Increased adrenergic tone resulting from cardiovascular stress leads to development of heart failure, in part, through chronic stimulation of β1 adrenergic receptors (βARs) on cardiac myocytes. Blocking these receptors is part of the basis for β-blocker therapy for heart failure. Recent data demonstrate that G protein-coupled receptors (GPCRs), including βARs, are activated intracellularly, although the biological significance is unclear. Here we investigated the functional role of Golgi βARs in rat cardiac myocytes and found they activate Golgi localized, prohypertrophic, phosphoinositide hydrolysis, that is not accessed by cell surface βAR stimulation. This pathway is accessed by the physiological neurotransmitter norepinephrine (NE) via an Oct3 organic cation transporter. Blockade of Oct3 or specific blockade of Golgi resident β1ARs prevents NE dependent cardiac myocyte hypertrophy. This clearly defines a pathway activated by internal GPCRs in a biologically relevant cell type and has implications for development of more efficacious β-blocker therapies.

Phospholipase C-epsilon (PLCϵ) plays a critical role in G-protein-coupled receptor-mediated inflammation. In addition to its ability to generate the second messengers inositol 1,4,5-trisphosphate and diacylglycerol, PLCϵ, unlike the other phospholipase C family members, is activated in a sustained manner. We hypothesized that the ability of PLCϵ to function as a guanine nucleotide exchange factor (GEF) for Rap1 supports sustained downstream signaling via feedback of Rap1 to the enzyme Ras-associating (RA2) domain. Using gene deletion and adenoviral rescue, we demonstrate that both the GEF (CDC25 homology domain) and RA2 domains of PLCϵ are required for long term protein kinase D (PKD) activation and subsequent induction of inflammatory genes. PLCϵ localization is largely intracellular and its compartmentalization could contribute to its sustained activation. Here we show that localization of PLCϵ to the Golgi is required for activation of PKD in this compartment as well as for subsequent induction of inflammatory genes. These data provide a molecular mechanism by which PLCϵ mediates sustained signaling and by which astrocytes mediate pathophysiological inflammatory responses.

Phosphatidylinositol (3,4,5) trisphosphate (PIP3)-dependent Rac exchanger 1 (P-Rex1) is a Rho guanine-nucleotide exchange factor that was originally discovered in neutrophils and is regulated by G protein βγ subunits and the lipid PIP3 in response to chemoattractants. P-Rex1 has also become increasingly recognized for its role in promoting metastasis of breast cancer, prostate cancer, and melanoma. Recent structural, biochemical, and biologic work has shown that binding of PIP3 to the pleckstrin homology (PH) domain of P-Rex1 is required for its activation in cells. Here, differential scanning fluorimetry was used in a medium-throughput screen to identify six small molecules that interact with the P-Rex1 PH domain and block binding of and activation by PIP3 Three of these compounds inhibit N-formylmethionyl-leucyl-phenylalanine induced spreading of human neutrophils as well as activation of the GTPase Rac2, both of which are downstream effects of P-Rex1 activity. Furthermore, one of these compounds reduces neutrophil velocity and inhibits neutrophil recruitment in response to inflammation in a zebrafish model. These results suggest that the PH domain of P-Rex1 is a tractable drug target and that these compounds might be useful for inhibiting P-Rex1 in other experimental contexts. SIGNIFICANCE STATEMENT: A set of small molecules identified in a thermal shift screen directed against the phosphatidylinositol (3,4,5) trisphosphate-dependent Rac exchanger 1 (P-Rex1) pleckstrin homology domain has effects consistent with P-Rex1 inhibition in neutrophils.

Activation of the small guanosine triphosphatase RhoA can promote cell survival in cultured cardiomyocytes and in the heart. We showed that the circulating lysophospholipid sphingosine 1-phosphate (S1P), a G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR) agonist, signaled through RhoA and phospholipase Cε (PLCε) to increase the phosphorylation and activation of protein kinase D1 (PKD1). Genetic deletion of either PKD1 or its upstream regulator PLCε inhibited S1P-mediated cardioprotection against ischemia/reperfusion injury. Cardioprotection involved PKD1-mediated phosphorylation and inhibition of the cofilin phosphatase Slingshot 1L (SSH1L). Cofilin 2 translocates to mitochondria in response to oxidative stress or ischemia/reperfusion injury, and both S1P pretreatment and SSH1L knockdown attenuated translocation of cofilin 2 to mitochondria. Cofilin 2 associates with the proapoptotic protein Bax, and the mitochondrial translocation of Bax in response to oxidative stress was also attenuated by S1P treatment in isolated hearts or by knockdown of SSH1L or cofilin 2 in cardiomyocytes. Furthermore, SSH1L knockdown, like S1P treatment, increased cardiomyocyte survival and preserved mitochondrial integrity after oxidative stress. These findings reveal a pathway initiated by GPCR agonist-induced RhoA activation, in which PLCε signals to PKD1-mediated phosphorylation of cytoskeletal proteins to prevent the mitochondrial translocation and proapoptotic function of cofilin 2 and Bax and thereby promote cell survival.