Protein Βγ Subunits Research Articles

Heterotrimeric G Protein Subunit Gαq is a Master Switch for Gβγ-Mediated Calcium Mobilization by Gi-Coupled GPCRs

Mechanisms that control mobilization of cytosolic calcium [Ca2+]i are key for regulation of numerous eukaryotic cell functions. One such paradigmatic mechanism involves activation of phospholipase Cβ (PLCβ) enzymes by G protein βγ subunits from activated Gαi-Gβγ heterotrimers. Here we report identification of a master switch to enable this control for PLCβ enzymes in living cells. We find that the Gαi-Gβγ-PLCβ-Ca2+ signaling module is entirely dependent on the presence of active Gαq. If Gαq is pharmacologically inhibited or genetically ablated, Gβγ can bind to PLCβ, but does not elicit Ca2+ signals. Removal of an auto-inhibitory linker that occludes the active site of the enzyme is required and sufficient to empower ‘stand-alone control’ of PLCβ by Gβγ. Dependence on Gαq of Gi-Gβγ-Ca2+ is a mechanism placing an entire signaling branch of G protein-coupled receptors (GPCRs) under hierarchical control of Gq, and changes our understanding of how Gi-GPCRs trigger [Ca2+]i via PLCβ enzymes.

κ-Opioid Receptor Modulation of GABAergic Inputs onto Ventrolateral Periaqueductal Gray Dopamine Neurons

The κ-opioid receptor (KOR) system has been implicated in the regulation of many behaviors including pain. While there are numerous studies suggesting KOR regulation of pain being mediated spinally, there have been reports of pain-like behaviors regulated by central KOR signaling. In particular, oxytocin-induced analgesia appears to be mediated by KOR receptors within the ventrolateral periaqueductal gray (vlPAG). We recently found that activation of dopamine (DA) neurons within the vlPAG is antinociceptive. In this study, we sought to determine the impact of KOR signaling on ­GABAergic inputs onto vlPAG DA neurons, and the mechanism through which KOR impacts these inputs. We found that activation of KOR reduced GABAergic transmission onto vlPAG DA neurons. In addition, our data suggest this effect is mediated presynaptically via the G protein βγ-subunit. They raise the possibility that KOR activation disinhibits ­vlPAG DA neurons, which could lead to altered regulation of pain-related behaviors.

Structural mechanism underlying G protein family-specific regulation of G protein-gated inwardly rectifying potassium channel

G protein-gated inwardly rectifying potassium channel (GIRK) plays a key role in regulating neurotransmission. GIRK is opened by the direct binding of the G protein βγ subunit (Gβγ), which is released from the heterotrimeric G protein (Gαβγ) upon the activation of G protein-coupled receptors (GPCRs). GIRK contributes to precise cellular responses by specifically and efficiently responding to the Gi/o-coupled GPCRs. However, the detailed mechanisms underlying this family-specific and efficient activation are largely unknown. Here, we investigate the structural mechanism underlying the Gi/o family-specific activation of GIRK, by combining cell-based BRET experiments and NMR analyses in a reconstituted membrane environment. We show that the interaction formed by the αA helix of Gαi/o mediates the formation of the Gαi/oβγ-GIRK complex, which is responsible for the family-specific activation of GIRK. We also present a model structure of the Gαi/oβγ-GIRK complex, which provides the molecular basis underlying the specific and efficient regulation of GIRK.

G protein \u03b2\u03b3 subunits play a critical role in the actions of amphetamine

