Receptor-mediated activation of heterotrimeric guanine nucleotide-binding proteins (G proteins) results in the dissociation of alpha from beta gamma subunits, thereby allowing both to regulate effectors. Little is known about the regions of effectors required for recognition of G beta gamma. A peptide encoding residues 956 to 982 of adenylyl cyclase 2 specifically blocked G beta gamma stimulation of adenylyl cyclase 2, phospholipase C-beta 3, potassium channels, and beta-adrenergic receptor kinase as well as inhibition of calmodulin-stimulated adenylyl cyclases, but had no effect on interactions between G beta gamma and G alpha o. Substitutions in this peptide identified a functionally important motif, Gln-X-X-Glu-Arg, that is also conserved in regions of potassium channels and beta-adrenergic receptor kinases that participate in G beta gamma interactions. Thus, the region defined by residues 956 to 982 of adenylyl cyclase 2 may contain determinants important for receiving signals from G beta gamma.

A BSTR ACTReceptors activate adenylyl cyclases through the G␣ s subunit. Previous studies from our laboratory have shown in certain cell types that express adenylyl cyclase 6 (AC6), heterologous desensitization included reduction of the capability of adenylyl cyclases to be stimulated by G␣ This region contains a conserved motif present in most adenylyl cyclases; however, the PKA phosphorylation site is unique to members of the AC6 family. These observations suggest a mechanism of how isoform selective regulatory diversity can be obtained within conserved regions involved in signal communication.Transmembrane signaling through the receptor-G s -adenylyl cyclase complex has long been studied as a model for signal transduction through heterotrimeric G proteins. Receptor activation of G s results in the dissociation of G␣ s from G␤␥, and the activated G␣ s stimulates adenylyl cyclase (1). Nine G␣ s -regulated adenylyl cyclases have currently been cloned, and the different isoforms of adenylyl cyclases are differentially regulated (2-4). One aspect of this differential regulation is their susceptibility to participate in heterologous desensitization. Previous studies from our laboratory have shown that in addition to receptor desensitization (5), the glucagonsensitive adenylyl cyclase can undergo desensitization at the level of G s (6) and adenylyl cyclase (7). Desensitization at the level of adenylyl cyclase is a cAMP-dependent process and was observed in hepatocytes and S49 lymphoma cells where a decrease in G␣ s -mediated adenylyl cyclase activities was seen. Cloning of adenylyl cyclase cDNAs from hepatocytes and S49 lymphoma cells indicated that adenylyl cyclase 6 (AC6) was present in both cells (7). We reasoned that the common sensitivity in hepatocytes and S49 cells of adenylyl cyclase activity to protein kinase A (PKA) could be caused by AC6. Hence we studied the effects of PKA on G␣ s regulation of AC6. MATERIALS AND METHODS Materials. [␣-32 P]ATP was from New England Nuclear. Purified catalytic subunit of PKA was purchased from Promega. Tissue culture reagents and fetal calf serum were from GIBCO. WIPTIDE and reagents for peptide synthesis were from Bachem. Anti-FLAG M2 column was from Kodak. All other reagents used were the highest grade available.Expression of Adenylyl Cyclases. AC1, AC2, and AC6 were tagged with the FLAG epitope at the N terminus. The FLAG tagged AC1 (8) and AC6 were constructed by using a strategy similar to that used for AC2 (9). The epitope-tagged adenylyl cyclases were expressed in Hi-5 cells by infection with recombinant baculovirus containing the required adenylyl cyclase insert. Hi-5 cells were infected with a multiplicity of 5-10, and membranes were prepared from infected cells 48 hrs after infection as described (9).PKA Treatment. Hi-5 cell membranes containing the recombinant adenylyl cyclase were treated for 15 min in a solution of 25 mM Tris⅐HCl, 10 mM MgCl 2 , 0.8 mM ATP, and protease inhibitor mixture (10) with and without 50-75 nM PKA catalytic subunit. After treatment the...

Regulation of basal activities of adenylyl cyclase (AC) 2 and 6, expressed in Sf9 cells by infection with recombinant baculovirus, was studied. An antipeptide antibody that recognizes AC2 and AC6 with equal sensitivity was used to establish that equivalent levels were expressed.

