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Side-chain-constrained amino acids are useful tools to modulate the biological properties of peptides. In this study, we applied side-chain constraints to apelin-13 (Ape13) by substituting the Pro12 and Phe13 positions, affecting the binding affinity and signaling profile on the apelin receptor (APJ). The residues 1Nal, Trp, and Aia were found to be beneficial substitutions for Pro12, and the resulting analogues displayed high affinity for APJ ( K i 0.08-0.18 nM vs Ape13 K i 0.7 nM). Besides, constrained (d-Tic) or α,α-disubstituted residues (Db z g; d-α-Me-Tyr(OBn)) were favorable for the Phe13 position. Compounds 47 (Pro12-Phe13 replaced by Aia-Phe, K i 0.08 nM) and 53 (Pro12-Phe13 replaced by 1Nal-Db z g, K i 0.08 nM) are the most potent Ape13 analogues activating the Gα 12 pathways ( 53 , EC 50 Gα 12 2.8 nM vs Ape13, EC 50 43 nM) known to date, displaying high affinity, resistance to ACE2 cleavage as well as improved pharmacokinetics in vitro ( t 1/2 5.8-7.3 h in rat plasma) and in vivo .

The adhesion G-protein-coupled receptor (GPCR) latrophilin 3 (ADGRL3) has been associated with increased risk of attention deficit hyperactivity disorder (ADHD) and substance use in human genetic studies. Knockdown in multiple species leads to hyperlocomotion and altered dopamine signaling. Thus, ADGRL3 is a potential target for treatment of neuropsychiatric disorders that involve dopamine dysfunction, but its basic signaling properties are poorly understood. Identification of adhesion GPCR signaling partners has been limited by a lack of tools to acutely activate these receptors in living cells. Here, we design a novel acute activation strategy to characterize ADGRL3 signaling by engineering a receptor construct in which we could trigger acute activation enzymatically. Using this assay, we found that ADGRL3 signals through G12/G13 and Gq, with G12/13 the most robustly activated. Gα 12/13 is a new player in ADGRL3 biology, opening up unexplored roles for ADGRL3 in the brain. Our methodological advancements should be broadly useful in adhesion GPCR research.

The G12/13 subfamily of heterotrimeric guanine nucleotide binding proteins comprises the α subunits Gα12 and Gα13, which transduce signals for cell growth, cytoskeletal rearrangements, and oncogenic transformation. In an increasing range of cancers, overexpressed Gα12 or Gα13 are implicated in aberrant cell proliferation and/or metastatic invasion. Although Gα12 and Gα13 bind non-redundant sets of effector proteins and participate in unique signalling pathways, the structural features responsible for functional differences between these α subunits are largely unknown. Invertebrates encode a single G12/13 homolog that participates in cytoskeletal changes yet appears to lack signalling to SRF (serum response factor), a transcriptional activator stimulated by mammalian Gα12 and Gα13 to promote growth and tumorigenesis. Our previous studies identified an evolutionarily divergent region in Gα12 for which replacement by homologous sequence from Drosophila melanogaster abolished SRF signalling, whereas the same invertebrate substitution was fully tolerated in Gα13 [Montgomery et al. (2014) Mol. Pharmacol. 85: 586]. These findings prompted our current approach of evolution-guided mutagenesis to identify fine structural features of Gα12 and Gα13 that underlie their respective SRF activation mechanisms. Our results identified two motifs flanking the α4 helix that play a key role in Gα12 signalling to SRF. We found the region encompassing these motifs to provide an interacting surface for multiple Gα12-specific target proteins that fail to bind Gα13. Adjacent to this divergent region, a highly-conserved domain was vital for SRF activation by both Gα12 and Gα13. However, dissection of this domain using invertebrate substitutions revealed different signalling mechanisms in these α subunits and identified Gα13-specific determinants of binding Rho-specific guanine nucleotide exchange factors. Furthermore, invertebrate substitutions in the C-terminal, α5 helical region were selectively disruptive to Gα12 signalling. Taken together, our results identify key structural features near the C-terminus that evolved after the divergence of Gα12 and Gα13, and should aid the development of agents to selectively manipulate signalling by individual α subunits of the G12/13 subfamily., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Despite recent advances in structural definition of GPCR-G protein complexes, the basis of receptor selectivity between G proteins remains unclear. The Gα 12 and Gα 13 subtypes together form the least studied group of heterotrimeric G proteins. G protein-coupled receptor 35 (GPR35) has been suggested to couple efficiently to Gα 13 but weakly to Gα 12 . Using combinations of cells genome-edited to not express G proteins and bioluminescence resonance energy transfer-based sensors, we confirmed marked selectivity of GPR35 for Gα 13 . Incorporating Gα 12 /Gα 13 chimeras and individual residue swap mutations into these sensors defined that selectivity between Gα 13 and Gα 12 was imbued largely by a single leucine-to-isoleucine variation at position G.H5.23. Indeed, leucine could not be substituted by other amino acids in Gα 13 without almost complete loss of GPR35 coupling. The critical importance of leucine at G.H5.23 for GPR35-G protein interaction was further demonstrated by introduction of this leucine into Gα q , resulting in the gain of coupling to GPR35. These studies demonstrate that Gα 13 is markedly the most effective G protein for interaction with GPR35 and that selection between Gα 13 and Gα 12 is dictated largely by a single conservative amino acid variation.-Mackenzie, A. E., Quon, T., Lin, L.-C., Hauser, A. S., Jenkins, L., Inoue, A., Tobin, A. B., Gloriam, D. E., Hudson, B. D., Milligan, G. Receptor selectivity between the G proteins Gα 12 and Gα 13 is defined by a single leucine-to-isoleucine variation.

