Direct regulation of p190RhoGEF by activated Rho and Rac GTPases.

Rho family GTPases regulate a wide range of cellular processes. This includes cellular dynamics where three subfamilies, Rho, Rac, and Cdc42, are known to regulate cell shape and migration though coordinate action. Activation of Rho proteins largely depends on Rho Guanine nucleotide Exchange Factors (RhoGEFs) through a catalytic Dbl homology (DH) domain linked to a pleckstrin homology (PH) domain that subserves various functions. The PH domains from Lbc RhoGEFs, which specifically activate RhoA, have been shown to bind to activated RhoA. Here, p190RhoGEF is shown to also bind Rac1·GTP. Crystal structures reveal that activated Rac1 and RhoA use their effector-binding surfaces to associate with the same hydrophobic surface on the PH domain. Both activated RhoA and Rac1 can stimulate exchange of nucleotide on RhoA by localization of p190RhoGEF to its substrate, RhoA·GDP, in vitro. The binding of activated RhoA provides a mechanism for positive feedback regulation as previously proposed for the family of Lbc RhoGEFs. In contrast, the novel interaction between activated Rac1 and p190RhoGEF reveals a potential mechanism for cross-talk regulation where Rac can directly effect stimulation of RhoA. The greater capacity of Rac1 to stimulate p190RhoGEF among the Lbc RhoGEFs suggests functional specialization.

Secondary PDZ domain-binding site on class B plexins enhances the affinity for PDZ-RhoGEF.

PDZ domains are abundant protein interaction modules and typically recognize a short motif at the C terminus of their ligands, with a few residues in the motif endowing the binding specificity. The sequence-based rules, however, cannot fully account for the specificity between the vast number of PDZ domains and ligands in the cell. Plexins are transmembrane receptors that regulate processes such as axon guidance and angiogenesis. Two related guanine nucleotide exchange factors (GEFs), PDZ-RhoGEF and leukemia-associated RhoGEF (LARG), use their PDZ domains to bind class B plexins and play critical roles in signaling. Here, we present the crystal structure of the full-length cytoplasmic region of PlexinB2 in complex with the PDZ domain of PDZ-RhoGEF. The structure reveals that, in addition to the canonical C-terminal motif/PDZ interaction, the 3D domain of PlexinB2 forms a secondary interface with the PDZ domain. Our biophysical and cell-based assays show that the secondary interface contributes to the specific interaction between plexin and PDZ-RhoGEF and to signaling by plexin in the cell. Formation of secondary interfaces may be a general mechanism for increasing affinity and specificity of modular domain-mediated interactions.

Regulated localization is sufficient for hormonal control of regulator of G protein signaling homology Rho guanine nucleotide exchange factors (RH-RhoGEFs).

The regulator of G protein signaling homology (RH) Rho guanine nucleotide exchange factors (RhoGEFs) (p115RhoGEF, leukemia-associated RhoGEF, and PDZ-RhoGEF) contain an RH domain and are specific GEFs for the monomeric GTPase RhoA. The RH domains interact specifically with the α subunits of G12 heterotrimeric GTPases. Activated Gα13 modestly stimulates the exchange activity of both p115RhoGEF and leukemia-associated RhoGEF but not PDZ-RhoGEF. Because all three RH-RhoGEFs can localize to the plasma membrane upon expression of activated Gα13, cellular localization of these RhoGEFs has been proposed as a mechanism for controlling their activity. We use a small molecule-regulated heterodimerization system to rapidly control the localization of RH-RhoGEFs. Acute localization of the proteins to the plasma membrane activates RhoA within minutes and to levels that are comparable with activation of RhoA by hormonal stimulation of G protein-coupled receptors. The catalytic activity of membrane-localized RhoGEFs is not dependent on activated Gα13. We further show that the conserved RH domains can rewire two different RacGEFs to activate Rac1 in response to a traditional activator of RhoA. Thus, RH domains act as independent detectors for activated Gα13 and are sufficient to modulate the activity of RhoGEFs by hormones via mediating their localization to substrate, membrane-associated RhoA.

Activated RhoA is a positive feedback regulator of the Lbc family of Rho guanine nucleotide exchange factor proteins.

The monomeric Rho GTPases are essential for cellular regulation including cell architecture and movement. A direct mechanism for hormonal regulation of the RhoA-type GTPases is their modulation by the G12 and G13 proteins via RH (RGS homology) containing RhoGEFs. In addition to the interaction of the G protein α subunits with the RH domain, activated RhoA also binds to the pleckstrin homology (PH) domain of PDZRhoGEF. The latter interaction is now extended to all seven members of the homologous Lbc family of RhoGEFs which includes the RH-RhoGEFs. This is evinced by direct measurements of binding or through effects on selected signaling pathways in cells. Overexpression of these PH domains alone can block RhoA-dependent signaling in cells to various extents. Whereas activated RhoA does not modulate the intrinsic activity of the RhoGEFs, activated RhoA associated with phospholipid vesicles can facilitate increased activity of soluble RhoGEFs on vesicle-delimited substrate (RhoA-GDP). This demonstrates feasibility of the hypothesis that binding of activated RhoA to the PH domains acts as a positive feedback mechanism. This is supported by cellular studies in which mutation of this binding site on PH strongly attenuates the stimulation of RhoA observed by overexpression of five of the RhoGEF DH-PH domains. This mutation is even more dramatic in the context of full-length p115RhoGEF. The utilization of this mechanism by multiple RhoGEFs suggests that this regulatory paradigm may be a common feature in the broader family of RhoGEFs.

