependent exocytosis is regulated by Galpha(12) and Galpha(13) through a Rho/Rho-associated kinase-dependent pathway.     

Rho-family GTPases modulate Ca(2+) -dependent ATP release from astrocytes

Previously, we reported that activation of G protein-coupled receptors (GPCR) in 1321N1 human astrocytoma cells elicits a rapid release of ATP that is partially dependent on a G(q)/phophospholipase C (PLC)/Ca(2+) mobilization signaling cascade. In this study we assessed the role of Rho-family GTPase signaling as an additional pathway for the regulation of ATP release in response to activation of protease-activated receptor-1 (PAR1), lysophosphatidic acid receptor (LPAR), and M3-muscarinic (M3R) GPCRs. Thrombin (or other PAR1 peptide agonists), LPA, and carbachol triggered quantitatively similar Ca(2+) mobilization responses, but only thrombin and LPA caused rapid accumulation of active GTP-bound Rho. The ability to elicit Rho activation correlated with the markedly higher efficacy of thrombin and LPA, relative to carbachol, as ATP secretagogues. Clostridium difficile toxin B and Clostridium botulinum C3 exoenzyme, which inhibit Rho-GTPases, attenuated the thrombin- and LPA-stimulated ATP release but did not decrease carbachol-stimulated release. Thus the ability of certain G(q)-coupled receptors to additionally stimulate Rho-GTPases acts to strongly potentiate a Ca(2+)-activated ATP release pathway. However, pharmacological inhibition of Rho kinase I/II or myosin light chain kinase did not attenuate ATP release. PAR1-induced ATP release was also reduced twofold by brefeldin treatment suggesting the possible mobilization of Golgi-derived, ATP-containing secretory vesicles. ATP release was also markedly repressed by the gap junction channel inhibitor carbenoxolone in the absence of any obvious thrombin-induced change in membrane permeability indicative of hemichannel gating.