Only three isoforms of adenylyl cyclase (EC 4.6.1.1) mRNAs (AC1, -2, and -5) are expressed at high levels in rat brain. ACI occurs predominantly in hippocampus and cerebellum, AC5 is restricted to the basal ganglia, whereas AC2 is more widely expressed, but at much lower levels. The distribution and abundance of adenylyl cyclase protein were examined by immunohistochemistry with an antiserum that recognizes a peptide sequence shared by all known mammalian adenylyl cyclase isoforms. The immunoreactivity in striatum and hippocampus could be readily interpreted within the context of previous in situ hybridization studies. However, extending the information that could be gathered by comparisons with in situ hybridization analysis, it was apparent that staining was confined to the neuropil-corresponding to immunoreactive dendrites and axon terminals. Electron microscopy indicated a remarkably selective subcellular distribution of adenylyl cyclase protein. In the CAl area of the hippocampus, the densest immunoreactivity was seen in postsynaptic densities in dendritic spine heads. Labeled presynaptic axon terminals were also observed, indicating the participation of adenylyl cyclase in the regulation of neurotransmitter release. The selective concentration of adenylyl cyclases at synaptic sites provides morphological data for understanding the pre-and postsynaptic roles of adenylyl cyclase in discrete neuronal circuits in rat brain. The apparent clustering of adenylyl cyclases, coupled with other data that suggest higher-order associations of regulatory elements including G proteins, N-methyl-D-aspartate receptors, and cAMP-dependent protein kinases, suggests not only that the primary structural information has been encoded to render the cAMP system responsive to the Ca2+-signaling system but also that higher-order strictures are in place to ensure that Ca2+ signals are economically delivered and propagated.In the central nervous system (CNS), adenylyl cyclases (EC 4.6.1.1) modulate many cellular processes in response to extraand intracellular signals, such as hormones, neurotransmitters, and Ca2+ (1-3). By being susceptible to more than one regulatory influence, adenylyl cyclases have also been suggested to serve as discriminators of convergent signals delivered by simultaneous activation of two independent inputs (4). The ability of adenylyl cyclase to function as a "coincidence detector" has been proposed to account for its role in the induction of long-term adaptive responses in neurons and synapses, such as long-term potentiation (LTP) in hippocampus (5, 6).Recent molecular studies have identified eight distinct isoforms of adenylyl cyclase (types I-VIII), which can be classified into distinct subfamilies based on their sequence similarities and their response to Ca2+ (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21). Of these, only three isoforms (types I, II, and V) occur at substantial levels in the CNS, based on in situ hybridization studies (1,18,19,(22)(23)(24)(25)(26)(27). The predominantly ...

Regulation of adenylyl cyclases 1, 2, and 6 by G␣ s was studied. All three mammalian adenylyl cyclases were expressed in insect (Sf9 or Hi-5) cells by baculovirus infection. Membranes containing the different adenylyl cyclases were stimulated by varying concentrations of mutant (Q227L) activated G␣ s expressed in reticulocyte lysates. G␣ s stimulation of AC1 involved a single site and had an apparent K act of 0.9 nM. G␣ s stimulation of AC2 was best explained by a non-interactive two site model with a "high affinity" site at 0.9 nM and a "low affinity" site at 15 nM. Occupancy of the high affinity site appears to be sufficient for G␤␥ stimulation of AC2. G␣ s stimulation of AC6 was also best explained by a two-site model with a high affinity site at 0.6 -0.8 nM and a low affinity site at 8 -22 nM; however, in contrast to AC2, only a model that assumed interactions between the two sites best fit the AC6 data. With 100 M forskolin, G␣ s stimulation of all three adenylyl cyclases showed very similar profiles. G␣ s stimulation in the presence of forskolin involved a single site with apparent K act of 0.1-0.4 nM. These observations indicate a conserved mechanism by which forskolin regulates G␣ s coupling to the different adenylyl cyclases. However, there are fundamental differences in the mechanism of G␣ s stimulation of the different adenylyl cyclases with AC2 and AC6 having multiple interconvertible sites. These mechanistic differences may provide an explanation for the varied responses by different cells and tissues to hormones that elevate cAMP levels.Nine adenylyl cyclases have been cloned and characterized (1-3). Though these adenylyl cyclase isoforms display distinct signal receiving capabilities from Ca 2ϩ (4, 5), G␤␥ (6), and protein kinase C (7), they all share the common capability to be stimulated by G␣ s . Due to its expected nature, regulation by G␣ s has received somewhat less attention than other modes of regulation. Little is known about the regions of adenylyl cyclases involved in G␣ s interactions. Early studies with the purified olfactory adenylyl cyclases showed that G␣ s could be covalently linked to adenylyl cyclase (8). As a prelude to the mapping of adenylyl cyclase regions involved in interactions with G␣ s , we have studied regulation of recombinant AC6, AC2, and AC1 by mutant (Q227L) activated G␣ s . Much to our surprise, we find G␣ s regulation of the three different adenylyl cyclase appears to be mechanistically different. Stimulation of AC1 is the simplest and appears to involve a single site of interaction. Stimulation of AC2 and AC6 appears to be best explained by multiple sites of interaction. This complex regulation is observable only in the absence of forskolin. In the presence of forskolin, stimulation by G␣ s of all three adenylyl cyclases tested appears to involve a single high affinity site. EXPERIMENTAL PROCEDURESMaterials-In vitro translation kits and Flexi rabbit reticulocyte lysates and reagents for RNA synthesis were from Promega (Madison, WI), and [␣-32 P]ATP was from ICN...