Förster Resonance Energy Transfer (FRET) provides a way to directly observe the activation of heterotrimeric G-proteins by G-protein coupled receptors (GPCRs). To this end, FRET based biosensors are made, employing heterotrimeric G-protein subunits tagged with fluorescent proteins. These FRET based biosensors complement existing, indirect, ways to observe GPCR activation. Here we report on the insertion of mTurquoise2 at several sites in the human Gα13 subunit, aiming to develop a FRET-based Gα13 activation biosensor. Three fluorescently tagged Gα13 variants were found to be functional based on i) plasma membrane localization and ii) ability to recruit p115-RhoGEF upon activation of the LPA2 receptor. The tagged Gα13 subunits were used as FRET donor and combined with cp173Venus fused to the Gγ2 subunit, as the acceptor. We constructed Gα13 biosensors by generating a single plasmid that produces Gα13-mTurquoise2, Gβ1 and cp173Venus-Gγ2. The Gα13 activation biosensors showed a rapid and robust response when used in primary human endothelial cells that were exposed to thrombin, triggering endogenous protease activated receptors (PARs). This response was efficiently inhibited by the RGS domain of p115-RhoGEF and from the biosensor data we inferred that this is due to GAP activity. Finally, we demonstrated that the Gα13 sensor can be used to dissect heterotrimeric G-protein coupling efficiency in single living cells. We conclude that the Gα13 biosensor is a valuable tool for live-cell measurements that probe spatiotemporal aspects of Gα13 activation.

The R7 regulator of G protein signaling family (R7-RGS) critically regulates nervous system development and function. Mice lacking all R7-RGS subtypes exhibit diverse neurological phenotypes, and humans bearing mutations in the retinal R7-RGS isoform RGS9-1 have vision deficits. Although each R7-RGS subtype forms heterotrimeric complexes with Gβ 5 and R7-RGS-binding protein (R7BP) that regulate G protein-coupled receptor signaling by accelerating deactivation of G i/o α-subunits, several neurological phenotypes of R7-RGS knock-out mice are not readily explained by dysregulated G i/o signaling. Accordingly, we used tandem affinity purification and LC-MS/MS to search for novel proteins that interact with R7-RGS heterotrimers in the mouse brain. Among several proteins detected, we focused on Gα 13 because it had not been linked to R7-RGS complexes before. Split-luciferase complementation assays indicated that Gα 13 in its active or inactive state interacts with R7-RGS heterotrimers containing any R7-RGS isoform. LARG (leukemia-associated Rho guanine nucleotide exchange factor (GEF)), PDZ-RhoGEF, and p115RhoGEF augmented interaction between activated Gα 13 and R7-RGS heterotrimers, indicating that these effector RhoGEFs can engage Gα 13 ·R7-RGS complexes. Because Gα 13 /R7-RGS interaction required R7BP, we analyzed phenotypes of neuronal cell lines expressing RGS7 and Gβ 5 with or without R7BP. We found that neurite retraction evoked by Gα 12/13 -dependent lysophosphatidic acid receptors was augmented in R7BP-expressing cells. R7BP expression blunted neurite formation evoked by serum starvation by signaling mechanisms involving Gα 12/13 but not Gα i/o These findings provide the first evidence that R7-RGS heterotrimers interact with Gα 13 to augment signaling pathways that regulate neurite morphogenesis. This mechanism expands the diversity of functions whereby R7-RGS complexes regulate critical aspects of nervous system development and function., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Even though GPCR signaling in human platelets is directly involved in hemostasis and thrombus formation, the sequence of events by which G protein activation leads to αIIbβ3 integrin activation (inside-out signaling) is not clearly defined. We previously demonstrated that a conformationally sensitive domain of one G protein, i.e. Gα13 switch region 1 (Gα13SR1), can directly participate in the platelet inside-out signaling process. Interestingly however, the dependence on Gα13SR1 signaling was limited to PAR1 receptors, and did not involve signaling through other important platelet GPCRs. Based on the limited scope of this involvement, and the known importance of G13 in hemostasis and thrombosis, the present study examined whether signaling through another switch region of G13, i.e. Gα13 switch region 2 (Gα13SR2) may represent a more global mechanism of platelet activation. Using multiple experimental approaches, our results demonstrate that Gα13SR2 forms a bi-molecular complex with the head domain of talin and thereby promotes β3 integrin activation. Moreover, additional studies provided evidence that Gα13SR2 is not constitutively associated with talin in unactivated platelets, but becomes bound to talin in response to elevated intraplatelet calcium levels. Collectively, these findings provide evidence for a novel paradigm of inside-out signaling in platelets, whereby β3 integrin activation involves the direct binding of the talin head domain to the switch region 2 sequence of the Gα13 subunit., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Cancer-associated point mutations in Ras, in particular, at glycine 12 and glycine 13, affect the normal cycle between inactive GDP-bound and active GTP-bound states. In this work, the role of G12V and G13V replacements in the GAP-stimulated intrinsic GTP hydrolysis reaction in Ras is studied using molecular dynamics (MD) simulations with quantum mechanics/molecular mechanics (QM/MM) potentials. A model molecular system was constructed by motifs of the relevant crystal structure (Protein Data Bank entry 1WQ1 ). QM/MM optimization of geometry parameters in the Ras-GAP-GTP complex and QM/MM-MD simulations were performed with a quantum subsystem comprising a large fraction of the enzyme active site. For the system with wild-type Ras, the conformations fluctuated near the structure ready to be involved in the efficient chemical reaction leading to the cleavage of the phosphorus-oxygen bond in GTP upon approach of the properly aligned catalytic water molecule. Dynamics of the system with the G13V mutant is characterized by an enhanced flexibility in the area occupied by the γ-phosphate group of GTP, catalytic water, and the side chains of Arg789 and Gln61, which should somewhat hinder fast chemical steps. Conformational dynamics of the system with the G12V mutant shows considerable displacement of the Gln61 side chain and catalytic water from their favorable arrangement in the active site that may lead to a marked reduction in the reaction rate. The obtained computational results correlate well with the recent kinetic measurements of the Ras-GAP-catalyzed hydrolysis of GTP.

