Browsing by Subject "G proteins"
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Item Open Access Characterization of Beta-arrestin-Modulated Lipid Kinase Activities for Diacylglycerol and Phosphatidylinositol 4-Phosphate(2007-05-10T15:22:51Z) Nelson, Christopher DavidThe study of arrestins as regulators of seven transmembrane receptor (7TMR) signaling has revealed multiple levels of complexity, initiating desensitization of G protein activity and coordination of receptor internalization via clathrin‐coated pits. Recently, β‐arrestins have also been shown to act as adaptor proteins, mediating G protein‐independent signaling as well as scaffolding of enzymes that degrade second messenger molecules. This latter function was demonstrated by β‐arrestins recruiting PDE4 phosphodiesterase to Gs‐coupled β2‐adrenergic receptors, enhancing metabolism of the second messenger cAMP. As β‐arrestins universally interact with members of the 7TMR superfamily, we sought to determine if this phenomenon of concerted desensitization might be applicable to additional receptor subtypes. We screened for β‐arrestin‐binding proteins among modulators of diacylglycerol and IP3 (second messengers downstream of Gq‐coupled 7TMRs). We observed β‐ arrestins constitutively interacted with members of the diacylglycerol kinase (DGK) family, which phosphorylate diacylglycerol to create phosphatidic acid. Furthermore, examining lipid extracts of 32P labeled cells separated by TLC, we observed that overexpression of β‐arrestin enhanced phosphatidic acid (PA) production after M1 muscarinic receptor stimulation. Conversely, depletion of β‐arrestins by RNA interference showed significantly decreased agonist‐stimulated PA accumulation. Additionally, overexpression of a β‐arrestin2 mutant that binds DGKs but not receptors served as a dominant negative for agonist‐dependent DGK activity. These results demonstrate a requirement for β‐arrestins in DGK translocation to the membrane, and specifically to activated 7TMRs, where concentrations of second messengers are at their highest. Phosphatidic acid is an effector for several enzymes, including the phosphatidylinositol 5‐kinases (PIP5K), which phosphorylate PIP to make PIP2. Thus, we hypothesized β‐arrestin‐targeted DGKs may regulate PIP5K activity. PIP5K Iα associated with β‐arrestin2 in an agonist‐dependent manner in HEK293 cells, and a β‐ arrestin2 mutant defective in receptor endocytosis (a PIP2‐dependent function) was impaired. Furthermore, knockdown of β‐arrestin2 by RNAi significantly decreased the amount of PIP5K Iα detected in receptor immunoprecipitates. In TLC assays, overexpressing both β‐arrestin2 and PIP5K Iα enhanced agonist‐stimulated PIP2 labeling, while either protein alone had no effect. These data support the concept of β‐ arrestin binding to 7TMRs and enriching local membrane concentrations of PA, which then stimulates production of PIP2, promoting receptor internalization.Item Open Access G Protein Signaling and Vein Graft Intimal Hyperplasia: Reduction of Intimal Hyperplasia in Vein Grafts by a Gbg Inhibitor(1998-08) Davies, MG; Huynh, TT; Fulton, GJ; Lefkowitz, RJ; Svendsen, E; Hagen, PO; Koch, WJAbstract—Vein grafting results in the development of intimal hyperplasia with accompanying changes in guanine nucleotide–binding (G) protein expression and function. Several serum mitogens that act through G protein–coupled receptors, such as lysophosphatidic acid, stimulate proliferative pathways that are dependent on the G protein βγ subunit (Gβγ)–mediated activation of p21ras. This study examines the role of Gβγ signaling in intimal hyperplasia by targeting a gene encoding a specific Gβγ inhibitor in an experimental rabbit vein graft model. This inhibitor, the carboxyl terminus of the β-adrenergic receptor kinase (βARKCT), contains a Gβγ-binding domain. Vein graft intimal hyperplasia was significantly reduced by 37% (P<0.01), and physiological studies demonstrated that the normal alterations in G protein coupling phenotypically seen in this model were blocked by βARKCT treatment. Thus, it appears that Gβγ-mediated pathways play a major role in intimal hyperplasia and that targeting inhibitors of Gβγ signaling offers novel intraoperative therapeutic modalities to inhibit the development of vein graft intimal hyperplasia and subsequent vein graft failure.Item