Receptor activation of heterotrimeric G proteins dissociates G␣ from the G␤␥ complex, allowing both to regulate effectors. Little is known about the effectorinteraction regions of G␤␥. We had used molecular modeling to dock a peptide encoding the region of residues 956-982 of adenylyl cyclase (AC) 2 onto G␤ to identify residues on G␤ that may interact with effectors. Based on predictions from the model, we synthesized peptides encoding sequences of residues 86-105 (G␤86-105) and 115-135 (G␤115-135) from G␤. The G␤86-105 peptide inhibited G␤␥ stimulation of AC2 and blocked G␤␥ inhibition of AC1 and by itself inhibited calmodulin-stimulated AC1, thus displaying partial agonist activity. Substitution of Met-101 with Asn in this peptide resulted in the loss of both the inhibitory and partial agonist activities. Most activities of the G␤115-135 peptide were similar to those of G␤86-105 but G␤115-135 was less efficacious in blocking G␤␥ inhibition of AC1. Substitution of Tyr-124 with Val in the G␤115-135 peptide diminished all of its activities. These results identify the region encoded by amino acids 84-143 of G␤ as a surface that is involved in transmitting signals to effectors.Heterotrimeric G proteins serve as signal transducers for a wide variety of receptors. Both G␣ and G␤␥ subunits can communicate receptor signals (1-5). Regions of G␤␥ complex involved in communicating the signal to effectors have not been well characterized. We had identified the region of residues 956-982 of adenylyl cyclase (AC) 2 as being involved in receiving signals from G␤␥ (6). By using the yeast twohybrid system, the AC2 region of residues 956-982 has been subsequently shown to interact with G␤ but not G␥ subunits (7). In recent studies we found that the peptide encoding residues 956-982 of AC2 can be crosslinked to G␤ when it is part of the free G␤␥ complex but not when it is part of the heterotrimer, indicating that the putative binding surface on G␤ for the AC2 peptide is occluded by interactions with G␣. On the basis of constraints deduced from the crosslinking studies and other biophysical criteria, we docked the AC2 peptide containing residues 956-982 onto the crystal structure of G␤ by using molecular modeling techniques (8). From this docking model, we have identified the regions of G␤ that are predicted to interact with the AC2 peptide. Herein we have tested whether peptides encoding the effector-interaction surface of G␤ predicted from the modeling (8) can modulate G␤␥ regulation of AC1 and AC2. MATERIALS AND METHODSMaterials. Reagents for peptide synthesis were from Bachem. [␣-32 P]ATP was from New England Nuclear. Tissue culture reagents and fetal calf serum was from GIBCO. All other chemicals used were the highest grade available.Peptide Synthesis. Peptides were synthesized on an Applied Biosystems peptide synthesizer (model 431A) and purified by HPLC on acetonitrile gradients. Purified peptides were lyophilized and stored at Ϫ20ЊC. When required peptides were dissolved in water to a final concentration of 1-3 mM....