Regions on adenylyl cyclase VII required for selective regulation by the G13 pathway.

Regulation of multiple adenylyl cyclases (AC) provides unique inputs to mediate the synthesis of cAMP, a ubiquitous second messenger that controls many aspects of cellular function. On stimulation by G(s), the activities of ACs can be further selectively modulated by other pathways to ensure precise control of intracellular cAMP responses to specific stimuli. Recently, we reported that one of the AC isoforms, AC7, is uniquely regulated by the G(13) pathway. To understand more fully the molecular mechanism of this regulation, we compared the regulation of AC7 with that of AC2 in bone marrow-derived macrophages devoid of AC7. Although both enzymes could fully restore regulation of cAMP by Gßγ, activation of the G(13) pathway preferentially synergized with AC7. Exchange of domains between the two isoforms indicates that the C1b domain and the N-terminus of the C1a domain are important for directing selective regulation of AC7 by the G(13) pathway. A mutagenesis screen identified more specific regions of AC7 that differentially mediate its regulation by distinct pathways.

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.

A global analysis of cross-talk in a mammalian cellular signalling network.

Cellular information processing requires the coordinated activity of a large network of intracellular signalling pathways. Cross-talk between pathways provides for complex non-linear responses to combinations of stimuli, but little is known about the density of these interactions in any specific cell. Here, we have analysed a large-scale survey of pathway interactions carried out by the Alliance for Cellular Signalling (AfCS) in RAW 264.7 macrophages. Twenty-two receptor-specific ligands were studied, both alone and in all pairwise combinations, for Ca2+ mobilization, cAMP synthesis, phosphorylation of many signalling proteins and for cytokine production. A large number of non-additive interactions are evident that are consistent with known mechanisms of cross-talk between pathways, but many novel interactions are also revealed. A global analysis of cross-talk suggests that many external stimuli converge on a relatively small number of interaction mechanisms to provide for context-dependent signalling.

Distinct roles of adenylyl cyclase VII in regulating the immune responses in mice.

The second messenger cAMP plays a critical role in regulating immune responses. Although well known for its immunosuppressive effect, cAMP is also required for the development of optimal immune responses. Thus, the regulation of this second messenger needs to be finely tuned and well balanced in a context dependent manner. To further understand the role of cAMP synthesis in the functions of the immune system, we focus on a specific adenylyl cyclase (AC) isoform, AC VII (AC7), which is highly expressed in the immune system. We show that mice deficient of AC7 are hypersensitive to LPS-induced endotoxic shock. Macrophages from AC7-deficient mice produce more of the proinflammatory cytokine, TNF-alpha, in response to LPS. The inability to generate intracellular cAMP response to serum factors, such as lysophosphatidic acid, is a potential cause for this phenotype. Thus, AC7 functions to control the extent of immune responses toward bacterial infection. However, it is also required for the optimal functions of B and T cells during adaptive immune responses. AC7 is the major isoform that regulates cAMP synthesis in both B and T cells. AC7-deficient mice display compromised Ab responses toward both T cell-independent and T cell-dependent Ags. The generation of memory T cells is also reduced. These results are the first to ascribe specific functions to an AC isoform in the immune system and emphasize the importance of cAMP synthesis by this isoform in shaping the immune responses.

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'-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.

Modeling species-specific diacylglycerol dynamics in the RAW 264.7 macrophage.

A mathematical model of the G protein signaling pathway in RAW 264.7 macrophages downstream of P2Y(6) receptors activated by the ubiquitous signaling nucleotide uridine 5'-diphosphate is developed. The model, which is based on time-course measurements of inositol trisphosphate, cytosolic calcium, and diacylglycerol, focuses particularly on differential dynamics of multiple chemical species of diacylglycerol. When using the canonical pathway representation, the model predicted that key interactions were missing from the current network structure. Indeed, the model suggested that accurate depiction of experimental observations required an additional branch to the signaling pathway. An intracellular pool of diacylglycerol is immediately phosphorylated upon stimulation of an extracellular receptor for uridine 5'-diphosphate and subsequently used to aid replenishment of phosphatidylinositol. As a result of sensitivity analysis of the model parameters, key predictions can be made regarding which of these parameters are the most sensitive to perturbations and are therefore most responsible for output uncertainty.

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.

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.

Regulation of Rho guanine nucleotide exchange factors by G proteins.

