Biophysical characterization of the CXC chemokine receptor 2 ligands.

The chemokines of the immune system act as first responders by operating as chemoattractants, directing immune cells to specific locations of inflamed tissues. This promiscuous network is comprised of 50 ligands and 18 receptors where the ligands may interact with the receptors in various oligomeric states i.e., monomers, homodimers, and heterodimers. Chemokine receptors are G-protein coupled receptors (GPCRs) present in the membrane of immune cells. The migration of immune cells occurs in response to a concentration gradient of the ligands. Chemotaxis of neutrophils is directed by CXC-ligand (CXCL) activation of the membrane bound CXC chemokine receptor 2 (CXCR2). CXCR2 plays an important role in human health and is linked to disorders such as autoimmune disorders, inflammation, and cancer. Yet, despite their important role, little is known about the biophysical characteristics controlling ligand:ligand and ligand:receptor interaction essential for biological activity. In this work, we study the homodimers of three of the CXCR2 cognate ligands, CXCL1, CXCL5, and CXCL8. The ligands share high structural integrity but a low sequence identity. We show that the sequence diversity has evolved different binding affinities and stabilities for the CXC-ligands resulting in diverse agonist/antagonist behavior. Furthermore, CXC-ligands fold through a three-state mechanism, populating a folded monomeric state before associating into an active dimer.

Stabilization of Interdomain Interactions in G protein α Subunits as a Determinant of Gαi Subtype Signaling Specificity.

Highly homologous members of the Gαi family, Gαi1-3, have distinct tissue distributions and physiological functions, yet their biochemical and functional properties are very similar. We recently identified PDZ-RhoGEF (PRG) as a novel Gαi1 effector that is poorly activated by Gαi2. In a proteomic proximity labeling screen we observed a strong preference for Gαi1 relative to Gαi2 with respect to engagement of a broad range of potential targets. We investigated the mechanistic basis for this selectivity using PRG as a representative target. Substitution of either the helical domain (HD) from Gαi1 into Gαi2 or substitution of a single amino acid, A230 in Gαi2 with the corresponding D in Gαi1, largely rescues PRG activation and interactions with other potential Gαi targets. Molecular dynamics simulations combined with Bayesian network models revealed that in the GTP bound state, separation at the HD-Ras-like domain (RLD) interface is more pronounced in Gαi2 than Gαi1. Mutation of A230 to D in Gαi2 stabilizes HD-RLD interactions via ionic interactions with R145 in the HD which in turn modify the conformation of Switch III. These data support a model where D229 in Gαi1 interacts with R144 and stabilizes a network of interactions between HD and RLD to promote protein target recognition. The corresponding A230 in Gαi2 is unable to stabilize this network leading to an overall lower efficacy with respect to target interactions. This study reveals distinct mechanistic properties that could underly differential biological and physiological consequences of activation of Gαi1 or Gαi2 by GPCRs.

Phospholipase C epsilon-1 (PLCƐ1) mediates macrophage activation and protection against tuberculosis.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) infects up to a quarter of the world's population. Although immune responses can control Mtb infection, 5%-10% of infected individuals can progress to active TB disease (progressors). A myriad of host factors regulate disease progression in TB and a better understanding of immune correlates of protection and disease is pivotal for the development of new therapeutics. Comparison of human whole blood transcriptomic metadata with that of macaque TB progressors and Mtb-infected diversity outbred mice (DO) led to the identification of differentially regulated gene (DEG) signatures, associated with TB progression or control. The current study assessed the function of Phospholipase C epsilon (PLCƐ1), the top downregulated gene across species in TB progressors, using a gene-specific knockout mouse model of Mtb infection and in vitro Mtb-infected bone marrow-derived macrophages. PLCƐ1 gene expression was downregulated in TB progressors across species. PLCε1 deficiency in the mouse model resulted in increased susceptibility to Mtb infection, coincident accumulation of lung myeloid cells, and reduced ability to mount antibacterial responses. However, PLCε1 was not required for the activation and accumulation of T cells in mice. Our results suggest an important early role for PLCƐ1 in shaping innate immune response to TB and may represent a putative target for host-directed therapy.

A PACAP-activated network for secretion requires coordination of Ca 2+ influx and Ca 2+ mobilization.

