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Timestamp: 2019-04-23 02:17:30+00:00

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You searched for subject:(Arrestin). Showing records 1 – 30 of 73 total matches.
1. Coffa, Sergio. Non-visual arrestins bind mitogen activated protein kinases and regulate their signaling.
▼ Arrestins are multifunctional signaling proteins, important for the regulation of signal transduction and the trafficking of G protein-coupled receptors (GPCRs). Recently, GPCR-arrestin interactions have been proposed to be necessary for activation of G-protein-independent signaling pathways, one of which is the activation of mitogen activated protein kinases (MAPKs). To investigate potential arrestin-MAPK interactions, we have used a variety of molecular tools including the co-expression of the individual domains of arrestin with single components of the c-Raf1-MEK1-ERK2 signaling cascade. We found that non-visual arrestins bind all three kinases, assembling c-Raf1, MEK1, and ERK2 along their short axis, with each kinase directly interacting with both domains. To further investigate the interactions between arrestins and MAPK, we used alanine-scanning mutagenesis of residues on the non-receptor-binding surface of arrestin that are conserved between arrestin-2 and arrestin-3. We found that the substitution of arginine 307 with an alanine significantly reduced arrestin-2 binding to c-Raf1, whereas the interactions of this mutant with active phosphorylated receptors and the downstream kinases MEK1 and ERK2 were not affected. In contrast to wild type arrestin-2, Arg307Ala mutant failed to rescue arrestin-dependent ERK1/2 activation in arrestin-2/3 knockout MEFs. Interestingly, alanine substitution of the homologous arrestin-3 residue (lysine 308) did not significantly affect c-Raf1 binding or its ability to promote ERK1/2 activation. Together, these findings suggest that the two non-visual arrestins perform the same function via distinct molecular mechanisms. To further elucidate arrestin-MAPK interactions, we performed in vitro binding assays using pure proteins, and demonstrated that ERK2 directly binds free arrestin-2 and arrestin-3, as well as receptor-associated arrestin-1, arrestin-2, and arrestin-3. We have also shown that the arrestin-2 and arrestin-3 association with beta2-adrenergic receptors (β2ARs) significantly enhances ERK2 binding, yet has virtually no effect upon arrestins interactions with the upstream kinases c-Raf1 and MEK1. Arrestins exist in three conformational states: free, receptor-bound, and microtubule (MT)-bound. Using conformationally biased arrestin mutants, we found that ERK2 prefers two conformations: MT-bound, mimicked by âconstitutively inactiveâ arrestin-Δ7, and receptor-bound, mimicked by âpre-activatedâ arrestin-3A. Both mutants were able to rescue arrestin-mediated ERK1/2 activation in arrestin-2/3 double knockout fibroblasts. Lastly, we found that the arrestin-2 interaction with c-Raf1 is enhanced by receptor binding, whereas the interaction between arrestin-3 and c-Raf1 is not, thus suggesting that the two non-visual arrestins execute similar functions via diverse mechanisms. Advisors/Committee Members: Charles Sanders (committee member), Brian E. Wadzinski (committee member), H. Alex Brown (committee member), Benjamin Spiller (chair), Vsevolod V. Gurevich (committee member).
Coffa, Sergio. “Non-visual arrestins bind mitogen activated protein kinases and regulate their signaling.” 2011. Doctoral Dissertation, Vanderbilt University. Accessed April 22, 2019. http://etd.library.vanderbilt.edu/available/etd-08042011-074210/ ;.
Coffa, Sergio. “Non-visual arrestins bind mitogen activated protein kinases and regulate their signaling.” 2011. Web. 22 Apr 2019.
Coffa S. Non-visual arrestins bind mitogen activated protein kinases and regulate their signaling. [Internet] [Doctoral dissertation]. Vanderbilt University; 2011. [cited 2019 Apr 22]. Available from: http://etd.library.vanderbilt.edu/available/etd-08042011-074210/ ;.
2. Blume, Lawrence Christopher. CANNABINOID RECEPTOR INTERACTING PROTEIN 1A (CRIP1A): EFFECTS ON CB1 RECEPTOR FUNCTION AND CELLULAR REGULATION.