Monomeric Rho GTPases regulate cellular dynamics through remodeling of the cytoskeleton, modulation of immediate signaling pathways, and longer-term regulation of gene transcription. One family of guanine nucleotide exchange factors for Rho proteins (RhoGEFs) provides a direct pathway for regulation of RhoA by cell surface receptors coupled to heterotrimeric G proteins. Some of these RhoGEFs also contain RGS domains that can attenuate signaling by the G(12) and G(13) proteins. The regulation provided by these RhoGEFs is defined by their selective regulation by specific G proteins, phosphorylation by kinases, and potential localization with signaling partners. Evidence of their physiological importance is derived from gene knockouts in Drosophila and mice. Current understanding of the basic regulatory mechanisms of these RhoGEFs is discussed. An overview of identified interactions with other signaling proteins suggests the growing spectrum of their involvement in numerous signaling pathways.

Crystal structures of the PH domains from Lbc family of RhoGEFs bound to activated RhoA GTPase.

The Pleckstrin homology (PH) domains from the Lbc family of Rho Guanine Nucleotide Exchange Factors (Lbc RhoGEFs) interact with activated Rho family GTPases. All 7 Lbc RhoGEFs associate directly with activated Rho GTPases via their PH domains. However, the binding affinities between the PH domains and the GTPases vary greatly. Here we present two crystal structures at resolutions of 1.4 Šand 2.0 Šof RhoA complexed with the PH domain from p114RhoGEF (PDB access code 6BCB) and AKAP-LbcRhoGEF (PDB access code 6BCA), respectively. These high resolution structures, together with the earlier structures of PDZRhoGEF-PH·RhoA and p190RhoGEF-PH·RhoA complexes, identify a highly conserved interface between the PH domains from Lbc-RhoGEFs and activated Rho GTPases. This manuscript is related to the manuscript titled "Direct Regulation of p190RhoGEF by Activated Rho and Rac GTPases" published in the Journal of Structural Biology.

Ribozyme- and siRNA-mediated suppression of RGS-containing RhoGEF proteins.

Given recent efforts to determine the sequence information on thousands of genes in the human genome, the current challenge is to identify the functions of these genes, including those encoding the regulator of G-protein signaling protein gene superfamily, and to establish their roles in particular signaling pathways in a native system. Increasingly, reverse genetic approaches are being used to address these questions. This article compares two powerful approaches [ribozyme and "short interfering" RNA (siRNA) techniques] under identical conditions for the first report on the suppression of endogenous RGS domain-containing RhoGEFs. The siRNA technique was found to be much more potent than ribozyme targeting at the same mRNA site of RGS-RhoGEFs. Also, the three siRNAs targeting LARG, PDZ-RhoGEF, and p115-RhoGEF are able to discriminate the closely related sequences within this RGS-RhoGEF gene family.

Zymosan activates protein kinase A via adenylyl cyclase VII to modulate innate immune responses during inflammation.

Pathogens use a variety of strategies to evade host immune defenses. A powerful way to suppress immune function is to increase intracellular concentrations of cAMP in host immune cells, which dampens inflammatory responses and prevents microbial killing. We found that the yeast cell wall extract, zymosan, is capable of increasing intracellular cAMP and activates the protein kinase A pathway in bone marrow derived macrophage (BMDM) cells from mice. This response is dependent on adenylyl cyclase type VII (AC7) and heterotrimeric G proteins, primarily G(12/13). Consequently, zymosan induced production of the inflammatory cytokine, TNFα, was much stronger in BMDMs from AC7 deficient mice compared to the response in wild type cells. In a model of zymosan induced peritonitis, mice deficient of AC7 in the myeloid lineage displayed prolonged inflammation. We propose that zymosan induced increases in cAMP and activation of PKA serve as a mechanism to dampen inflammatory responses in host cells, which consequently favors the survival of microbes. This would also help explain a well documented phenomenon, that the ability of zymosan to stimulate inflammatory cytokine responses via TLR2 appears to be weaker than other stimuli of TLR2.

Modulation of a GEF switch: autoinhibition of the intrinsic guanine nucleotide exchange activity of p115-RhoGEF.

p115-RhoGEF (p115) belongs to the family of RGS-containing guanine nucleotide exchange factors for Rho GTPases (RGS-RhoGEFs) that are activated by G12 class heterotrimeric G protein α subunits. All RGS-RhoGEFs possess tandemly linked Dbl-homology (DH) and plekstrin-homology (PH) domains, which bind and catalyze the exchange of GDP for GTP on RhoA. We have identified that the linker region connecting the N-terminal RGS-homology (RH) domain and the DH domain inhibits the intrinsic guanine nucleotide exchange (GEF) activity of p115, and determined the crystal structures of the DH/PH domains in the presence or absence of the inhibitory linker region. An N-terminal extension of the canonical DH domain (the GEF switch), which is critical to GEF activity, is well folded in the crystal structure of DH/PH alone, but becomes disordered in the presence of the linker region. The linker region is completely disordered in the crystal structure and partially disordered in the molecular envelope calculated from measurements of small angle x-ray scattering (SAXS). It is possible that Gα subunits activate p115 in part by relieving autoinhibition imposed by the linker region.

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.