Abnormal levels of dopamine (DA) are thought to contribute to several neurological and psychiatric disorders including drug addiction. Extracellular DA levels are regulated primarily via reuptake by the DA transporter (DAT). Amphetamine, a potent psychostimulant, increases extracellular DA by inducing efflux through DAT. Recently, we discovered that G protein βγ subunits (Gβγ) interact with DAT, and that in vitro activation of Gβγ promotes DAT-mediated efflux. Here, we investigated the role of Gβγ in the actions of amphetamine in DA neurons in culture, ex vivo nucleus accumbens (NAc), and freely moving rats. Activation of Gβγ with the peptide myr-Ser-Ile-Arg-Lys-Ala-Leu-Asn-Ile-Leu-Gly-Tyr-Pro-Asp-Tyr-Asp (mSIRK) in the NAc potentiated amphetamine-induced hyperlocomotion, but not cocaine-induced hyperlocomotion, and systemic or intra-accumbal administration of the Gβγ inhibitor gallein attenuated amphetamine-induced, but not cocaine-induced hyperlocomotion. Infusion into the NAc of a TAT-fused peptide that targets the Gβγ-binding site on DAT (TAT-DATct1) also attenuated amphetamine-induced but not cocaine-induced hyperlocomotion. In DA neurons in culture, inhibition of Gβγ with gallein or blockade of the Gβγ–DAT interaction with the TAT-DATct1 peptide decreased amphetamine-induced DA efflux. Furthermore, activation of Gβγ with mSIRK potentiated and inhibition of Gβγ with gallein reduced amphetamine-induced increases of extracellular DA in the NAc in vitro and in freely moving rats. Finally, systemic or intra-accumbal inhibition of Gβγ with gallein blocked the development of amphetamine-induced, but not cocaine-induced place preference. Collectively, these results suggest that interaction between Gβγ and DAT plays a critical role in the actions of amphetamine and presents a novel target for modulating the actions of amphetamine in vivo.

G protein–coupled receptors differentially regulate glycosylation and activity of the inwardly rectifying potassium channel Kir7.1

Kir7.1 is an inwardly rectifying potassium channel with important roles in the regulation of the membrane potential in retinal pigment epithelium, uterine smooth muscle, and hypothalamic neurons. Regulation of G protein-coupled inwardly rectifying potassium (GIRK) channels by G protein-coupled receptors (GPCRs) via the G protein βγ subunits has been well characterized. However, how Kir channels are regulated is incompletely understood. We report here that Kir7.1 is also regulated by GPCRs, but through a different mechanism. Using Western blotting analysis, we observed that multiple GPCRs tested caused a striking reduction in the complex glycosylation of Kir7.1. Further, GPCR-mediated reduction of Kir7.1 glycosylation in HEK293T cells did not alter its expression at the cell surface but decreased channel activity. Of note, mutagenesis of the sole Kir7.1 glycosylation site reduced conductance and open probability, as indicated by single-channel recording. Additionally, we report that the L241P mutation of Kir7.1 associated with Lebers congenital amaurosis (LCA), an inherited retinal degenerative disease, has significantly reduced complex glycosylation. Collectively, these results suggest that Kir7.1 channel glycosylation is essential for function, and this activity within cells is suppressed by most GPCRs. The melanocortin-4 receptor (MC4R), a GPCR previously reported to induce ligand-regulated activity of this channel, is the only GPCR tested that does not have this effect on Kir7.1.

Investigating the Role of G Protein βγ in Kv7-Dependent Relaxations of the Rat Vasculature.

Objective- In renal arteries, inhibitors of G protein βγ subunits (Gβγ) reduce Kv7 activity and inhibit Kv7-dependent receptor-mediated vasorelaxations. However, the mechanisms underlying receptor-mediated relaxation are artery specific. Consequently, the aim of this study was to ascertain the role of Gβγ in Kv7-dependent vasorelaxations of the rat vasculature. Approach and Results- Isometric tension recording was performed in isolated rat renal, mesenteric, and cerebral arteries to study isoproterenol and calcitonin gene-related peptide relaxations. Kv7.4 was knocked down via morpholino transfection while inhibition of Gβγ was investigated with gallein and M119K. Proximity ligation assay was performed on isolated myocytes to study the association between Kv7.4 and G protein β subunits or signaling intermediaries. Isoproterenol or calcitonin gene-related peptide-induced relaxations were attenuated by Kv7.4 knockdown in all arteries studied. Inhibition of Gβγ with gallein or M119K had no effect on isoproterenol-mediated relaxations in mesenteric artery but had a marked effect on calcitonin gene-related peptide-induced responses in mesenteric artery and cerebral artery and isoproterenol responses in renal artery. Isoproterenol increased association with Kv7.4 and Rap1a in mesenteric artery which were not sensitive to gallein, whereas in renal artery, isoproterenol increased Kv7.4-AKAP (A-kinase anchoring protein) associations in a gallein-sensitive manner. Conclusions- The Gβγ-Kv7 relationship differs between vessels and is an essential requirement for AKAP, but not Rap-mediated regulation of the channel.