Chromaffin cells of the adrenal medulla transduce sympathetic nerve activity into stress hormone secretion. The two neurotransmitters principally responsible for coupling cell stimulation to secretion are acetylcholine and pituitary adenylate activating polypeptide (PACAP). In contrast to acetylcholine, PACAP evokes a persistent secretory response from chromaffin cells. However, the mechanisms by which PACAP acts are poorly understood. Here, it is shown that PACAP induces sustained increases in cytosolic Ca 2+ which are disrupted when Ca 2+ influx through L-type channels is blocked or internal Ca 2+ stores are depleted. PACAP liberates stored Ca 2+ via inositol trisphosphate receptors (IP3Rs) on the endoplasmic reticulum (ER), thereby functionally coupling Ca 2+ mobilization to Ca 2+ influx and supporting Ca 2+ -induced Ca 2+ -release. These Ca 2+ influx and mobilization pathways are unified by an absolute dependence on phospholipase C epsilon (PLCε) activity. Thus, the persistent secretory response that is a defining feature of PACAP activity, in situ , is regulated by a signaling network that promotes sustained elevations in intracellular Ca 2+ through multiple pathways.

Internalized ß2-Adrenergic Receptors Inhibit Subcellular Phospholipase C-Dependent Cardiac Hypertrophic Signaling.

Chronically elevated neurohumoral drive, and particularly elevated adrenergic tone leading to ß-adrenergic receptor (ß-AR) overstimulation in cardiac myocytes, is a key mechanism involved in the progression of heart failure. ß1-AR and ß2-ARs are the two major subtypes of ß-ARs present in the human heart, however, they elicit different or even opposite effects on cardiac function and hypertrophy. For example, chronic activation of ß1ARs drives detrimental cardiac remodeling while ß2AR signaling is protective. The underlying molecular mechanisms for cardiac protection through ß2ARs remain unclear. Here we show that ß2-AR protects against hypertrophy through inhibition of PLCε signaling at the Golgi apparatus. The mechanism for ß2AR-mediated PLC inhibition requires internalization of ß2AR, activation of Gi and Gßγ subunit signaling at endosomes and ERK activation. This pathway inhibits both angiotensin II and Golgi-ß1-AR-mediated stimulation of phosphoinositide hydrolysis at the Golgi apparatus ultimately resulting in decreased PKD and HDAC5 phosphorylation and protection against cardiac hypertrophy. This reveals a mechanism for ß2-AR antagonism of the PLCε pathway that may contribute to the known protective effects of ß2-AR signaling on the development of heart failure.

Stabilization of Interdomain Interactions in G protein αi Subunits Determines Gαi Subtype Signaling Specificity.

Highly homologous members of the Gαi family, Gαi1-3, have distinct tissue distributions and physiological functions, yet the functional properties of these proteins with respect to GDP/GTP binding and regulation of adenylate cyclase are very similar. We recently identified PDZ-RhoGEF (PRG) as a novel Gαi1 effector, however, it is poorly activated by Gαi2. Here, in a proteomic proximity labeling screen we observed a strong preference for Gαi1 relative to Gαi2 with respect to engagement of a broad range of potential targets. We investigated the mechanistic basis for this selectivity using PRG as a representative target. Substitution of either the helical domain (HD) from Gαi1 into Gαi2 or substitution of a single amino acid, A230 in Gαi2 to the corresponding D in Gαi1, largely rescues PRG activation and interactions with other Gαi targets. Molecular dynamics simulations combined with Bayesian network models revealed that in the GTP bound state, dynamic separation at the HD-Ras-like domain (RLD) interface is prevalent in Gαi2 relative to Gαi1 and that mutation of A230s4h3.3 to D in Gαi2 stabilizes HD-RLD interactions through formation of an ionic interaction with R145HD.11 in the HD. These interactions in turn modify the conformation of Switch III. These data support a model where D229s4h3.3 in Gαi1 interacts with R144HD.11 stabilizes a network of interactions between HD and RLD to promote protein target recognition. The corresponding A230 in Gαi2 is unable to form the "ionic lock" to stabilize this network leading to an overall lower efficacy with respect to target interactions. This study reveals distinct mechanistic properties that could underly differential biological and physiological consequences of activation of Gαi1 or Gαi2 by GPCRs.

A Small Contribution to a Large System: The Leptin Receptor Complex.