▼ Cannabis, or marijuana, is the most commonly used illicit drug in the United States. The CB1 cannabinoid receptor (CB1R) has been extensively studied since the initial characterization of its involvement in mediating the psychotropic effects of marijuana. CB1R is one of the most abundantly expressed G protein coupled receptors (GPCR) in the CNS and has been targeted therapeutically for multiple diseases; however side effect profiles for CB1R have posed difficulty in the development of clinically successful CB1R drugs. The identification of GPCR interacting proteins has provided additional insight into the fine-tuning and regulation of numerous GPCR's. The Cannabinoid Receptor Interacting Protein 1a (CRIP1a) binds to the extreme carboxy terminus of CB1R, and has been shown to alter CB1R-mediated neuronal function. The mechanisms by which CRIP1a regulates CB1R activity and trafficking have not yet been identified; therefore the focus of this dissertation is to examine the cellular effects of CRIP1a on CB1R function and cellular trafficking.
Blume, Lawrence Christopher. “CANNABINOID RECEPTOR INTERACTING PROTEIN 1A (CRIP1A): EFFECTS ON CB1 RECEPTOR FUNCTION AND CELLULAR REGULATION.” 2014. Thesis, Wake Forest University. Accessed April 22, 2019. http://hdl.handle.net/10339/39334.
Blume, Lawrence Christopher. “CANNABINOID RECEPTOR INTERACTING PROTEIN 1A (CRIP1A): EFFECTS ON CB1 RECEPTOR FUNCTION AND CELLULAR REGULATION.” 2014. Web. 22 Apr 2019.
Blume LC. CANNABINOID RECEPTOR INTERACTING PROTEIN 1A (CRIP1A): EFFECTS ON CB1 RECEPTOR FUNCTION AND CELLULAR REGULATION. [Internet] [Thesis]. Wake Forest University; 2014. [cited 2019 Apr 22]. Available from: http://hdl.handle.net/10339/39334.
3. Huang, Shun-Ping. Exploring alternative roles of visual arrestin 1 in photoreceptor synaptic regulation and deciphering the molecular pathway of retinal degeneration using mouse knockout technology.
▼ In the G-protein-coupled receptor (GPCR) phototransduction cascade, visual Arrestin1 (Arr1) binds to and deactivates phosphorylated light-activated opsins, a process that is critical for effective recovery and normal vision. In this dissertation study, we discovered a novel synaptic interaction between Arr1 and N-ethylmaleimide sensitive factor (NSF) that is enhanced in a dark environment when photoreceptors are depolarized and the rate of exocytosis is elevated. In the photoreceptor synapse, NSF functions to sustain a higher rate of exocytosis, in addition to the compensatory endocytosis to retrieve and to recycle vesicle membrane and synaptic proteins. Not only does Arr1 bind to the junction of NSF N-terminal and first ATPase domains in an ATP-dependent manner, but Arr1 also enhances both NSF ATPase and NSF disassembly activities. In mouse retinas with no Arr1 expression, the expression levels of NSF and other synapse-enriched genes are markedly reduced and lead to a substantial decrease in the exocytosis rate. This study demonstrates a vital modulatory role of Arr1 in the photoreceptor synapse and provides key insights into the potential molecular mechanisms of inherited retinal diseases, such as Oguchi disease and Arr1-associated retinitis pigmentosa.; Arr1-/- mice develop a light-dependent retinal degeneration and a light-independent cone dystrophy. We observed increased terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), activation of phosphorylated signal transducer and activator of transcription 3 (pSTAT3) in the Janus kinase (JAK)-STAT3 pathway, reactive gliosis, and cone dystrophy in Arr1-/- mice. To further explore the molecular pathways leading to Arr1-/ – related light-independent cone dystrophy, we compared controls and Arr1-/- with Affymetrix exon array analysis and observed in Arr1-/- up-regulated retinal transcripts including serping1, C4b, C3 and C3a receptor 1 (C3ar1), annexin A1(anxa1), oncostatin M receptor, STAT3, endothelin2, and glial fibrillary acidic protein (GFAP). Top canonical pathways reveal several potential pathways involved in this cone dystrophy phenotype including complement system activation, acute phase response signaling, oncostatin M signaling and JAK3-STAT3 activation. Our data support Arr1 is crucial for regulating an endogenous defense mechanism to ensure survival and normal function of cone photoreceptors. Advisors/Committee Members: Craft, Cheryl M. (Committee Chair), Hinton, David R. (Committee Member), Sampath, Alapakkam P. (Committee Member), Mircheff, Austin K. (Committee Member), Ko, Chien-Ping (Committee Member).