Open Access Sex in Cryptococcus: Signaling, Mating-type Locus Evolution and Gene Silencing(2008-02-26) Hsueh, Yen-PingFungi have a genetically controlled sex determination system, which is governed by a small, sex-specific region in the genome called the mating-type locus (MAT). In the basidiomycetous yeast Cryptococcus neoformans, the pathogen that causes cryptococcal meningitis and cryptococcosis, sex has been associated with virulence. To further understand how sex is genetically regulated in C. neoformans, we focused our studies on the evolution of the MAT locus and molecular dissection of the pheromone signaling pathway that controls sexual development. Two MAT-linked meiotic recombination hotspots that likely drove the assembly and rearrangement of MAT were identified. Fine mapping through the integration of genetic markers established that two hotspots, one on each side of the MAT locus, are located in an ~10 kb and ~5 kb region. Plotting the G + C content along MAT and the flanking regions revealed a strong association between the location of these two hotspots and a high G + C content. By deletion and insertion of the G + C rich region, we demonstrated that the high G + C rich region is required but not sufficient to induce recombination. On the other hand, to provide direct experimental evidence to support the previously proposed model for the evolution of MAT, we sought to recapitulate the ancestral tetrapolar, and the intermediate tripolar mating systems of C. neoformans by manipulating the MAT structure to model a tetrapolar system. In the two modified "a" and "α" strains, the sex-determining genes SXI1α or SXI2a residing at the MAT locus were disrupted and the wild-type allele of these two genes was then reintroduced at another genomic location (URA5) that is unlinked to MAT. Our results show that C. neoformans can complete the sexual cycle with a tetrapolar mating configuration and the transitional tripolar state might be under strong negative selection pressure, which could have facilitated the transition from a tripolar state to the final bipolar mating system.
The MAT locus is the major determinant of the sexual identity of a cell, but several signaling pathways, including the pheromone signaling pathway, are required to regulate mating and sexual development. Many components of the pheromone signaling pathway have been identified; however, it is less clear what lies upstream of the MAPK cascade. To address this question, we studied the role of two Gα subunits (Gpa2, Gpa3) in mating and concluded that they share both redundant and divergent roles in mating. gpa2 gpa3 double mutants, but neither gpa2 nor gpa3 single mutants, are sterile in bilateral crosses. In their GTP-bound form, they signal in opposition: Gpa2 promotes mating whereas Gpa3 inhibits. Furthermore, we also studied the functions of a novel upstream component Cpr2, a pheromone receptor-like gene, in pheromone signaling and sexual development. All lines of evidence suggest that Cpr2 is a constitutive ligand-independent receptor that, when expressed, engages the same G-proteins and activates the same pheromone signaling pathway as the canonical ligand-activated pheromone receptors. Expression of Cpr2 is induced post cell fusion during mating, and likely introduces a positive feedback loop to allow a self-perpetuating signaling state to enable efficient mating. Cells lacking this receptor are fertile, but produce abnormal filamentous structures. Overexpression of CPR2 in a or α cells strongly enhances fruiting, an alternative same-sex mating process in C. neoformans. Therefore, Cpr2 establishes a new paradigm for a naturally occurring constitutively active GPCR that governs cell fate in fungi.
Finally, we described a sex-induced silencing (SIS) phenomenon in C. neoformans. Using genetic approaches, we showed that SIS is triggered by a tandem insertion of a transgene during the sexual cycle. Interestingly, only a proportion of progeny carrying the transgene are silenced. Gene deletion, RIP, or DNA methylation do not contribute to SIS but the RNAi machinery is required. In conclusion, these studies provide further understanding of sex in C. neoformans from different perspectives, which invites comparisons to other fungal and even more broadly, eukaryotic pathogens to address the role of sex in evolution.