Obesity is a classified epidemic, increasing the risk of secondary diseases such as diabetes, inflammation, cardiovascular disease, and cancer. The pleiotropic hormone leptin is the proposed link for the gut-brain axis controlling nutritional status and energy expenditure. Research into leptin signaling provides great promise toward discovering therapeutics for obesity and its related diseases targeting leptin and its cognate leptin receptor (LEP-R). The molecular basis underlying the human leptin receptor complex assembly remains obscure, due to the lack of structural information regarding the biologically active complex. In this work, we investigate the proposed receptor binding sites in human leptin utilizing designed antagonist proteins combined with AlphaFold predictions. Our results show that binding site I has a more intricate role in the active signaling complex than previously described. We hypothesize that the hydrophobic patch in this region engages a third receptor forming a higher-order complex, or a new LEP-R binding site inducing allosteric rearrangement.

Phospholipase C-ε defines a PACAP-stimulated pathway for secretion in the chromaffin cell.

Adrenomedullary chromaffin cells respond to splanchnic (sympathetic) nerve stimulation by releasing stress hormones into the circulation. The signal for hormone secretion is encoded in the neurotransmitters - especially acetylcholine (ACh) and pituitary adenylate cyclase activating polypeptide (PACAP) - that are released into the splanchnic-chromaffin cell synapse. However, functional differences in the effects of ACh and PACAP on the chromaffin cell secretory response are not well defined. Here, selective agonists of PACAP receptors or nicotinic and muscarinic acetylcholine receptors were applied to chromaffin cells. The major differences in the effects of these agents were not on exocytosis, per se, but rather on the steps upstream of exocytosis. In almost every respect, the properties of individual fusion events triggered by PACAP and cholinergic agonists were similar. On the other hand, the properties of the Ca2+ transients evoked by PACAP differed in several ways from those evoked by muscarinic and nicotinic receptor stimulation. A defining feature of the PACAP-stimulated secretory pathway was its dependence on signaling through exchange protein directly activated by cAMP (Epac) and PLCε. However, the absence of PLCε did not disrupt Ca2+ transients evoked by cholinergic agonists. Accordingly, inhibition of Epac activity did not disrupt secretion triggered by acetylcholine or specific agonists of muscarinic and nicotinic receptors. Thus, PACAP and acetylcholine stimulate chromaffin cell secretion via separate and independent pathways. This feature of stimulus-secretion coupling may be important for sustaining hormone release from the adrenal medulla under conditions associated with the sympathetic stress response.

PACAP and acetylcholine cause distinct Ca2+ signals and secretory responses in chromaffin cells.

The adrenomedullary chromaffin cell transduces chemical messages into outputs that regulate end organ function throughout the periphery. At least two important neurotransmitters are released by innervating preganglionic neurons to stimulate exocytosis in the chromaffin cell-acetylcholine (ACh) and pituitary adenylate cyclase activating polypeptide (PACAP). Although PACAP is widely acknowledged as an important secretagogue in this system, the pathway coupling PACAP stimulation to chromaffin cell secretion is poorly understood. The goal of this study is to address this knowledge gap. Here, it is shown that PACAP activates a Gαs-coupled pathway that must signal through phospholipase C ε (PLCε) to drive Ca2+ entry and exocytosis. PACAP stimulation causes a complex pattern of Ca2+ signals in chromaffin cells, leading to a sustained secretory response that is kinetically distinct from the form stimulated by ACh. Exocytosis caused by PACAP is associated with slower release of peptide cargo than exocytosis stimulated by ACh. Importantly, only the secretory response to PACAP, not ACh, is eliminated in cells lacking PLCε expression. The data show that ACh and PACAP, acting through distinct signaling pathways, enable nuanced and variable secretory outputs from chromaffin cells.

Phospholipase Cε insufficiency causes ascending aortic aneurysm and dissection.

