Regulation of cAMP responses by the G12/13 pathway converges on adenylyl cyclase VII

Regulation of intracellular cyclic adenosine 3’, 5’-monophosphate (cAMP) by multiple pathways enables differential function of this ubiquitous second messenger in a context dependent manner. Modulation of Gs-stimulated intracellular cAMP has long been known to be modulated by the Gi and Gq/Ca2+ pathways. Recently, the G13 pathway was also shown to facilitate cAMP responses in murine macrophage cells. We report here that this synergistic regulation of cAMP synthesis by the Gs and G13 pathways is mediated by a specific isoform of adenylyl cyclase, AC7. Furthermore, this signaling paradigm exists in several hematopoietic lineages and can be recapitulated by exogenous expression of AC7 in HEK 293 cells. Mechanistic characterization of this synergistic interaction indicates that it occurs downstream of receptor activation and it can be mediated by the alpha subunit of either G12 or G13. Our results demonstrate that AC7 is a specific downstream effector of the G12/13 pathway

Structure of the p115RhoGEF rgRGS domain-Galpha13/i1 chimera complex suggests convergent evolution of a GTPase activator

p115RhoGEF, a guanine nucleotide exchange factor (GEF) for Rho GTPase, is also a GTPase-activating protein (GAP) for G12 and G13 heterotrimeric Galpha subunits. The GAP function of p115RhoGEF resides within the N-terminal region of p115RhoGEF (the rgRGS domain), which includes a module that is structurally similar to RGS (regulators of G-protein signaling) domains. We present here the crystal structure of the rgRGS domain of p115RhoGEF in complex with a chimera of Galpha13 and Galphai1. Two distinct surfaces of rgRGS interact with Galpha. The N-terminal betaN-alphaN hairpin of rgRGS, rather than its RGS module, forms intimate contacts with the catalytic site of Galpha. The interface between the RGS module of rgRGS and Galpha is similar to that of a Galpha-effector complex, suggesting a role for the rgRGS domain in the stimulation of the GEF activity of p115RhoGEF by Galpha13

Structure of the rgRGs domain of p115RhoGEF

p115RhoGEF, a guanine nucleotide exchange factor for Rho GTPase, is also a GTPase activating protein (GAP) for G12 and G13 heterotrimeric Gα subunits. Near its N-terminus, p115RhoGEF contains a domain (rgRGS) with remote sequence identity to RGS (regulators of G protein signaling) domains. The rgRGS domain is necessary but not sufficient for the GAP activity of p115RhoGEF. The 1.9 Å resolution crystal structure of the rgRGS domain shows structural similarity to RGS domains but possesses a C-terminal extension that folds into a layer of helices that pack against the hydrophobic core of the domain. Mutagenesis experiments show that rgRGS may form interactions with Gα13 that are analogous to those in complexes of RGS proteins with their Gα substrates

Recognition of the activated states of Galpha13 by the rgRGS domain of PDZRhoGEF

G12 class heterotrimeric G proteins stimulate RhoA activation by RGS-RhoGEFs. However, p115RhoGEF is a GTPase Activating Protein (GAP) toward Galpha13, whereas PDZRhoGEF is not. We have characterized the interaction between the PDZRhoGEF rgRGS domain (PRG-rgRGS) and the alpha subunit of G13 and have determined crystal structures of their complexes in both the inactive state bound to GDP and the active states bound to GDP*AlF (transition state) and GTPgammaS (Michaelis complex). PRG-rgRGS interacts extensively with the helical domain and the effector-binding sites on Galpha13 through contacts that are largely conserved in all three nucleotide-bound states, although PRG-rgRGS has highest affinity to the Michaelis complex. An acidic motif in the N terminus of PRG-rgRGS occupies the GAP binding site of Galpha13 and is flexible in the GDP*AlF complex but well ordered in the GTPgammaS complex. Replacement of key residues in this motif with their counterparts in p115RhoGEF confers GAP activity

Activation of p115-RhoGEF requires direct association of Gα13 and the Dbl homology domain