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., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Heterotrimeric G proteins, composed of α, β, and γ subunits, are activated by exchange of GDP for GTP on the Gα subunit. Canonically, Gα is stimulated by the guanine-nucleotide exchange factor (GEF) activity of ligand-bound G protein-coupled receptors. However, Gα subunits may also be activated in a noncanonical manner by members of the Ric-8 family, cytoplasmic proteins that also act as GEFs for Gα subunits. We used a signaling pathway active during Drosophila gastrulation as a model system to study Ric-8/Gα interactions. A component of this pathway, the Drosophila Gα12/13 subunit, Concertina (Cta), is necessary to trigger actomyosin contractility during gastrulation events. Ric-8 mutants exhibit similar gastrulation defects to Cta mutants. Here we use a novel tissue culture system to study a signaling pathway that controls cytoskeletal rearrangements necessary for cellular morphogenesis. We show that Ric-8 regulates this pathway through physical interaction with Cta and preferentially interacts with inactive Cta and directs its localization within the cell. We also use this system to conduct a structure-function analysis of Ric-8 and identify key residues required for both Cta interaction and cellular contractility.

Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous cancer composed of at least 2 molecular subtypes that differ in gene expression and distribution of mutations. Recently, application of genome/exome sequencing and RNA-seq to DLBCL has revealed numerous genes that are recurrent targets of somatic point mutation in this disease. Here we provide a whole-genome-sequencing-based perspective of DLBCL mutational complexity by characterizing 40 de novo DLBCL cases and 13 DLBCL cell lines and combining these data with DNA copy number analysis and RNA-seq from an extended cohort of 96 cases. Our analysis identified widespread genomic rearrangements including evidence for chromothripsis as well as the presence of known and novel fusion transcripts. We uncovered new gene targets of recurrent somatic point mutations and genes that are targeted by focal somatic deletions in this disease. We highlight the recurrence of germinal center B-cell-restricted mutations affecting genes that encode the S1P receptor and 2 small GTPases (GNA13 and GNAI2) that together converge on regulation of B-cell homing. We further analyzed our data to approximate the relative temporal order in which some recurrent mutations were acquired and demonstrate that ongoing acquisition of mutations and intratumoral clonal heterogeneity are common features of DLBCL. This study further improves our understanding of the processes and pathways involved in lymphomagenesis, and some of the pathways mutated here may indicate new avenues for therapeutic intervention.

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.

Vascular injury initiates rapid platelet activation that is critical for hemostasis, but it also may cause thrombotic diseases, such as myocardial infarction or ischemic stroke. Reorganizations of the platelet cytoskeleton are crucial for platelet shape change and secretion and are thought to involve activation of the small GTPase RhoA. In this study, we analyzed the in vitro and in vivo consequences of megakaryocyte- and platelet-specific RhoA gene deletion in mice. We found a pronounced macrothrombocytopenia in RhoA-deficient mice, with platelet counts of approximately half that of wild-type controls. The mutant cells displayed an altered shape but only a moderately reduced life span. Shape change of RhoA-deficient platelets in response to G(13)-coupled agonists was abolished, and it was impaired in response to G(q) stimulation. Similarly, RhoA was required for efficient secretion of α and dense granules downstream of G(13) and G(q). Furthermore, RhoA was essential for integrin-mediated clot retraction but not for actomyosin rearrangements and spreading of activated platelets on fibrinogen. In vivo, RhoA deficiency resulted in markedly prolonged tail bleeding times but also significant protection in different models of arterial thrombosis and in a model of ischemic stroke. Together, these results establish RhoA as an important regulator of platelet function in thrombosis and hemostasis.

RH-RhoGEFs are a family of guanine nucleotide exchange factors that contain a regulator of G protein signaling homology (RH) domain. The heterotrimeric G protein Gα(13) stimulates the guanine nucleotide exchange factor (GEF) activity of RH-RhoGEFs, leading to activation of RhoA. The mechanism by which Gα(13) stimulates the GEF activity of RH-RhoGEFs, such as p115RhoGEF, has not yet been fully elucidated. Here, specific residues in Gα(13) that mediate activation of p115RhoGEF are identified. Mutation of these residues significantly impairs binding of Gα(13) to p115RhoGEF as well as stimulation of GEF activity. These data suggest that the exchange activity of p115RhoGEF is stimulated allosterically by Gα(13) and not through its interaction with a secondary binding site. A crystal structure of Gα(13) bound to the RH domain of p115RhoGEF is also presented, which differs from a previously crystallized complex with a Gα(13)-Gα(i1) chimera. Taken together, these data provide new insight into the mechanism by which p115RhoGEF is activated by Gα(13).