Phospholipase Cε (PLCε) is a phospholipase C isoform with a wide range of physiological functions. It has been implicated in aortic valve disorders, but its role in frequently associated aortic disease remains unclear. To determine the role of PLCε in thoracic aortic aneurysm and dissection (TAAD) we used PLCε-deficient mice, which develop aortic valve insufficiency and exhibit aortic dilation of the ascending thoracic aorta and arch without histopathological evidence of injury. Fourteen days of infusion of Plce1+/+ and Plce1-/- mice with angiotensin II (ANG II), which induces aortic dilation and dissection, led to sudden death secondary to ascending aortic dissection in 43% of Plce1-/- versus 5% of Plce1+/+ mice (P < 0.05). Medial degeneration and TAAD were detected in 80% of Plce1-/- compared with 10% of Plce1+/+ mice (P < 0.05) after 4 days of ANG II. Treatment with ANG II markedly increased PLCε expression within the ascending aortic adventitia. Total RNA sequencing demonstrated marked upregulation of inflammatory and fibrotic pathways mediated by interleukin-1ß, interleukin-6, and tumor necrosis factor-α. In silico analysis of whole exome sequences of 258 patients with type A dissection identified 5 patients with nonsynonymous PLCE1 variants. Our data suggest that PLCε deficiency plays a role in the development of TAAD and aortic insufficiency.NEW & NOTEWORTHY We describe a novel phenotype by which PLCε deficiency predisposes to aortic valve insufficiency and ascending aortic aneurysm, dissection, and sudden death in the setting of ANG II-mediated hypertension. We demonstrate PLCE1 variants in patients with type A aortic dissection and aortic insufficiency, suggesting that PLCE1 may also play a role in human aortic disease. This finding is of very high significance because it has not been previously demonstrated that PLCε directly mediates aortic dissection.

Coincident Regulation of PLCß Signaling by Gq-Coupled and µ-Opioid Receptors Opposes Opioid-Mediated Antinociception.

Pain management is an important problem worldwide. The current frontline approach for pain management is the use of opioid analgesics. The primary analgesic target of opioids is the µ-opioid receptor (MOR). Deletion of phospholipase Cß3 (PLCß3) or selective inhibition of Gßγ regulation of PLCß3 enhances the potency of the antinociceptive effects of morphine suggesting a novel strategy for achieving opioid-sparing effects. Here we investigated a potential mechanism for regulation of PLC signaling downstream of MOR in human embryonic kidney 293 cells and found that MOR alone could not stimulate PLC but rather required a coincident signal from a Gq-coupled receptor. Knockout of PLCß3 or pharmacological inhibition of its upstream regulators, Gßγ or Gq, ex vivo in periaqueductal gray slices increased the potency of the selective MOR agonist [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin acetate salt in inhibiting presynaptic GABA release. Finally, inhibition of Gq- G protein-coupled receptor coupling in mice enhanced the antinociceptive effects of morphine. These data support a model where Gq and Gßγ-dependent signaling cooperatively regulate PLC activation to decrease MOR-dependent antinociceptive potency. Ultimately, this could lead to identification of new non-MOR targets that would allow for lower-dose utilization of opioid analgesics. SIGNIFICANCE STATEMENT: Previous work demonstrated that deletion of phospholipase Cß3 (PLCß3) in mice potentiates µ-opioid receptor (MOR)-dependent antinociception. How PLCß3 is regulated downstream of MOR had not been clearly defined. We show that PLC-dependent diacylglycerol generation is cooperatively regulated by MOR-Gßγ and Gq-coupled receptor signaling through PLCß3 and that blockade of either Gq-signaling or Gßγ signaling enhances the potency of opioids in ex vivo brain slices and in vivo. These results reveal potential novel strategies for improving opioid analgesic potency and safety.

Subcellular ß-Adrenergic Receptor Signaling in Cardiac Physiology and Disease.

ABSTRACT: Adrenergic receptors are critical regulators of cardiac function with profound effects on cardiac output during sympathetic stimulation. Chronic stimulation of the adrenergic system of the heart under conditions of cardiac stress leads to cardiac dysfunction, hypertrophy, and ultimately failure. Emerging data have revealed that G protein-coupled receptors in intracellular compartments are functionally active and regulate distinct cellular processes from those at the cell surface. ß2 adrenergic receptors internalize onto endosomes in various cell types where they have recently been shown to continue to stimulate cAMP production to selectively regulate gene expression. Other studies have identified ß1 adrenergic receptors at the nuclear envelope and the Golgi apparatus. Here, we discuss data on signaling by ß1 and ß2 adrenergic receptors in the heart and the possible influence of their subcellular locations on their divergent physiological functions in cardiac myocytes and in cardiac pathology. Understanding the relative roles of these receptors at these locations could have a significant impact on pharmacological targeting of these receptors for the treatment of heart failure and cardiac diseases.

A naturally occurring membrane-anchored Gαs variant, XLαs, activates phospholipase Cß4.