RGS-containing RhoGEFs (RGS-RhoGEFs) represent a direct link between the G(12) class of heterotrimeric G proteins and the monomeric GTPases. In addition to the canonical Dbl homology (DH) and pleckstrin homology domains that carry out the guanine nucleotide exchange factor (GEF) activity toward RhoA, these RhoGEFs also possess RGS homology (RH) domains that interact with activated α subunits of G(12) and G(13). Although the GEF activity of p115-RhoGEF (p115), an RGS-RhoGEF, can be stimulated by Gα(13), the exact mechanism of the stimulation has remained unclear. Using combined studies with small angle x-ray scattering, biochemistry, and mutagenesis, we identify an additional binding site for activated Gα(13) in the DH domain of p115. Small angle x-ray scattering reveals that the helical domain of Gα(13) docks onto the DH domain, opposite to the surface of DH that binds RhoA. Mutation of a single tryptophan residue in the α3b helix of DH reduces binding to activated Gα(13) and ablates the stimulation of p115 by Gα(13). Complementary mutations at the predicted DH-binding site in the αB-αC loop of the helical domain of Gα(13) also affect stimulation of p115 by Gα(13). Although the GAP activity of p115 is not required for stimulation by Gα(13), two hydrophobic motifs in RH outside of the consensus RGS box are critical for this process. Therefore, the binding of Gα(13) to the RH domain facilitates direct association of Gα(13) to the DH domain to regulate its exchange activity. This study provides new insight into the mechanism of regulation of the RGS-RhoGEF and broadens our understanding of G protein signaling

Mapping the Galpha13 binding interface of the rgRGS domain of p115RhoGEF

Structural requirements for function of the Rho GEF (guanine nucleotide exchange factor) regulator of G protein signaling (rgRGS) domains of p115RhoGEF and homologous exchange factors differ from those of the classical RGS domains. An extensive mutagenesis analysis of the p115RhoGEF rgRGS domain was undertaken to determine its functional interface with the Galpha(13) subunit. Results indicate that there is global resemblance between the interaction surface of the rgRGS domain with Galpha(13) and the interactions of RGS4 and RGS9 with their Galpha substrates. However, there are distinct differences in the distribution of functionally critical residues between these structurally similar surfaces and an additional essential requirement for a cluster of negatively charged residues at the N terminus of rgRGS. Lack of sequence conservation within the N terminus may also explain the lack of GTPase-activating protein (GAP) activity in a subset of the rgRGS domains. For all mutations, loss of functional GAP activity is paralleled by decreases in binding to Galpha(13). The same mutations, when placed in the context of the p115RhoGEF molecule, produce deficiencies in GAP activity as observed with the rgRGS domain alone but show no attenuation of the regulation of Rho exchange activity by Galpha(13). This suggests that the rgRGS domain may serve a structural or allosteric role in the regulation of the nucleotide exchange activity of p115RhoGEF on Rho by Galpha(13)

Activated RhoA binds to the pleckstrin homology (PH) domain of PDZ-RhoGEF, a potential site for autoregulation

Guanine nucleotide exchange factors (GEFs) catalyze exchange of GDP for GTP by stabilizing the nucleotide-free state of the small GTPases through their Dbl homology/pleckstrin homology (DH.PH) domains. Unconventionally, PDZ-RhoGEF (PRG), a member of the RGS-RhoGEFs, binds tightly to both nucleotide-free and activated RhoA (RhoA.GTP). We have characterized the interaction between PRG and activated RhoA and determined the structure of the PRG-DH.PH-RhoA.GTPgammaS (guanosine 5\u27-O-[gamma-thio]triphosphate) complex. The interface bears striking similarity to a GTPase-effector interface and involves the switch regions in RhoA and a hydrophobic patch in PRG-PH that is conserved among all Lbc RhoGEFs. The two surfaces that bind activated and nucleotide-free RhoA on PRG-DH.PH do not overlap, and a ternary complex of PRG-DH.PH bound to both forms of RhoA can be isolated by size-exclusion chromatography. This novel interaction between activated RhoA and PH could play a key role in regulation of RhoGEF activity in vivo

Use of a cAMP BRET Sensor to Characterize a Novel Regulation of cAMP by the Sphingosine 1-Phosphate/G13 Pathway