G protein-coupled receptors (GPCRs) represent a large family of seven transmembrane receptors, which communicate extracellular signals into the cellular lumen. The human genome contains 720-800 GPCRs, and their diverse signal characteristics are determined by their specific tissue and subcellular expression profiles, as well as their coupling profile to the various G protein families (G(s), G(i), G(q), G(12)). The G protein coupling pattern links GPCR activation to the specific downstream effector pathways. G(12/13) signalling of GPCRs has been studied only recently in more detail, and involves activation of RhoGTPase nucleotide exchange factors (RhoGEFs). Four mammalian RhoGEFs regulated by G(12/13) proteins are known: p115-RhoGEF, PSD-95/Disc-large/ZO-1 homology-RhoGEF, leukemia-associated RhoGEF and lymphoid blast crisis-RhoGEF. These link GPCRs to activation of the small monomeric GTPase RhoA, and other downstream effectors. Misregulated G(12/13) signalling is involved in multiple pathophysiological conditions such as cancer, cardiovascular diseases, arterial and pulmonary hypertension, and bronchial asthma. Specific targeting of G(12/13) signalling-related diseases of GPCRs hence provides novel therapeutic approaches. Assays to quantitatively measure GPCR-mediated activation of G(12/13) are only emerging, and are required to understand the G(12/13)-linked pharmacology. The review gives an overview of G(12/13) signalling of GPCRs with a focus on RhoGEF proteins as the immediate mediators of G(12/13) activation.

The transient protein-protein interactions induced by guanine nucleotide-dependent conformational changes of G proteins play central roles in G protein-coupled receptor-mediated signaling systems. Leukemia-associated RhoGEF (LARG), a guanine nucleotide exchange factor for Rho, contains an RGS homology (RH) domain and Dbl homology/pleckstrin homology (DH/PH) domains and acts both as a GTPase-activating protein (GAP) and an effector for Galpha(13). However, the molecular mechanism of LARG activation upon Galpha(13) binding is not yet well understood. In this study, we analyzed the Galpha(13)-LARG interaction using cellular and biochemical methods, including a surface plasmon resonance (SPR) analysis. The results obtained using various LARG fragments demonstrated that active Galpha(13) interacts with LARG through the RH domain, DH/PH domains, and C-terminal region. However, an alanine substitution at the RH domain contact position in Galpha(13) resulted in a large decrease in affinity. Thermodynamic analysis revealed that binding of Galpha(13) proceeds with a large negative heat capacity change (DeltaCp degrees ), accompanied by a positive entropy change (DeltaS degrees ). These results likely indicate that the binding of Galpha(13) with the RH domain triggers conformational rearrangements between Galpha(13) and LARG burying an exposed hydrophobic surface to create a large complementary interface, which facilitates complex formation through both GAP and effector interfaces, and activates the RhoGEF. We propose that LARG activation is regulated by an induced-fit mechanism through the GAP interface of Galpha(13).

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.

Galpha12 and Galpha13 transduce signals from G protein-coupled receptors to RhoA through RhoGEFs containing an RGS homology (RH) domain, such as p115 RhoGEF or leukemia-associated RhoGEF (LARG). The RH domain of p115 RhoGEF or LARG binds with high affinity to active forms of Galpha12 and Galpha13 and confers specific GTPase-activating protein (GAP) activity, with faster GAP responses detected in Galpha13 than in Galpha12. At the same time, Galpha13, but not Galpha12, directly stimulates the RhoGEF activity of p115 RhoGEF or nonphosphorylated LARG in reconstitution assays. In order to better understand the molecular mechanism by which Galpha13 regulates RhoGEF activity through interaction with RH-RhoGEFs, we sought to identify the region(s) of Galpha13 involved in either the GAP response or RhoGEF activation. For this purpose, we generated chimeras between Galpha12 and Galpha13 subunits and characterized their biochemical activities. In both cell-based and reconstitution assays of RhoA activation, we found that replacing the carboxyl-terminal region of Galpha12 (residues 267-379) with that of Galpha13 (residues 264-377) conferred gain-of-function to the resulting chimeric subunit, Galpha12C13. The inverse chimera, Galpha13C12, exhibited basal RhoA activation which was similar to Galpha12. In contrast to GEF assays, GAP assays showed that Galpha12C13 or Galpha13C12 chimeras responded to the GAP activity of p115 RhoGEF or LARG in a manner similar to Galpha12 or Galpha13, respectively. We conclude from these results that the carboxyl-terminal region of Galpha13 (residues 264-377) is essential for its RhoGEF stimulating activity, whereas the amino-terminal alpha helical and switch regions of Galpha12 and Galpha13 are responsible for their differential GAP responses to the RH domain.

Many cellular signaling pathways share regulation by protein phosphatase-2A (PP2A), a widely expressed serine/threonine phosphatase, and the heterotrimeric G protein Galpha(12). PP2A activity is altered in carcinogenesis and in some neurodegenerative diseases. We have identified binding of Galpha(12) with the Aalpha subunit of PP2A, a trimeric enzyme composed of A (scaffolding), B (regulatory), and C (catalytic) subunits and demonstrated that Galpha(12) stimulated phosphatase activity (J Biol Chem 279: 54983-54986, 2004). We now show in substrate-velocity analysis using purified PP2A that V(max) was stimulated 3- to 4-fold by glutathione transferase (GST)-Galpha(12) with little effect on K(m) values. To identify the binding domains mediating the Aalpha-Galpha(12) interaction, an extensive mutational analysis was performed. Well-characterized mutations of Aalpha were expressed in vitro and tested for binding to GST-Galpha(12) in pull-down assays. Galpha(12) binds to Aalpha along repeats 7 to 10, and PP2A B subunits are not necessary for binding. To identify where Aalpha binds to Galpha(12), a series of 61 Galpha(12) mutants were engineered to contain the sequence Asn-Ala-Ala-Ile-Arg-Ser (NAAIRS) in place of 6 consecutive amino acids. Mutant Galpha(12) proteins were individually expressed in human embryonic kidney cells and analyzed for interaction with GST or GST-Aalpha in pull-down assays. The Aalpha binding sites were localized to regions near the N and C termini of Galpha(12). The expression of constitutively activated Galpha(12) (QLalpha(12)) in Madin Darby canine kidney cells stimulated PP2A activity as determined by decreased phosphorylation of tyrosine 307 on the catalytic subunit. Based on crystal structures of Galpha(12) and PP2A Aalpha, a model describing the binding surfaces and potential mechanisms of Galpha(12)-mediated PP2A activation is presented.