Small molecule disruption of G protein βγ subunit signaling reprograms human macrophage phenotype and prevents autoimmune myocarditis in rats.

The purpose of this study was to determine whether blocking of G protein βγ (Gβγ) signaling halts heart failure (HF) progression by macrophage phenotype manipulation. Cardiac Gβγ signaling plays a crucial role in HF pathogenesis. Previous data suggested that inhibiting Gβγ signaling reprograms T helper cell 1 (Th1) and Th2 cytokines, suggesting that Gβγ might be a useful drug target for treating HF. We investigated the efficacy of a small molecule Gβγ inhibitor, gallein, in a clinically relevant, experimental autoimmune myocarditis (EAM) model of HF as well as in human macrophage phenotypes in vitro. In the myocardium of HF patients, we observed that G protein coupled receptor kinase (GRK)2 levels were down-regulated compared with healthy controls. In rat EAM, treatment with gallein effectively improved survival and cardiac function, suppressed cardiac remodeling, and further attenuated myocardial protein expression of GRK2 as well as high mobility group box (HMGB)1 and its cascade signaling proteins. Furthermore, gallein effectively inhibited M1 polarization and promoted M2 polarization in vivo in the EAM heart and in vitro in human monocyte-derived macrophages. Taken together, these data suggest that the small molecule Gβγ inhibitor, gallein, could be an important pharmacologic therapy for HF as it can switch the phenotypic reprogramming from M1 to M2 phenotype in a rat model of EAM heart and in human macrophages.

G protein βγ subunits directly interact with and activate phospholipase CΕ

Phospholipase C (PLC) enzymes hydrolyze membrane phosphatidylinositol 4,5 bisphosphate (PIP2) and regulate Ca2+ and protein kinase signaling in virtually all mammalian cell types. Chronic activation of the PLCϵ isoform downstream of G protein-coupled receptors (GPCRs) contributes to the development of cardiac hypertrophy. We have previously shown that PLCϵ-catalyzed hydrolysis of Golgi-associated phosphatidylinositol 4-phosphate (PI4P) in cardiac myocytes depends on G protein βγ subunits released upon stimulation with endothelin-1. PLCϵ binds and is directly activated by Ras family small GTPases, but whether they directly interact with Gβγ has not been demonstrated. To identify PLCϵ domains that interact with Gβγ, here we designed various single substitutions and truncations of WT PLCϵ and tested them for activation by Gβγ in transfected COS-7 cells. Deletion of only a single domain in PLCϵ was not sufficient to completely block its activation by Gβγ, but blocked activation by Ras. Simultaneous deletion of the C-terminal RA2 domain and the N-terminal CDC25 and cysteine-rich domains completely abrogated PLCϵ activation by Gβγ, but activation by the GTPase Rho was retained. In vitro reconstitution experiments further revealed that purified Gβγ directly interacts with a purified fragment of PLCϵ (PLCϵ-PH-RA2) and increases PIP2 hydrolysis. Deletion of the RA2 domain decreased Gβγ binding and eliminated Gβγ stimulation of PIP2 hydrolysis. These results provide first evidence that Gβγ directly interacts with PLCϵ and yield insights into the mechanism by which βγ subunits activate PLCϵ.