Huang, Shun-Ping. “Exploring alternative roles of visual arrestin 1 in photoreceptor synaptic regulation and deciphering the molecular pathway of retinal degeneration using mouse knockout technology.” 2010. Doctoral Dissertation, University of Southern California. Accessed April 22, 2019. http://digitallibrary.usc.edu/cdm/compoundobject/collection/p15799coll127/id/370201/rec/2648.
Huang, Shun-Ping. “Exploring alternative roles of visual arrestin 1 in photoreceptor synaptic regulation and deciphering the molecular pathway of retinal degeneration using mouse knockout technology.” 2010. Web. 22 Apr 2019.
Huang S. Exploring alternative roles of visual arrestin 1 in photoreceptor synaptic regulation and deciphering the molecular pathway of retinal degeneration using mouse knockout technology. [Internet] [Doctoral dissertation]. University of Southern California; 2010. [cited 2019 Apr 22]. Available from: http://digitallibrary.usc.edu/cdm/compoundobject/collection/p15799coll127/id/370201/rec/2648.
4. Robinson, Kelly Ann. Arrestin Interactions with Rhodopsin in the Squid Visual System.
Light activation of squid rhodopsin results in stimulation of the Gq signalling cascade. Activated rhodopsin (metarhodopsin) is a target for squid arrestin and squid rhodopsin kinase which are involved in the inactivation of metarhodopsin. The aim of this project is to characterize the interaction between squid rhodopsin and arrestin, and the role of phosphorylation on their interactions. We determined the affinity of arrestin for metarhodopsin to be 32nM. Two mutations to the polar core did not decrease the affinity of arrestin for metarhodopsin, suggesting a difference in basal structure of squid arrestin compared to other arrestins. Serine392 and Serine397 in the C-terminus of squid arrestin were phosphorylated by squid rhodopsin kinase. Arrestin phosphorylation decreased the affinity of arrestin binding to metarhodopsin, while metarhodopsin phosphorylation increased the dissociation of the two proteins. Further studies are required to identify mechanisms of metarhodopsin and arrestin dephosphorylation in the squid visual system.
Advisors/Committee Members: Mitchell, Jane A, Pharmacology.
Robinson, Kelly Ann. “Arrestin Interactions with Rhodopsin in the Squid Visual System.” 2015. Masters Thesis, University of Toronto. Accessed April 22, 2019. http://hdl.handle.net/1807/69696.
Robinson, Kelly Ann. “Arrestin Interactions with Rhodopsin in the Squid Visual System.” 2015. Web. 22 Apr 2019.
Robinson KA. Arrestin Interactions with Rhodopsin in the Squid Visual System. [Internet] [Masters thesis]. University of Toronto; 2015. [cited 2019 Apr 22]. Available from: http://hdl.handle.net/1807/69696.
5. Deming, Janise D. Cone arrestin 4 contributes to vision, cone health, and desensitization of the dopamine receptor D4.
