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 G protein-coupled receptors.

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.

Epigenetic regulation of arginine vasopressin receptor 2 expression by PAX2 and Pax transcription interacting protein.

Renal arginine vasopressin receptor 2 (AVPR2) plays a crucial role in osmoregulation. Engagement of ligand with AVPR2 results in aquaporin 2 movement to the apical membrane and water reabsorption from the urinary filtrate. Despite this essential role, little is known about transcriptional regulation of Avpr2. Here, we identify novel roles for PAX2, a transcription factor crucial for kidney development, and its adaptor protein, Pax transcription interacting protein (PTIP), for epigenetic regulation of Avpr2 and thus body water balance. Chromatin immunoprecipitation (ChIP) from murine inner medulla cells (IMCD-3) identified the minimal DNA-binding region of PAX2 on the Avpr2 promoter. Regulation of Avpr2 by PAX2 was confirmed using a heterologous DNA expression system. PAX2 recruits the adaptor protein PTIP and its associated histone methyltransferase (HMT) complex to Avpr2 promoter, imposing epigenetic marks on this region and throughout the coding sequence that modulate Avpr2 gene transcription. Reduction of PAX2 or PTIP protein levels by siRNA prevented histone lysine methylation and expression of Avpr2. ChIP using mouse or human kidneys determined that PAX2 is highly enriched in the AVPR2 promoter alongside PTIP and HMT proteins, leading to high levels of histone H3 lysine trimethylation within the promoter and throughout the gene. In conclusion, PAX2 provides locus specificity for PTIP, allowing the HMT complex to impart epigenetic changes at the Avpr2 locus and regulate Avpr2 transcription. These finding have major implications for understanding regulation of body water balance.NEW & NOTEWORTHY The transcription factor PAX2 plays an indispensable role in kidney development. In the adult kidney, we identified the first described protein this protein regulates. PAX2 and its interacting partner Pax transcription interacting protein recruit a histone methyltransferase complex to the promoter and epigentically regulate the expression of arginine vasopressin receptor 2, a protein that plays a crucial role in osmoregulation in the distal tubule.

Pax2 and Pax8 Proteins Regulate Urea Transporters and Aquaporins to Control Urine Concentration in the Adult Kidney.

BACKGROUND: As the glomerular filtrate passes through the nephron and into the renal medulla, electrolytes, water, and urea are reabsorbed through the concerted actions of solute carrier channels and aquaporins at various positions along the nephron and in the outer and inner medulla. Proliferating stem cells expressing the nuclear transcription factor Pax2 give rise to renal epithelial cells. Pax2 expression ends once the epithelial cells differentiate into mature proximal and distal tubules, whereas expression of the related Pax8 protein continues. The collecting tubules and renal medulla are derived from Pax2-positive ureteric bud epithelia that continue to express Pax2 and Pax8 in adult kidneys. Despite the crucial role of Pax2 in renal development, functions for Pax2 or Pax8 in adult renal epithelia have not been established. METHODS: To examine the roles of Pax2 and Pax8 in the adult mouse kidney, we deleted either Pax2, Pax8, or both genes in adult mice and examined the resulting phenotypes and changes in gene expression patterns. We also explored the mechanism of Pax8-mediated activation of potential target genes in inner medullary collecting duct cells. RESULTS: Mice with induced deletions of both Pax2 and Pax8 exhibit severe polyuria that can be attributed to significant changes in the expression of solute carriers, such as the urea transporters encoded by Slc14a2, as well as aquaporins within the inner and outer medulla. Furthermore, Pax8 expression is induced by high-salt levels in collecting duct cells and activates the Slc14a2 gene by recruiting a histone methyltransferase complex to the promoter. CONCLUSIONS: These data reveal novel functions for Pax proteins in adult renal epithelia that are essential for retaining water and concentrating urine.

The Groucho-associated phosphatase PPM1B displaces Pax transactivation domain interacting protein (PTIP) to switch the transcription factor Pax2 from a transcriptional activator to a repressor.

Pax genes encode developmental regulatory proteins that specify cell lineages and tissues in metazoans. Upon binding to DNA through the conserved paired domain, Pax proteins can recruit both activating and repressing complexes that imprint distinct patterns of histone methylation associated with either gene activation or silencing. How the switch from Pax-mediated activation to repression is regulated remains poorly understood. In this report, we identify the phosphatase PPM1B as an essential component of the Groucho4 repressor complex that is recruited by Pax2 to chromatin. PPM1B can dephosphorylate the Pax2 activation domain and displace the adaptor protein PTIP, thus inhibiting H3K4 methylation and gene activation. Loss of PPM1B prevents Groucho-mediated gene repression. Thus, PPM1B helps switch Pax2 from a transcriptional activator to a repressor protein. This can have profound implications for developmental regulation by Pax proteins and suggests a model for imprinting specific epigenetic marks depending on the availability of co-factors.