Gγ identity dictates efficacy of Gβγ signaling and macrophage migration

G protein βγ subunit (Gβγ) is a major signal transducer and controls processes ranging from cell migration to gene transcription. Despite having significant subtype heterogeneity and exhibiting diverse cell- and tissue-specific expression levels, Gβγ is often considered a unified signaling entity with a defined functionality. However, the molecular and mechanistic basis of Gβγ's signaling specificity is unknown. Here, we demonstrate that Gγ subunits, bearing the sole plasma membrane (PM)-anchoring motif, control the PM affinity of Gβγ and thereby differentially modulate Gβγ effector signaling in a Gγ-specific manner. Both Gβγ signaling activity and the migration rate of macrophages are strongly dependent on the PM affinity of Gγ. We also found that the type of C-terminal prenylation and five to six pre-CaaX motif residues at the PM-interacting region of Gγ control the PM affinity of Gβγ. We further show that the overall PM affinity of the Gβγ pool of a cell type is a strong predictor of its Gβγ signaling-activation efficacy. A kinetic model encompassing multiple Gγ types and parameterized for empirical Gβγ behaviors not only recapitulated experimentally observed signaling of Gβγ, but also suggested a Gγ-dependent, active-inactive conformational switch for the PM-bound Gβγ, regulating effector signaling. Overall, our results unveil crucial aspects of signaling and cell migration regulation by Gγ type-specific PM affinities of Gβγ.

Two independent but synchronized Gβγ subunit–controlled pathways are essential for trailing-edge retraction during macrophage migration

Chemokine-induced directional cell migration is a universal cellular mechanism and plays crucial roles in numerous biological processes, including embryonic development, immune system function, and tissue remodeling and regeneration. During the migration of a stationary cell, the cell polarizes, forms lamellipodia at the leading edge (LE), and triggers the concurrent retraction of the trailing edge (TE). During cell migration governed by inhibitory G protein (Gi)-coupled receptors (GPCRs), G protein βγ (Gβγ) subunits control the LE signaling. Interestingly, TE retraction has been linked to the activation of the small GTPase Ras homolog family member A (RhoA) by the Gα12/13 pathway. However, it is not clear how the activation of Gi-coupled GPCRs at the LE orchestrates the TE retraction in RAW264.7 macrophages. Here, using an optogenetic approach involving an opsin to activate the Gi pathway in defined subcellular regions of RAW cells, we show that in addition to their LE activities, free Gβγ subunits also govern TE retraction by operating two independent, yet synchronized, pathways. The first pathway involves RhoA activation, which prevents dephosphorylation of the myosin light chain, allowing actomyosin contractility to proceed. The second pathway activates phospholipase Cβ and induces myosin light chain phosphorylation to enhance actomyosin contractility through increasing cytosolic calcium. We further show that both of these pathways are essential, and inhibition of either one is sufficient to abolish the Gi-coupled GPCR-governed TE retraction and subsequent migration of RAW cells.

G protein βγ subunits inhibit TRPM3 ion channels in sensory neurons.

Transient receptor potential (TRP) ion channels in peripheral sensory neurons are functionally regulated by hydrolysis of the phosphoinositide PI(4,5)P2 and changes in the level of protein kinase mediated phosphorylation following activation of various G protein coupled receptors. We now show that the activity of TRPM3 expressed in mouse dorsal root ganglion (DRG) neurons is inhibited by agonists of the Gi-coupled µ opioid, GABA-B and NPY receptors. These agonist effects are mediated by direct inhibition of TRPM3 by Gβγ subunits, rather than by a canonical cAMP mediated mechanism. The activity of TRPM3 in DRG neurons is also negatively modulated by tonic, constitutive GPCR activity as TRPM3 responses can be potentiated by GPCR inverse agonists. GPCR regulation of TRPM3 is also seen in vivo where Gi/o GPCRs agonists inhibited and inverse agonists potentiated TRPM3 mediated nociceptive behavioural responses.

Pharmacological and Activated Fibroblast Targeting of Gβγ-GRK2 After Myocardial Ischemia Attenuates Heart Failure Progression