The first 15 years, or so, brought the realization that there existed a G protein coupled signal transduction mechanism by which hormone receptors regulate adenylyl cyclases and the light receptor rhodopsin activates visual phosphodiesterase. Three G proteins, Gs, Gi and transducin (T) had been characterized as alphabetagamma heterotrimers, and Gsalpha-GTP and Talpha-GTP had been identified as the sigaling arms of Gs and T. These discoveries were made using classical biochemical approaches, and culminated in the purification of these G proteins. The second 15 years, or so, are the subject of the present review. This time coincided with the advent of powerful recombinant DNA techniques. Combined with the classical approaches, the field expanded the repertoire of G proteins from 3 to 16, discovered the superfamily of seven transmembrane G protein coupled receptors (GPCRs) -- which is not addressed in this article -- and uncovered an amazing repertoire of effector functions regulated not only by alphaGTP complexes but also by betagamma dimers. Emphasis is placed in presenting how the field developed with the hope of conveying why many of the new findings were made.

In Caenorhabditis elegans adults, the single Rho GTPase orthologue, RHO-1, stimulates neurotransmitter release at synapses. We show that one of the pathways acting upstream of RHO-1 in acetylcholine (ACh)-releasing motor neurons depends on Galpha12 (GPA-12), which acts via the single C. elegans RGS RhoGEF (RHGF-1). Activated GPA-12 has the same effect as activated RHO-1, inducing the accumulation of diacylglycerol and the neuromodulator UNC-13 at release sites, and increased ACh release. We showed previously that RHO-1 stimulates ACh release by two separate pathways-one that requires UNC-13 and a second that does not. We show here that a non-DAG-binding-UNC-13 mutant that partially blocks increased ACh release by activated RHO-1 completely blocks increased ACh release by activated GPA-12. Thus, the upstream GPA-12/RHGF-1 pathway stimulates only a subset of RHO-1 downstream effectors, suggesting that either the RHO-1 effectors require different levels of activated RHO-1 for activation or there are two distinct pools of RHO-1 within C. elegans neurons.

Integrin-mediated adhesion is a crucial step in lymphocyte extravasation and homing. We show here that not only the chemokines CXCL12 and CXCL13 but also the lysophospholipids sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) enhance adhesion of murine follicular and marginal zone B cells to ICAM-1 in vitro. This process involves clustering of integrin LFA-1 and is blocked by pertussis toxin, suggesting that G(i) family G-proteins are involved. In addition, lysophospholipid-induced adhesion on ICAM-1 depends on Rho and Rhokinase, indicative of an involvement of G(12)/G(13), possibly also G(q)/G(11) family G-proteins. We used G(12)/G(13)- or G(q)/G(11)-deficient B cells to study the role of these G-protein families in lysophospholipid-induced adhesion and found that the pro-adhesive effects of LPA and S1P are completely abrogated in G(12)/G(13)-deficient marginal zone B cells, reduced in G(12)/G(13)-deficient follicular B cells, and normal in G(q)/G(11)-deficient B cells. We also show that loss of lysophospholipid-induced adhesion results in disinhibition of migration in response to the follicular chemokine CXCL13, which might contribute to the abnormal localization of splenic B cell populations observed in B cell-specific G(12)/G(13)-deficient mice in vivo. Taken together, this study shows that lysophospholipids regulate integrin-mediated adhesion of splenic B cells to ICAM-1 through G(i) and G(12)/G(13) family G-proteins but not through G(q)/G(11).

The signaling effects of lysophospholipids such as lysophosphatidic acid (LPA) are mediated by G protein-coupled receptors (GPCRs). There are currently four LPA receptors known as LPA(1-4). Genetic deletion studies have identified essential biological functions for LPA receptors in mice. However, these studies have also revealed phenotypes consistent with the existence of as yet unidentified receptors. Toward identifying new LPA receptors, we have screened collections of GPCR cDNAs using reverse transfection and cell-based assays. Here we report an interim result of one screen to identify receptors that produced LPA-dependent changes in cell shape: the orphan receptor GPR92 has properties of a new LPA receptor. Sequence analyses of human GPR92 and its mouse homolog have approximately 35% amino acid identity with LPA4/GPR23. The same cell-based approaches that were used to identify and/or characterize LPA(1-4), particularly heterologous expression in B103 cells or RH7777 cells, were utilized and compared with known LPA receptors. Retroviral-mediated expression of epitope-tagged receptors was further combined with G protein minigenes and pharmacological intervention, along with calcium imaging and whole-cell patch clamp electrophysiology. LPA-dependent receptor internalization following exposure to LPA but not related lysophospholipids was observed. Furthermore, LPA induced concentration-dependent activation of G(12/13) and G(q) and increased cAMP levels. Specific [3H]LPA binding was detected in cell membranes heterologously expressing GPR92 but not control membranes. Northern blot and reverse transcriptase-PCR studies indicated a broad low level of expression in many tissues including embryonic brain and enrichment in small intestine and sensory dorsal root ganglia, as well as embryonic stem cells. These results support GPR92 as a fifth LPA receptor, LPA5, which likely has distinct physiological functions in view of its expression pattern.