Extra-large stimulatory Gα (XLαs) is a large variant of G protein αs subunit (Gαs) that uses an alternative promoter and thus differs from Gαs at the first exon. XLαs activation by G protein-coupled receptors mediates cAMP generation, similarly to Gαs; however, Gαs and XLαs have been shown to have distinct cellular and physiological functions. For example, previous work suggests that XLαs can stimulate inositol phosphate production in renal proximal tubules and thereby regulate serum phosphate levels. In this study, we show that XLαs directly and specifically stimulates a specific isoform of phospholipase Cß (PLCß), PLCß4, both in transfected cells and with purified protein components. We demonstrate that neither the ability of XLαs to activate cAMP generation nor the canonical G protein switch II regions are required for PLCß stimulation. Furthermore, this activation is nucleotide independent but is inhibited by Gßγ, suggesting a mechanism of activation that relies on Gßγ subunit dissociation. Surprisingly, our results indicate that enhanced membrane targeting of XLαs relative to Gαs confers the ability to activate PLCß4. We also show that PLCß4 is required for isoproterenol-induced inositol phosphate accumulation in osteocyte-like Ocy454 cells. Taken together, we demonstrate a novel mechanism for activation of phosphoinositide turnover downstream of Gs-coupled receptors that may have a critical role in endocrine physiology.

A network of Gαi signaling partners is revealed by proximity labeling proteomics analysis and includes PDZ-RhoGEF.

G protein­coupled receptors (GPCRs) that couple to the Gαi family of G proteins are key regulators of cell and tissue physiology. Our previous work has revealed new roles for Gαi in regulating the migration of neutrophils and fibrosarcoma cells downstream of activated chemoattractant receptors. Here, we used an intact cell proximity­based labeling coupled to tandem mass tag (TMT)­based quantitative proteomics analysis to identify proteins that selectively interacted with the GTP-bound form of Gαi1. Multiple targets were identified and validated with a BioID2-tagged, constitutively active Gαi1 mutant, suggesting a network of interactions for activated GαI proteins in intact cells. We showed that active Gαi1, but not Gαi2, stimulated one candidate protein, PDZ-RhoGEF (PRG), despite more than 85% sequence identity between the G proteins. We also demonstrated in primary human neutrophils that active Gαi likely regulated the polarization of phosphorylated myosin light chain, a process critical for migration, through the activation of PRG. The identification and characterization of new targets directly or indirectly regulated by Gαi will aid in the investigation of the functional roles of Gαi-coupled GPCRs in multiple biological processes.

Uveal melanoma-associated mutations in PLCß4 are constitutively activating and promote melanocyte proliferation and tumorigenesis.

Activating mutations in Gαq/11 proteins are frequent in uveal melanoma, the most common eye cancer arising from the uveal tract. A small proportion of uveal melanomas have a D630Y mutation in phospholipase C ß4 (PLCß4), an effector of Gαq/11. Here, we found that the D630Y mutation in PLCß4 results in a high level of constitutive PLCß4 activity. Mutations at the corresponding position in other PLC isoforms also resulted in constitutive activity, revealing an unrecognized mechanism underlying PLC activation. In cultured human uveal melanoma cell lines, inhibition of PLC suppressed proliferation in Gαq/11-dependent cells. Furthermore, we found that PLCß4(D630Y) mediated proliferation in cutaneous melanocytes and the growth of melanomas in mice. These results are consistent with PLCß4(D630Y) driving oncogenic signaling downstream of Gαq/11.

A universal allosteric mechanism for G protein activation.

G proteins play a central role in signal transduction and pharmacology. Signaling is initiated by cell-surface receptors, which promote guanosine triphosphate (GTP) binding and dissociation of Gα from the Gßγ subunits. Structural studies have revealed the molecular basis of subunit association with receptors, RGS proteins, and downstream effectors. In contrast, the mechanism of subunit dissociation is poorly understood. We use cell signaling assays, molecular dynamics (MD) simulations, and biochemistry and structural analyses to identify a conserved network of amino acids that dictates subunit release. In the presence of the terminal phosphate of GTP, a glycine forms a polar network with an arginine and glutamate, putting torsional strain on the subunit binding interface. This "G-R-E motif" secures GTP and, through an allosteric link, discharges the Gßγ dimer. Replacement of network residues prevents subunit dissociation regardless of agonist or GTP binding. These findings reveal the molecular basis of the final committed step of G protein activation.