BackgroundCardiac fibroblasts are a critical cell population responsible for myocardial extracellular matrix homeostasis. Upon injury or pathological stimulation, these cells transform to an activated myofibroblast state and play a fundamental role in myocardial fibrosis and remodeling. Chronic sympathetic overstimulation, a hallmark of heart failure (HF), induces pathological signaling through G protein βγ (Gβγ) subunits and their interaction with G protein−coupled receptor kinase 2 (GRK2). ObjectivesThis study investigated the hypothesis that Gβγ-GRK2 inhibition and/or ablation after myocardial injury would attenuate pathological myofibroblast activation and cardiac remodeling. MethodsThe therapeutic potential of small molecule Gβγ-GRK2 inhibition, alone or in combination with activated fibroblast- or myocyte-specific GRK2 ablation—each initiated after myocardial ischemia−reperfusion (I/R) injury—was investigated to evaluate the possible salutary effects on post-I/R fibroblast activation, pathological remodeling, and cardiac dysfunction. ResultsSmall molecule Gβγ-GRK2 inhibition initiated 1 week post-injury was cardioprotective in the I/R model of chronic HF, including preservation of cardiac contractility and a reduction in cardiac fibrotic remodeling. Systemic small molecule Gβγ-GRK2 inhibition initiated 1 week post-I/R in cardiomyocyte-restricted GRK2 ablated mice (also post-I/R) still demonstrated significant cardioprotection, which suggested a potential protective role beyond the cardiomyocyte. Inducible ablation of GRK2 in activated fibroblasts (i.e., myofibroblasts) post-I/R injury demonstrated significant functional cardioprotection with reduced myofibroblast transformation and fibrosis. Systemic small molecule Gβγ-GRK2 inhibition initiated 1 week post-I/R provided little to no further protection in mice with ablation of GRK2 in activated fibroblasts alone. Finally, Gβγ-GRK2 inhibition significantly attenuated activation characteristics of failing human cardiac fibroblasts isolated from end-stage HF patients. ConclusionsThese findings suggested consideration of a paradigm shift in the understanding of the therapeutic role of Gβγ-GRK2 inhibition in treating HF and the potential therapeutic role for Gβγ-GRK2 inhibition in limiting pathological myofibroblast activation, interstitial fibrosis, and HF progression.

Abstract 422: Small Molecule and Activated Fibroblast Targeting of the Gβγ-GRK2 Interface After Myocardial Ischemia Attenuates Heart Failure Progression

Cardiac fibroblasts are a critical cell population responsible for myocardial extracellular matrix homeostasis. Upon injury or pathologic stimulation, these cells transform to an activated myofibroblast state and play a fundamental role in myocardial fibrosis and remodeling. Chronic sympathetic overstimulation, a hallmark of heart failure, induces pathologic signaling through G protein βγ subunits and their interaction with G protein-coupled receptor kinase 2 (GRK2). We hypothesized that Gβγ-GRK2 inhibition/ablation after myocardial injury would attenuate pathologic myofibroblast activation and cardiac remodeling. The therapeutic potential of small molecule Gβγ-GRK2 inhibition alone or in combination with activated fibroblast- or myocyte-specific GRK2 ablation, each initiated after myocardial ischemia/reperfusion (I/R) injury, was investigated to evaluate possible salutary effects on post-I/R fibroblast activation, pathologic remodeling and cardiac function. Small molecule Gβγ-GRK2 inhibition initiated one week post-injury was cardioprotective in the I/R model of chronic heart failure, including preservation of cardiac contractility and reduction in cardiac fibrotic remodeling. Systemic small molecule Gβγ-GRK2 inhibition initiated one week post-I/R in cardiomyocyte-restricted GRK2 ablated mice (also post-I/R) demonstrated additional cardioprotection, suggesting a potential protective role beyond the cardiomyocyte. Inducible ablation of GRK2 in activated fibroblasts (i.e. myofibroblasts) post-I/R injury demonstrated significant functional cardioprotection with reduced myofibroblast transformation and fibrosis. Systemic small molecule Gβγ-GRK2 inhibition initiated one week post-I/R provided little to no further protection in mice with ablation of GRK2 in activated fibroblasts alone. Finally, Gβγ-GRK2 inhibition significantly attenuated activation characteristics of failing human cardiac fibroblasts isolated from end stage heart failure patients. These findings suggest a potential therapeutic role for Gβγ-GRK2 inhibition in limiting pathologic myofibroblast activation, interstitial fibrosis and heart failure progression.

G-Protein-coupled receptors: Decoding mixed signals.