Although the prognosis for patients with early-stage breast cancer has improved, the therapeutic options for patients with locally advanced and metastatic disease are limited. To improve the treatment of these patients, the molecular mechanisms underlying breast cancer invasion and metastasis must be understood. In this study, we report that signaling through the G12 family of heterotrimeric G proteins (Galpha12 and Galpha13) promotes breast cancer cell invasion. Moreover, we demonstrate that inhibition of G12 signaling reduces the metastatic dissemination of breast cancer cells in vivo. Finally, we demonstrate that the expression of Galpha12 is significantly up-regulated in the earliest stages of breast cancer, implying that amplification of G12 signaling may be an early event in breast cancer progression. Taken together, these observations identify the G12 family proteins as important regulators of breast cancer invasion and suggest that these proteins may be targeted to limit invasion- and metastasis-induced patient morbidity and mortality.

The oncogenic G(12/13) subfamily of heterotrimeric G proteins transduces extracellular signals that regulate the actin cytoskeleton, cell cycle progression, and gene transcription. Previously, structural analyses of fully functional G alpha(12/13) subunits have been hindered by insufficient amounts of homogeneous, functional protein. Herein, we report that substitution of the N-terminal helix of G alpha(i1) for the corresponding region of G alpha12 or G alpha13 generated soluble chimeric subunits (G alpha(i/12) and G alpha(i/13)) that could be purified in sufficient amounts for crystallographic studies. Each chimera bound guanine nucleotides, G betagamma subunits, and effector proteins and exhibited GAP responses to p115RhoGEF and leukemia-associated RhoGEF. Like their wild-type counterparts, G alpha(i/13), but not G alpha(i/12), stimulated the activity of p115RhoGEF. Crystal structures of the G alpha(i/12) x GDP x AlF4(-) and G alpha(i/13) x GDP complexes were determined using diffraction data extending to 2.9 and 2.0 A, respectively. These structures reveal not only the native structural features of G alpha12 and G alpha13 subunits, which are expected to be important for their interactions with GPCRs and effectors such as G alpha-regulated RhoGEFs, but also novel conformational changes that are likely coupled to GTP hydrolysis in the G alpha(12/13) class of heterotrimeric G proteins.

The ERM (ezrin, radixin, moesin) proteins function as cross-linkers between cell membrane and cytoskeleton by binding to membrane proteins via their N-terminal domain and to F-actin via their C-terminal domain. Previous studies from our laboratory have shown that the alpha-subunit of heterotrimeric G(13) protein induces conformational activation of radixin via interaction with its N-terminal domain (Vaiskunaite, R., Adarichev, V., Furthmayr, H., Kozasa, T., Gudkov, A., and Voyno-Yasenetskaya, T. A. (2000) J. Biol. Chem. 275, 26206-26212). In the present study, we tested whether radixin can regulate Galpha(13)-mediated signaling pathways. We determined the effects of the N-terminal domain (amino acids 1-318) and C-terminal domain (amino acids 319-583) of radixin on serum response element (SRE)-dependent gene transcription initiated by a constitutively activated Galpha(13)Q226L. The N-terminal domain potentiated SRE activation induced by Galpha(13)Q226L; RhoGDI inhibited this effect. Surprisingly, the C-terminal domain also stimulated the SRE-dependent gene transcription. When co-transfected with Galpha(13)Q226L, the C-terminal domain of radixin synergistically stimulated the SRE activation; RhoGDI inhibited this effect. Using in vivo pull-down assays, we have determined that the C-terminal domain of radixin activated Rac1 but not RhoA or Cdc42 proteins. By contrast, Galpha(13)Q226L activated RhoA but not Rac1 or Cdc42. We have also shown that both the C-terminal domain of radixin and Galpha(13)Q226L can stimulate Ca(2+)/calmodulin-dependent kinase, CaMKII. Activated mutant that mimics the phosphorylated state of radixin (T564E) stimulated Rac1, induced the phosphorylation of CaMKII, and stimulated SRE-dependent gene transcription. Down-regulation of endogenous radixin using small interference RNA inhibited SRE-dependent gene transcription and phosphorylation of CaMKII induced by Galpha(13)Q226L. Overall, our results indicated that radixin via its C-terminal domain mediates SRE-dependent gene transcription through activation of Rac1 and CaMKII. In addition, the radixin-CaMKII signaling pathway is involved in Galpha(13)-mediated SRE-dependent gene transcription, suggesting that radixin could be involved in novel signaling pathway regulated by G(13) protein.

Scaffolding proteins play a critical role in conferring specificity and fidelity to signaling pathways. The JNK-interacting leucine zipper protein (JLP) has been identified as a scaffolding protein involved in linking components of the JNK signaling module. Galpha(12) and Galpha(13), the alpha-subunits of heterotrimeric G proteins G12 and G13, respectively, stimulate the JNK module in diverse cell types. Here, we report that Galpha(13) physically interacts with JLP, and this interaction enhances Galpha(13)-mediated JNK activation. We also demonstrate endogenous interaction between JLP and Galpha(13) in MCF-7 cells. JLP interaction is specific to the G12 family of alpha-subunits via its C-terminal domain (termed GID-JLP), spanning amino acids 1165-1307, and this interaction is more pronounced with the mutationally or functionally activated form of Galpha(13) compared to that of wild-type Galpha(13). The presence of a ternary complex consisting of Galpha(13), JLP, and JNK suggests a role for JLP in tethering Galpha(13) to the signaling components involved in JNK activation. Coexpression of GID-JLP disrupts ternary complex formation in addition to attenuating Galpha(13)-stimulated JNK activity. These findings identify JLP as a novel scaffolding protein in the Galpha(13)-mediated JNK signaling pathway.