Landscape genetics and population structure in Valley Oak (Quercus lobata Née).

PREMISE OF THE STUDY: Although long-distance pollen movement is common in wind-pollinated trees, barriers to gene flow may occur in species that have discontinuous ranges or are confined to certain habitat types. We investigated the genetic structure of Quercus lobata Née populations throughout much of their range in California. We assessed the connectivity of populations and determined if barriers to gene flow occurred, and if so, if they corresponded to landscape features. METHODS: We collected leaf samples from 270 trees from 12 stands of Quercus lobata and genotyped these trees using eight polymorphic microsatellite loci. Genetic structure and clustering was evaluated using genetic distance methods, Bayesian clustering approaches, and network analysis of spatial genetic structure. KEY RESULTS: The southernmost population of Quercus lobata sampled from the Santa Monica area comprised a separate genetic cluster from the rest of the species, suggesting that Transverse Ranges such as the San Gabriel Mountains limit gene flow. Population differentiation among the other sites was small but significant. Network analysis reflected higher connectivity among populations along the Central Coast range, with few connections spanning the dry, low Central Valley. CONCLUSIONS: While long distance pollen movement has been shown to be common in oaks, on larger spatial scales, topographic features such as mountain ranges and the large, flat Central Valley of California limit gene flow. Such landscape features explain gene flow patterns much better than geographic distance alone.

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 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.

HNF1A is a novel oncogene that regulates human pancreatic cancer stem cell properties.

The biological properties of pancreatic cancer stem cells (PCSCs) remain incompletely defined and the central regulators are unknown. By bioinformatic analysis of a human PCSC-enriched gene signature, we identified the transcription factor HNF1A as a putative central regulator of PCSC function. Levels of HNF1A and its target genes were found to be elevated in PCSCs and tumorspheres, and depletion of HNF1A resulted in growth inhibition, apoptosis, impaired tumorsphere formation, decreased PCSC marker expression, and downregulation of POU5F1/OCT4 expression. Conversely, HNF1A overexpression increased PCSC marker expression and tumorsphere formation in pancreatic cancer cells and drove pancreatic ductal adenocarcinoma (PDA) cell growth. Importantly, depletion of HNF1A in xenografts impaired tumor growth and depleted PCSC marker-positive cells in vivo. Finally, we established an HNF1A-dependent gene signature in PDA cells that significantly correlated with reduced survivability in patients. These findings identify HNF1A as a central transcriptional regulator of PCSC properties and novel oncogene in PDA.

Screening for asymptomatic atrial fibrillation while monitoring the blood pressure at home: trial of regular versus irregular pulse for prevention of stroke (TRIPPS 2.0).

Asymptomatic atrial fibrillation (AF) is a common cause of strokes. Physician screening for AF has been recommended. Home screening for AF may increase the likelihood of detecting asymptomatic AF in patients at risk for stroke because of AF. The aim of this study was to assess the feasibility and accuracy of screening for AF when taking home blood pressure (BP) measurements using an automatic AF-detecting BP monitor. Subjects aged >64 years or those with hypertension, diabetes, congestive heart failure, or previous stroke were enrolled by their primary physicians and given the AF-BP monitor and an electrocardiographic event monitor to use at home for 30 days. The AF-BP monitor reading was compared with the electrocardiographic reading to calculate the sensitivity and specificity of the device for detecting AF. A total of 160 subjects were enrolled, with 10 withdrawing, 1 excluded, and 10 with no AF-BP monitor logs or electrocardiographic recordings. Of the 139 subjects included, 14 had known AF. There was a total of 3,316 days with AF-BP monitor readings and electrocardiographic readings. On the basis of the initial daily AF-BP monitor readings, the AF-BP monitor demonstrated sensitivity of 99.2% and specificity of 92.9% for detecting AF. Two subjects with no histories of AF had AF-BP monitor readings of AF that were confirmed by the electrocardiographic monitor. One of these subjects was started on warfarin. In conclusion, home screening for asymptomatic AF while taking BP measurements can be performed accurately. This can be used to detect new AF, allowing treatment with anticoagulation to reduce the future risk for stroke.