Cysteinyl leukotrienes (cysLTs) are important mediators of cell trafficking and innate immune responses, involved in the pathogenesis of inflammatory processes, i.e., atherosclerosis, pulmonary fibrosis, and bronchial asthma. The aim of this study was to examine the regulation of cysLT signaling by IFN-γ in human primary endothelial cells. IFN-γ increased cysLT receptor 2 (CysLTR2) mRNA expression and CysLTR2-specific calcium signaling in endothelial cells. IFN-γ signaled through Jak/STAT1, as both AG490, a Jak2 inhibitor, and expression of a STAT1 dominant-negative construct, significantly inhibited CysLTR2 mRNA expression in response to IFN-γ. To determine mechanisms of IFN-γ-induced CysLTR2 expression, the human CysLTR2 gene structure was characterized. The CysLTR2 gene has a TATA-less promoter, with multiple transcription start sites. It consists of six variably spliced exons. Eight different CysLTR2 transcripts were identified in endothelial and monocytic cells. Gene reporter assay showed potent basal promoter activity of a putative CysLTR2 promoter region. However, there were no significant changes in gene reporter and mRNA t1/2 assays in response to IFN-γ, suggesting transcriptional control of CysLTR2 mRNA up-regulation by IFN-γ response motifs localized outside of the cloned CysLTR2 promoter region. Stimulation of endothelial cells by cysLTs induced mRNA and protein expression of early growth response genes 1, 2, and 3 and cycloxygenase-2. This response was mediated by CysLTR2 coupled to Gq/11, activation of phospholipase C, and inositol-1,4,5-triphosphate, and was enhanced further 2- to 5-fold by IFN-γ stimulation. Thus, IFN-γ induces CysLTR2 expression and enhances cysLT-induced inflammatory responses.

Background Acute Respiratory Distress Syndrome (ARDS) is associated with increased pulmonaryvascular permeability. In the lung, transient receptor potential vanilloid 4 (TRPV4), a Ca2+-permeable cation channel, is a regulator of endothelial permeability and pulmonary edema. We performed a Phase I, placebocontrolled, doubleblind, randomized, parallel group, proofofmechanism study to investigate the effects of TRPV4 channel blocker, GSK2798745, on pulmonaryvascular barrier permeability using a model of lipopolysaccharide (LPS)induced lung inflammation. Methods Healthy participants were randomized 1:1 to receive 2 single doses of GSK2798745 or placebo, 12 hours apart. Two hours after the first dose, participants underwent bronchoscopy and segmental LPS instillation. Total protein concentration and neutrophil counts were measured in bronchoalveolar lavage (BAL) samples collected before and 24 hours after LPS challenge, as markers of barrier permeability and inflammation, respectively. The primary endpoint was baseline adjusted total protein concentration in BAL at 24 hours after LPS challenge. A Bayesian framework was used to estimate the posterior probability of any percentage reduction (GSK2798745 relative to placebo). Safety endpoints included the incidence of adverse events (AEs), vital signs, 12-lead electrocardiogram, clinical laboratory and haematological evaluations, and spirometry. Results Forty-seven participants were dosed and 45 completed the study (22 on GSK2798745 and 23 on placebo). Overall, GSK2798745 was well tolerated. Small reductions in mean baseline adjusted BAL total protein (~ 9%) and neutrophils (~ 7%) in the LPS-challenged segment were observed in the GSK2798745 group compared with the placebo group; however, the reductions did not meet pre-specified success criteria of at least a 95% posterior probability that the percentage reduction in the mean 24hours post LPS BAL total protein level (GSK2798745 relative to placebo) exceeded zero. Median plasma concentrations of GSK2798745 were predicted to inhibit TRPV4 on lung vascular endothelial cells by ~ 7085% during the 24 hours after LPS challenge; median ureacorrected BAL concentrations of GSK2798745 were 3.0 to 8.7fold higher than those in plasma. Conclusions GSK2798745 did not affect segmental LPS-induced elevation of BAL total protein or neutrophils, despite blood and lung exposures that were predicted to be efficacious.