G protein-coupled receptors (GPCRs) form a large superfamily of membrane proteins that play an essential role in modulating many vital physiological events, such as cell communication, neurotransmission, sensory perception, and chemotaxis. Understanding of the 3D (dimensional) structures of these receptors and their binding interactions with G proteins will help in the design of drugs for the treatment of GPCR-related diseases. By means of the approach of structural bioinformatics, the 3D structures of human alpha-13 subunit of guanine nucleotide-binding protein (G alpha 13) and human thromboxane A2 (TXA2) receptor were developed. The former plays an important role in the control of cell growth that may serve as a prototypical G protein; the latter is a target for nitric oxide-mediated desensitization that may serve as a prototypical GPCR. On the basis of the 3D models, their coupling interactions were investigated via docking studies. It has been found that the two proteins are coupled with each other mainly through the interaction between the minigene of G alpha 13 and the 3rd intracellular loop of the TXA2 receptor, consistent with the existing deduction in the literatures. However, it has also been observed via a close view that some residues of the TXA2 receptor that are sequentially far away but spatially quite close to the loop region are also involved in forming hydrogen bonds with the minigene of G alpha 13. These findings may provide useful information for conducting mutagenesis and reveal the molecular mechanism how the human TXA2 receptor interact with G alpha 13 to activate intracellular signaling. The findings may also provide useful insights for stimulating new therapeutic approaches by manipulating the interaction of the receptor with the relevant G proteins.

The involvement of heterotrimeric G proteins in the regulation of adherens junction function is unclear. We identified alphaSNAP as an interactive partner of G alpha12 using yeast two-hybrid screening. Glutathione S-transferase pull-down assays showed the selective interaction of alphaSNAP with G alpha12 in COS-7 as well as in human umbilical vein endothelial cells. Using domain swapping experiments, we demonstrated that the N-terminal region of G alpha12 (1-37 amino acids) was necessary and sufficient for its interaction with alphaSNAP. G alpha13 with its N-terminal extension replaced by that of G alpha12 acquired the ability to bind to alphaSNAP, whereas G alpha12 with its N terminus replaced by that of G alpha13 lost this ability. Using four point mutants of alphaSNAP, which alter its ability to bind to the SNARE complex, we determined that the convex rather than the concave surface of alphaSNAP was involved in its interaction with G alpha12. Co-transfection of human umbilical vein endothelial cells with G alpha12 and alphaSNAP stabilized VE-cadherin at the plasma membrane, whereas down-regulation of alphaSNAP with siRNA resulted in the loss of VE-cadherin from the cell surface and, when used in conjunction with G alpha12 overexpression, decreased endothelial barrier function. Our results demonstrate a direct link between the alpha subunit of G12 and alphaSNAP, an essential component of the membrane fusion machinery, and implicate a role for this interaction in regulating the membrane localization of VE-cadherin and endothelial barrier function.

The G alpha subunits of the G12 family of heterotrimeric G proteins, G alpha12 and G alpha13, are closely related in sequences and some effectors, but they often act through different pathways or bind to different proteins. We have examined subcellular distribution of these two G proteins and found that endogenous G alpha12 and G alpha13 localize in membrane and cytoplasmic fractions, respectively. Exogenously expressed G alpha12 and G alpha13 also localize in membrane and cytoplasmic fractions, respectively, in COS-7 cells. Stimulation of lysophosphatidic acid receptor coupled to G alpha13 markedly promotes the translocation of G alpha13 from cytoplasm to membrane. This different localization of G alpha12 and G alpha13 may explain some of the nonoverlapping actions of G alpha12 and G alpha13.

AKAP-Lbc is a novel member of the A-kinase anchoring protein (AKAPs) family, which functions as a cAMP-dependent protein kinase (PKA)-targeting protein as well as a guanine nucleotide exchange factor (GEF) for RhoA. We recently demonstrated that AKAP-Lbc Rho-GEF activity is stimulated by the alpha-subunit of the heterotrimeric G protein G(12), whereas phosphorylation of AKAP-Lbc by the anchored PKA induces the recruitment of 14-3-3, which inhibits its GEF function. In the present report, using co-immunoprecipitation approaches, we demonstrated that AKAP-Lbc can form homo-oligomers inside cells. Mutagenesis studies revealed that oligomerization is mediated by two adjacent leucine zipper motifs located in the C-terminal region of the anchoring protein. Most interestingly, disruption of oligomerization resulted in a drastic increase in the ability of AKAP-Lbc to stimulate the formation of Rho-GTP in cells under basal conditions, suggesting that oligomerization maintains AKAP-Lbc in a basal-inactive state. Based on these results and on our previous findings showing that AKAP-Lbc is inactivated through the association with 14-3-3, we investigated the hypothesis that AKAP-Lbc oligomerization might be required for the regulatory action of 14-3-3. Most interestingly, we found that mutants of AKAP-Lbc impaired in their ability to undergo oligomerization were completely resistant to the inhibitory effect of PKA and 14-3-3. This suggests that 14-3-3 can negatively regulate the Rho-GEF activity of AKAP-Lbc only when the anchoring protein is in an oligomeric state. Altogether, these findings provide a novel mechanistic explanation of how oligomerization can regulate the activity of exchange factors of the Dbl family.

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.