A new mechanism of functional crosstalk between two distinct G-protein-coupled receptors (GPCRs)—the parathyroid hormone receptor (PTHR) and β2-adrenergic receptor (β2 Ar)—that occurs at the level of G protein βγ subunits and a specific adenylyl cyclase isoform is identified. This crosstalk augments cAMP signaling by the PTHR from endosomes, and thus promotes the actions of PTH ligands in bone target cells.

PREX1 integrates G protein-coupled receptor and phosphoinositide 3-kinase signaling to promote glioblastoma invasion.

A defining feature of the brain cancer glioblastoma is its highly invasive nature. When glioblastoma cells are isolated from patients using serum free conditions, they accurately recapitulate this invasive behaviour in animal models. The Rac subclass of Rho GTPases has been shown to promote invasive behaviour in glioblastoma cells isolated in this manner. However the guanine nucleotide exchange factors responsible for activating Rac in this context have not been characterized previously. PREX1 is a Rac guanine nucleotide exchange factor that is synergistically activated by binding of G protein αγ subunits and the phosphoinositide 3-kinase pathway second messenger phosphatidylinositol 3,4,5 trisphosphate. This makes it of particular interest in glioblastoma, as the phosphoinositide 3-kinase pathway is aberrantly activated by mutation in almost all cases. We show that PREX1 is expressed in glioblastoma cells isolated under serum-free conditions and in patient biopsies. PREX1 promotes the motility and invasion of glioblastoma cells, promoting Rac-mediated activation of p21-associated kinases and atypical PKC, which have established roles in cell motility. Glioblastoma cell motility was inhibited by either inhibition of phosphoinositide 3-kinase or inhibition of G protein βγ subunits. Motility was also inhibited by the generic dopamine receptor inhibitor haloperidol or a combination of the selective dopamine receptor D2 and D4 inhibitors L-741,626 and L-745,870. This establishes a role for dopamine receptor signaling via G protein βγ subunits in glioblastoma invasion and shows that phosphoinositide 3-kinase mutations in glioblastoma require a context of basal G protein–coupled receptor activity in order to promote this invasion.

Inhibition of G Protein βγ Subunit Signaling Abrogates Nephritis in Lupus-Prone Mice.

Despite considerable advances in the understanding of systemic lupus erythematosus (SLE), there is still an urgent need for new and more targeted treatment approaches. We previously demonstrated that small-molecule blockade of G protein βγ subunit (Gβγ) signaling inhibits acute inflammation through inhibition of chemokine receptor signal transduction. We undertook this study to determine whether inhibition of Gβγ signaling ameliorates disease in a mouse model of SLE. Lupus-prone (NZB × NZW)F1 female mice were prophylactically or therapeutically treated with the small-molecule Gβγ inhibitor gallein. Tissue samples were analyzed by flow cytometry and immunohistochemistry. The development and extent of nephritis were assessed by monitoring proteinuria and by immunohistochemical analysis. Serum immunoglobulin levels were measured by enzyme-linked immunosorbent assay, and total IgG and anti-double-stranded DNA (anti-dsDNA) antibody-secreting cells were measured by enzyme-linked immunospot assay. Gallein inhibited accumulation of T cells and germinal center (GC) B cells in the spleen. Both prophylactic and therapeutic treatment reduced GC size, decreased antibody-secreting cell production in the spleen, and markedly decreased accumulation of autoreactive anti-dsDNA antibody-secreting cells in kidneys. Gallein also reduced immune complex deposition in kidneys. Finally, gallein treatment dramatically inhibited kidney inflammation, prevented glomerular damage, and decreased proteinuria. Mechanistically, gallein inhibited immune cell migration and signaling in response to chemokines in vitro, which suggests that its mechanisms of action in vivo are inhibition of migration of immune cells to sites of inflammation and inhibition of immune cell maturation. Overall, these data demonstrate the potential use of gallein or novel inhibitors of Gβγ signaling in SLE treatment.

