Constraining the Side Chain of C-Terminal Amino Acids in Apelin-13 Greatly Increases Affinity, Modulates Signaling, and Improves the Pharmacokinetic Profile.

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 (Ki 0.08-0.18 nM vs Ape13 Ki 0.7 nM). Besides, constrained (d-Tic) or α,α-disubstituted residues (Dbzg; d-α-Me-Tyr(OBn)) were favorable for the Phe13 position. Compounds 47 (Pro12-Phe13 replaced by Aia-Phe, Ki 0.08 nM) and 53 (Pro12-Phe13 replaced by 1Nal-Dbzg, Ki 0.08 nM) are the most potent Ape13 analogues activating the Gα12 pathways (53, EC50 Gα12 2.8 nM vs Ape13, EC50 43 nM) known to date, displaying high affinity, resistance to ACE2 cleavage as well as improved pharmacokinetics in vitro (t1/2 5.8-7.3 h in rat plasma) and in vivo.

G12/13 is activated by acute tethered agonist exposure in the adhesion GPCR ADGRL3.

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. Gα12/13 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.

Divergent C-terminal motifs in Gα12 and Gα13 provide distinct mechanisms of effector binding and SRF activation.

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.

Receptor selectivity between the G proteins Gα12 and Gα13 is defined by a single leucine-to-isoleucine variation.

Despite recent advances in structural definition of GPCR-G protein complexes, the basis of receptor selectivity between G proteins remains unclear. The Gα12 and Gα13 subtypes together form the least studied group of heterotrimeric G proteins. G protein-coupled receptor 35 (GPR35) has been suggested to couple efficiently to Gα13 but weakly to Gα12. 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 Gα13. Incorporating Gα12/Gα13 chimeras and individual residue swap mutations into these sensors defined that selectivity between Gα13 and Gα12 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 Gα13 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 Gαq, resulting in the gain of coupling to GPR35. These studies demonstrate that Gα13 is markedly the most effective G protein for interaction with GPR35 and that selection between Gα13 and Gα12 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 Gα12 and Gα13 is defined by a single leucine-to-isoleucine variation.

A FRET-based biosensor for measuring Gα13 activation in single cells.

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, Gß1 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.

Regulation of neurite morphogenesis by interaction between R7 regulator of G protein signaling complexes and G protein subunit Gα13.

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 Gß5 and R7-RGS-binding protein (R7BP) that regulate G protein-coupled receptor signaling by accelerating deactivation of Gi/o α-subunits, several neurological phenotypes of R7-RGS knock-out mice are not readily explained by dysregulated Gi/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 Gα13 because it had not been linked to R7-RGS complexes before. Split-luciferase complementation assays indicated that Gα13 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 Gα13 and R7-RGS heterotrimers, indicating that these effector RhoGEFs can engage Gα13·R7-RGS complexes. Because Gα13/R7-RGS interaction required R7BP, we analyzed phenotypes of neuronal cell lines expressing RGS7 and Gß5 with or without R7BP. We found that neurite retraction evoked by Gα12/13-dependent lysophosphatidic acid receptors was augmented in R7BP-expressing cells. R7BP expression blunted neurite formation evoked by serum starvation by signaling mechanisms involving Gα12/13 but not Gαi/o These findings provide the first evidence that R7-RGS heterotrimers interact with Gα13 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.

Gα13 Switch Region 2 Binds to the Talin Head Domain and Activates αIIbß3 Integrin in Human Platelets.

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 αIIbß3 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 ß3 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 ß3 integrin activation involves the direct binding of the talin head domain to the switch region 2 sequence of the Gα13 subunit.

Modeling the role of G12V and G13V Ras mutations in the Ras-GAP-catalyzed hydrolysis reaction of guanosine triphosphate.

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.

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.

Drosophila Ric-8 interacts with the Gα12/13 subunit, Concertina, during activation of the Folded gastrulation pathway.

Heterotrimeric G proteins, composed of α, ß, 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.

Mutational and structural analysis of diffuse large B-cell lymphoma using whole-genome sequencing.

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.

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.

Megakaryocyte-specific RhoA deficiency causes macrothrombocytopenia and defective platelet activation in hemostasis and thrombosis.

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.

Identification of critical residues in G(alpha)13 for stimulation of p115RhoGEF activity and the structure of the G(alpha)13-p115RhoGEF regulator of G protein signaling homology (RH) domain complex.

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).

Regulation of RhoGEF proteins by G12/13-coupled receptors.

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.

Activation of leukemia-associated RhoGEF by Galpha13 with significant conformational rearrangements in the interface.

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).

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.

Distinct regions of Galpha13 participate in its regulatory interactions with RGS homology domain-containing RhoGEFs.

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.

Domains necessary for Galpha12 binding and stimulation of protein phosphatase-2A (PP2A): Is Galpha12 a novel regulatory subunit of PP2A?

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.