The Galpha(12/13) family of heterotrimeric G proteins modulate multiple cellular processes including regulation of the actin cytoskeleton. Galpha(12/13) interact with several cytoskeletal/scaffolding proteins, and in a yeast two-hybrid screen with Galpha(12), we detected an interaction with the scaffolding subunit (Aalpha) of the Ser/Thr phosphatase, protein phosphatase 2A (PP2A). PP2A dephosphorylates multiple substrates including tau, a microtubule-associated protein that is hyperphosphorylated in neurofibrillary tangles. The interaction of Aalpha and Galpha(12) was confirmed by coimmunoprecipitation studies in transfected COS cells and by glutathione S-transferase (GST)-Galpha(12) pull-downs from cell lysates of primary neurons. The interaction was specific for Aalpha and Galpha(12) and was independent of Galpha(12) conformation. Endogenous Aalpha and Galpha(12) colocalized by immunofluorescent microscopy in Caco-2 cells and in neurons. In vitro reconstitution of GST-Galpha(12) or recombinant Galpha(12) with PP2A core enzyme resulted in approximately 300% stimulation of PP2A activity that was not detected with other Galpha subunits and was similar with GTPgammaS- and GDP-liganded Galpha(12). When tau and active kinase (Cdk5 and p25) were cotransfected in to COS cells, there was robust tau phosphorylation. Co-expression of wild type or QLalpha(12) with tau and the active kinase resulted in 60 +/- 15% reductions in tau phosphorylation. In primary cortical neurons stimulated with lysophosphatitic acid, a 50% decrease in tau phosphorylation was observed. The Galpha(12) effect on tau phosphorylation was inhibited by the PP2A inhibitor, okadaic acid (50 nm), in COS cells and neurons. Taken together, these findings reveal novel, direct regulation of PP2A activity by Galpha(12) and potential in vivo modulation of PP2A target proteins including tau.

The alpha-subunit of G proteins of the G(12/13) family stimulate Rho by their direct binding to the RGS-like (RGL) domain of a family of Rho guanine nucleotide exchange factors (RGL-RhoGEFs) that includes PDZ-RhoGEF (PRG), p115RhoGEF, and LARG, thereby regulating cellular functions as diverse as shape and movement, gene expression, and normal and aberrant cell growth. The structural features determining the ability of G alpha(12/13) to bind RGL domains and the mechanism by which this association results in the activation of RGL-RhoGEFs are still poorly understood. Here, we explored the structural requirements for the functional interaction between G alpha(13) and RGL-RhoGEFs based on the structure of RGL domains and their similarity with the area by which RGS4 binds the switch region of G alpha(i) proteins. Using G alpha(i2), which does not bind RGL domains, as the backbone in which G alpha(13) sequences were swapped or mutated, we observed that the switch region of G alpha(13) is strictly necessary to bind PRG, and specific residues were identified that are critical for this association, likely by contributing to the binding surface. Surprisingly, the switch region of G alpha(13) was not sufficient to bind RGL domains, but instead most of its GTPase domain is required. Furthermore, membrane localization of G alpha(13) and chimeric G alpha(i2) proteins was also necessary for Rho activation. These findings revealed the structural features by which G alpha(13) interacts with RGL domains and suggest that molecular interactions occurring at the level of the plasma membrane are required for the functional activation of the RGL-containing family of RhoGEFs.

Galpha(12), the alpha-subunit of G protein G12, is ubiquitously expressed and it has been identified as a putative "causative oncogene" of soft-tissue sarcomas. Overexpression of wild-type or GTPase-deficient mutant of Galpha(12) (Galpha(12)Q229L or Galpha(12)QL) leads to the oncogenic transformation of NIH3T3 cells. Galpha(12)QL-tramsformed NIH3T3 cells show a distinct oncogenic phenotype defined by increased cell proliferation, anchorage-independent growth, reduced growth-factor dependency, attenuation of apoptotic signals, and neoplastic cytoskeletal changes. In this study, the genes contributing to the reduced growth-factor dependency of Galpha(12)QL-NIH3T3 cells were identified by transcription profiling of serum-starved Galpha(12)QL-transformed NIH3T3 (Galpha(12)QL-NIH3T3) cells. Results from these studies indicate that Galpha(12)QL stimulates the expression of genes that promote cell growth. The increased expressions of growth-promoting genes in Galpha(12)QL-NIH3T3 cells were validated by semiquantitative reverse transcription-polymerase chain reaction and immunoblot analyses. Further studies aimed at investigating the critical role of two of such upregulated genes, namely PDGFRalpha and JAK3, indicated that the inhibition of PDGFRalpha or JAK3 activity-attenuated Galpha(12)QL-mediated serum-independent cell proliferation. These studies point to possible novel autocrine and/or paracrine control mechanisms involving PDGFRalpha and JAK3 in Galpha(12)-mediated proliferation and oncogenesis.

G alpha 12/13-mediated pathways have been shown to be involved in various fundamental cellular functions in mammalian cells such as axonal guidance, apoptosis, and chemotaxis. Here, we identified a homologue of Rho-guanine nucleotide exchange factor (GEF) in Caenorhabditis elegans (CeRhoGEF), which functions downstream of gpa-12, the C. elegans homologue of G alpha 12/13. CeRhoGEF contains a PSD-95/Dlg/ZO-1 domain and a regulator of G protein signaling (RGS) domain upstream of the Dbl homology-pleckstrin homology region similar to mammalian RhoGEFs with RGS domains, PSD-95/Dlg/ZO-1-RhoGEF and leukemia-associated RhoGEF. It has been shown in mammalian cells that these RhoGEFs interact with activated forms of G alpha 12 or G alpha 13 through their RGS domains. We demonstrated by coimmunoprecipitation that the RGS domain of CeRhoGEF interacts with GPA-12 in an AIF4- activation-dependent manner and confirmed that the Dbl homology-pleckstrin homology domain of CeRhoGEF was capable of Rho-dependent signaling. These results proved conservation of the G alpha 12-RhoGEF pathway in C. elegans. Expression of DsRed or GFP under the control of the promoter of CeRhoGEF or gpa-12 revealed an overlap of their expression patterns in ventral cord motor neurons and several neurons in the head. RNA-mediated gene interference for CeRhoGEF and gpa-12 resulted in similar phenotypes such as embryonic lethality and sensory and locomotive defects in adults. Thus, the G alpha 12/13-RhoGEF pathway is likely to be involved in embryonic development and neuronal function in C. elegans.