Abstract 428: Pharmacologic and Activated Fibroblast-specific Gβγ-GRK2 Inhibition is Protective Following Ischemia Reperfusion Injury

Heart failure is a devastating disease characterized by chamber remodeling, interstitial fibrosis and reduced ventricular compliance. Cardiac fibroblasts are responsible for extracellular matrix homeostasis, however upon injury or pathologic stimulation, these cells transform to a myofibroblast phenotype and play a fundamental role in myocardial fibrosis and remodeling. Chronic sympathetic overstimulation induces excess signaling through G protein βγ subunits and ultimately the pathologic activation of G protein-coupled receptor kinase 2 (GRK2). We hypothesized that Gβγ-GRK2 inhibition plays an important role in the cardiac fibroblast to attenuate pathologic myofibroblast activation and cardiac remodeling. To investigate this hypothesis, mice were subjected to ischemia/reperfusion (I/R) injury and treated with the small molecule Gβγ-GRK2 inhibitor gallein. While animals receiving vehicle demonstrated a reduction in overall cardiac function as measured by echocardiography, mice treated with gallein exhibited nearly complete preservation of cardiac function and reduced fibrotic scar formation. We next sought to establish the cell specificity of this compound by treating inducible cardiomyocyte- and activated fibroblast-specific GRK2 knockout mice post-I/R. Although we observed modest restoration in cardiac function in cardiomyocyte-specific GRK2 null mice, treatment of these mice with gallein resulted in further protection against myocardial dysfunction following injury, suggesting a functional role in other cardiac cell types, including fibroblasts. Activated fibroblast-specific GRK2 knockout mice were also subjected to ischemia/reperfusion injury; these animals displayed preserved myocardial function and reduced collagen deposition compared to littermate controls following injury. Furthermore, systemic Gβγ-GRK2 inhibition by gallein did not appear to confer further protection over activated fibroblast-specific GRK2 ablation alone. In summary, these findings suggest a potential therapeutic role for Gβγ-GRK2 inhibition in limiting pathologic myofibroblast activation, interstitial fibrosis and heart failure progression.

G Protein-Coupled Receptor-G-Protein βγ-Subunit Signaling Mediates Renal Dysfunction and Fibrosis in Heart Failure.

Development of CKD secondary to chronic heart failure (CHF), known as cardiorenal syndrome type 2 (CRS2), clinically associates with organ failure and reduced survival. Heart and kidney damage in CRS2 results predominantly from chronic stimulation of G protein-coupled receptors (GPCRs), including adrenergic and endothelin (ET) receptors, after elevated neurohormonal signaling of the sympathetic nervous system and the downstream ET system, respectively. Although we and others have shown that chronic GPCR stimulation and the consequent upregulated interaction between the G-protein βγ-subunit (Gβγ), GPCR-kinase 2, and β-arrestin are central to various cardiovascular diseases, the role of such alterations in kidney diseases remains largely unknown. We investigated the possible salutary effect of renal GPCR-Gβγ inhibition in CKD developed in a clinically relevant murine model of nonischemic hypertrophic CHF, transverse aortic constriction (TAC). By 12 weeks after TAC, mice developed CKD secondary to CHF associated with elevated renal GPCR-Gβγ signaling and ET system expression. Notably, systemic pharmacologic Gβγ inhibition by gallein, which we previously showed alleviates CHF in this model, attenuated these pathologic renal changes. To investigate a direct effect of gallein on the kidney, we used a bilateral ischemia-reperfusion AKI mouse model, in which gallein attenuated renal dysfunction, tissue damage, fibrosis, inflammation, and ET system activation. Furthermore, in vitro studies showed a key role for ET receptor-Gβγ signaling in pathologic fibroblast activation. Overall, our data support a direct role for GPCR-Gβγ in AKI and suggest GPCR-Gβγ inhibition as a novel therapeutic approach for treating CRS2 and AKI.