ß-arrestin mediates communication between plasma membrane and intracellular GPCRs to regulate signaling.

It has become increasingly apparent that G protein-coupled receptor (GPCR) localization is a master regulator of cell signaling. However, the molecular mechanisms involved in this process are not well understood. To date, observations of intracellular GPCR activation can be organized into two categories: a dependence on OCT3 cationic channel-permeable ligands or the necessity of endocytic trafficking. Using CXC chemokine receptor 4 (CXCR4) as a model, we identified a third mechanism of intracellular GPCR signaling. We show that independent of membrane permeable ligands and endocytosis, upon stimulation, plasma membrane and internal pools of CXCR4 are post-translationally modified and collectively regulate EGR1 transcription. We found that ß-arrestin-1 (arrestin 2) is necessary to mediate communication between plasma membrane and internal pools of CXCR4. Notably, these observations may explain that while CXCR4 overexpression is highly correlated with cancer metastasis and mortality, plasma membrane localization is not. Together these data support a model where a small initial pool of plasma membrane-localized GPCRs are capable of activating internal receptor-dependent signaling events.

Activation of Phospholipase C ß by Gßγ and Gαq Involves C-Terminal Rearrangement to Release Autoinhibition.

Phospholipase C (PLC) enzymes hydrolyze phosphoinositide lipids to inositol phosphates and diacylglycerol. Direct activation of PLCß by Gαq and/or Gßγ subunits mediates signaling by Gq and some Gi coupled G-protein-coupled receptors (GPCRs), respectively. PLCß isoforms contain a unique C-terminal extension, consisting of proximal and distal C-terminal domains (CTDs) separated by a flexible linker. The structure of PLCß3 bound to Gαq is known, however, for both Gαq and Gßγ; the mechanism for PLCß activation on membranes is unknown. We examined PLCß2 dynamics on membranes using hydrogen-deuterium exchange mass spectrometry (HDX-MS). Gßγ caused a robust increase in dynamics of the distal C-terminal domain (CTD). Gαq showed decreased deuterium incorporation at the Gαq binding site on PLCß. In vitro Gßγ-dependent activation of PLC is inhibited by the distal CTD. The results suggest that disruption of autoinhibitory interactions with the CTD leads to increased PLCß hydrolase activity.

Hypertension induces glomerulosclerosis in phospholipase C-ε1 deficiency.

Loss-of-function mutations in phospholipase C-ε1 (PLCE1) have been detected in patients with nephrotic syndrome, but other family members with the same mutation were asymptomatic, suggesting additional stressor are required to cause the full phenotype. Consistent with these observations, we determined that global Plce1-deficient mice have histologically normal glomeruli and no albuminuria at baseline. Angiotensin II (ANG II) is known to induce glomerular damage in genetically susceptible individuals. Therefore, we tested whether ANG II enhances glomerular damage in Plce1-deficient mice. ANG II increased blood pressure equally in Plce1-deficient and wild-type littermates. Additionally, it led to 20-fold increased albuminuria and significantly more sclerotic glomeruli in Plce1-deficient mice compared with wild-type littermates. Furthermore, Plce1-deficient mice demonstrated diffuse mesangial expansion, podocyte loss, and focal podocyte foot process effacement. To determine whether these effects are mediated by hypertension and hyperfiltration, rather than directly through ANG II, we raised blood pressure to a similar level using DOCA + salt + uninephrectomy and norepinephrine. This caused a fivefold increase in albuminuria in Plce1-deficient mice and a significant increase in the number of sclerotic glomeruli. Consistent with previous findings in mice, we detected strong PLCE1 transcript expression in podocytes using single cell sequencing of human kidney tissue. In hemagglutinin-tagged Plce1 transgenic mice, Plce1 was detected in podocytes and also in glomerular arterioles using immunohistochemistry. Our data demonstrate that Plce1 deficiency in mice predisposes to glomerular damage secondary to hypertensive insults.

Discovery of Small Molecules That Target the Phosphatidylinositol (3,4,5) Trisphosphate (PIP3)-Dependent Rac Exchanger 1 (P-Rex1) PIP3-Binding Site and Inhibit P-Rex1-Dependent Functions in Neutrophils.

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 ßγ 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.