Regulation of intracellular cyclic adenosine 3',5'-monophosphate (cAMP) is integral in mediating cell growth, cell differentiation, and immune responses in hematopoietic cells. To facilitate studies of cAMP regulation we developed a BRET (bioluminescence resonance energy transfer) sensor for cAMP, CAMYEL (cAMP sensor using YFP-Epac-RLuc), which can quantitatively and rapidly monitor intracellular concentrations of cAMP in vivo. This sensor was used to characterize three distinct pathways for modulation of cAMP synthesis stimulated by presumed Gs-dependent receptors for isoproterenol and prostaglandin E2. Whereas two ligands, uridine 5'-diphosphate and complement C5a, appear to use known mechanisms for augmentation of cAMP via Gq/calcium and Gi, the action of sphingosine 1-phosphate (S1P) is novel. In these cells, S1P, a biologically active lysophospholipid, greatly enhances increases in intracellular cAMP triggered by the ligands for Gs-coupled receptors while having only a minimal effect by itself. The enhancement of cAMP by S1P is resistant to pertussis toxin and independent of intracellular calcium. Studies with RNAi and chemical perturbations demonstrate that the effect of S1P is mediated by the S1P2 receptor and the heterotrimeric G13 protein. Thus in these macrophage cells, all four major classes of G proteins can regulate intracellular cAMP

Deciphering Signaling Outcomes from a System of Complex Networks

Cellular signal transduction machinery integrates information from multiple inputs to actuate discrete cellular behaviors. Interaction complexity exists when an input modulates the output behavior that results from other inputs. To address whether this machinery is iteratively complex—that is, whether increasing numbers of inputs produce exponential increases in discrete cellular behaviors—we examined the modulated secretion of six cytokines from macrophages in response to up to five-way combinations of an agonist of Toll-like receptor 4, three cytokines, and conditions that activated the cyclic adenosine monophosphate pathway. Although all of the selected ligands showed synergy in paired combinations, few examples of nonadditive outputs were found in response to higher-order combinations. This suggests that most potential interactions are not realized and that unique cellular responses are limited to discrete subsets of ligands and pathways that enhance specific cellular functions

Purification from Sf9 cells and characterization of recombinant Gq alpha and G11 alpha. Activation of purified phospholipase C isozymes by G alpha subunits

Members of the Gq alpha subfamily of heterotrimeric guanine nucleotide-binding proteins (G proteins) activate phospholipase C (PLC). The complementary DNAs (cDNAs) for the G protein alpha subunits Gq alpha and G11 alpha were expressed in insect (Sf9) cells using recombinant baculovirus. Active, nonaggregated, and membrane-associated protein was generated only when the alpha subunit cDNA was expressed together with cDNAs encoding G protein beta and gamma subunits. Recombinant alpha subunits (rGq alpha and rG11 alpha) were purified by three-step procedures, as was a PLC-activating alpha subunit(s) endogenous to Sf9 cells. Guanosine 5'-3-(thio)triphosphate (GTP gamma S) activated rGq alpha and rG11 alpha with an apparent K0.5 of 30 microM; similarly high concentrations of the nucleotide were required to observe [35S]GTP gamma S binding to rGq alpha. Activated rGq alpha and rG11 alpha each stimulated all three isoforms of purified PLC-beta with the rank order of potency PLC-beta 1 = PLC-beta 3 > or = PLC-beta 2; both alpha subunits also stimulated PLC-beta 1 and PLC-beta 3 to a much greater extent (10-fold) than they did PLC-beta 2. In contrast, activated rGq alpha and rG11 alpha failed to stimulate either PLC-delta 1 or PLC- gamma 1. Recombinant Gi alpha 1, Gi alpha 2, Gi alpha 3, Go alpha (A), Gs alpha, and Gz alpha all failed to stimulate any of the isoforms of PLC. The apparent affinities of rGq alpha and rG11 alpha for PLC-beta 1 and their capacities to activate the enzyme were similar to values observed for purified brain Gq alpha/11 alpha. Purified brain beta gamma subunits also stimulated the three isoforms of PLC-beta. The capacities of rGq alpha and rG11 alpha to activate PLC-beta 1 and PLC- beta 3 greatly exceeded those of beta gamma, whereas Gq alpha, G11 alpha and beta gamma were roughly equiefficacious with PLC-beta 2; the alpha subunits were more potent than beta gamma in all cases. The effects of alpha and beta gamma together were nonadditive for both PLC- beta 1 and PLC-beta 2. These results demonstrate that Gq alpha and G11 alpha specifically and selectively stimulate beta isoforms of PLC and confirm the idea that these members of the Gq alpha subfamily of G proteins are physiological regulators of this signaling pathway