Structural and Functional Characterization of the Metastatic RhoGEF P‐Rex1 and its Regulation by PtdIns(3,4,5)P3: Towards Inhibitory Small Molecule Development

Phosphatidylinositol 3,4,5‐trisphosphate (PIP3)‐dependent Rac exchanger 1 (P‐Rex1) is a Rho guanine‐nucleotide exchange factor (RhoGEF) that regulates cell motility and is strongly associated with cancer metastasis. P‐Rex1 is synergistically recruited to the cell membrane and activated by PIP3 and heterotrimeric G protein βγ (Gβγ) subunits, positioning the enzyme downstream of multiple classes of cell surface receptors that control processes such as cytoskeleton rearrangement and cell migration. P‐Rex1 has thus become an attractive therapeutic target for the suppression of cancer metastasis. However, development of inhibitors against P‐Rex1 is hindered by the fact that its regulatory mechanisms are poorly understood. My primary goal is to define the molecular basis for regulation of P‐Rex1 by PIP3 and Gβγ using X‐ray crystallography and biochemical and cell‐based assays, with the aim of identifying the surfaces of P‐Rex1 that are important for interaction with these molecules as well as their mechanisms of activation. Thus far, I have determined crystal structures of the tandem Dbl homology (DH)/pleckstrin homology (PH) domain catalytic core of P‐Rex1 in complexes with its substrate small GTPases Cdc42 and Rac1, as well as of the independent PH domain in complex with Ins(1,3,4,5)P4, a soluble analog of PIP3. Using site‐directed mutagenesis, we have shown that the PH domain is necessary and sufficient for PIP3‐dependent activation. Interestingly, mutation of residues within the PIP3 binding site does not abrogate membrane binding, suggesting that PIP3 activates P‐Rex1 through an allosteric mechanism as opposed to simple recruitment to the cell membrane. These studies have also opened the door to medium‐throughput screens anticipated to identify small molecule probes that compete with physiological regulators of P‐Rex1. These compounds could potentially be developed into molecules that block PIP3 binding, thereby inhibiting P‐Rex1 activation in cells and, ultimately, blocking cancer metastasis.Support or Funding InformationThis work was supported by an American Cancer Society – Michigan Cancer Research Fund Postdoctoral Fellowship (PF‐14‐224‐01‐DMC) to J.C. and an American Heart Association Undergraduate Student Research Program Award (14UFEL20510027) to E.D.

Abstract 14173: Pharmacologic and Activated Fibroblast-Specific Gβγ-GRK2 Inhibition is Protective Following Ischemia Reperfusion Injury

Heart failure is a devastating disease characterized by chamber remodeling, interstitial fibrosis and reduced ventricular compliance. Cardiac fibroblasts are largely responsible for extracellular matrix homeostasis, however upon injury or pathologic stimulation, these cells transition to a myofibroblast phenotype and play a fundamental role in myocardial fibrosis and remodeling. Chronic sympathetic overstimulation induces excess signaling through G protein βγ subunits and ultimately results in pathologic G protein-coupled receptor kinase 2 (GRK2)-mediated β-adrenergic receptor downregulation. We hypothesized that Gβγ-GRK2 inhibition plays an important role in the cardiac fibroblast to attenuate pathologic myofibroblast differentiation, interstitial fibrosis and the overall progression of heart failure. To investigate this hypothesis, mice were subjected to ischemia/reperfusion injury and treated with the small molecule Gβγ-GRK2 inhibitor gallein for 4 weeks. Animals receiving vehicle demonstrated severe cardiac dilatation and deterioration in overall cardiac function as measured by percent fractional shortening and ejection fraction. Remarkably, mice treated with gallein exhibited nearly complete preservation of cardiac function, along with reduced scar formation and fibrotic marker expression, suggesting a direct effect within the cardiac fibroblasts. To further explore possible mechanisms, activated fibroblast-restricted GRK2 knockout mice (GRK2fl x PeriostinMCM) were subjected to 4 weeks of ischemia/reperfusion injury. These animals displayed preserved myocardial function and reduced eccentric hypertrophy and collagen deposition compared to GRK2fl littermate controls. Finally, systemic Gβγ-GRK2 inhibition by gallein may not confer further protection over activated fibroblast-specific GRK2 ablation alone. In summary, these findings suggest a potential therapeutic role for Gβγ-GRK2 inhibition in limiting pathologic myofibroblast activation, interstitial fibrosis and heart failure progression. We believe this fibroblast-targeted approach will lead to the refinement of existing targets and compounds, and possibly the generation of novel therapeutics for the treatment of heart failure.