▼ In this dissertation, I investigated classical and alternative roles of Cone Arrestin 4 (ARR4). First, my collaborators and I described visual phenotypes of Arr4 null mice. Subsequently, we discovered a unique arrestin‐driven desensitization mechanism of dopamine receptor D4 (DRD4), a G‐protein coupled receptor (GPCR) involved in specific retinal physiological pathways. GPCRs are responsible for many biological processes, including but not limited to phototransduction, olfaction, sympathetic and parasympathetic signaling. Classical GPCR signaling occurs through a canonical activation pathway, followed by desensitization with the binding of an arrestin. ❧ Two closely related visual arrestins (ARR) play a critical role in shutoff of rod and cone phototransduction. When electrophysiological responses are measured for a single mouse cone photoreceptor, ARR1 expression can substitute for ARR4 in cone pigment desensitization; however, each arrestin may also contribute its own, unique role to modulate other cellular functions. In the first research report included in this dissertation (Deming et al. 2015, IOVS, in press), I worked with our collaborators to characterize the retinal phenotype of Arr4 null (Arr4-/-) mice compared to age‐matched control, wild‐type mice using a combination of optokinetic tracking (OKT), electroretinography (ERG), immunohistochemistry and immunoblot analysis. When 2 month‐old Arr4-/- mice were compared to controls, they had diminished visual acuity and contrast sensitivity, yet enhanced ERG flicker response and higher photopic ERG b-wave amplitudes. In contrast, in older Arr4-/- mice, all ERG amplitudes were significantly reduced in magnitude compared to age‐matched controls. Furthermore, in older Arr4-/- mice the total cone numbers decreased and cone opsin protein immunoreactive expression levels were significantly reduced, while overall rod photoreceptor outer nuclear layer thickness was unchanged. Our study demonstrates that Arr4-/- mice display distinct retinal phenotypic differences when compared to controls, suggesting that ARR4 modulates essential functions in high acuity vision and downstream cellular signaling pathways that are not fulfilled or substituted by the co‐expression of ARR1, despite its high expression levels in all mouse cones. Without normal ARR4 expression levels, cones slowly degenerate with increasing age, making this a new model to study age‐related cone dystrophy. ❧ In the second research project included in this dissertation (Deming et al., 2015, Adv. Exp. Med. Biol., in press), my collaborators and I studied the DRD4, which plays an essential role in normal vision. In order to study DRD4 expression in vivo, it is important to have reliable antibodies that are specific for DRD4 for both immunoblot and immunohistochemical (IHC) applications. We tested six antibodies raised against DRD4 peptides in vitro, using transfected mammalian cells, and in vivo, using mouse retinas. Three Santa Cruz (SC) antibodies, D-16, N-20, and R-20, were successful in IHC of transfected DRD4;… Advisors/Committee Members: Craft, Cheryl M. (Committee Chair), Hinton, David R. (Committee Member), Chen, Jeannie (Committee Member).
Deming, Janise D. “Cone arrestin 4 contributes to vision, cone health, and desensitization of the dopamine receptor D4.” 2015. Doctoral Dissertation, University of Southern California. Accessed April 22, 2019. http://digitallibrary.usc.edu/cdm/compoundobject/collection/p15799coll3/id/584780/rec/1575.
Deming, Janise D. “Cone arrestin 4 contributes to vision, cone health, and desensitization of the dopamine receptor D4.” 2015. Web. 22 Apr 2019.
Deming JD. Cone arrestin 4 contributes to vision, cone health, and desensitization of the dopamine receptor D4. [Internet] [Doctoral dissertation]. University of Southern California; 2015. [cited 2019 Apr 22]. Available from: http://digitallibrary.usc.edu/cdm/compoundobject/collection/p15799coll3/id/584780/rec/1575.
6. Mayer, Daniel. Molecular basis for the recognition of the phosphorylation pattern in the C-terminal tail of GPCRs by arrestins.
▼ Arrestins are responsible for desensitization and internalization of activated, phosphorylated G protein-coupled receptors (GPCRs). Over the years, it became clear that they have the potential to activate certain signaling pathways which are independent from G protein signaling. The Arrestins family only comprises four members, but they interact with hundreds of GPCRs. Several studies suggested that downstream signaling by Arrestins is modulated by the phosphorylation patterns created by a small number of GPCR-specific kinases (GRKs). They are capable of introducing specific phosphorylation motifs on the cytosolic side of the receptor, which are either located at the third intracellular loop (ICL3) or at the C terminal tail. These phosphorylation motifs are ligand- and cell-dependent, which shows the highly dynamic regulatory potential of this system. Here, I used a library of synthetic phosphorylated peptides derived from the bovine Rhodopsin C-terminus to study their influence on the activation of Arrestins. I obtained a high resolution view of the conformational changes induced by different phosphorylation patterns in Arrestin-1 by NMR. In combination with other biophysical techniques, this allowed me to identify a pattern of two phosphorylations sites near the distal part of the C-terminus that are the KEY sites for the activation of Arrestins. Other motifs were found as well that may further modulate the conformational changes in Arrestins. This could explain how distinct phosphorylation patterns are capable of regulating a variety of downstream signaling events. I also showed that Arrestins differ in their potential to interact with phosphorylated C-tails which highlights the non-redundancy of β-Arrestins. Advisors/Committee Members: Schertler, Gebhard F. X., Veprintsev, Dmitry, Meier, Beat, Damberger, Fred.
Mayer, Daniel. “Molecular basis for the recognition of the phosphorylation pattern in the C-terminal tail of GPCRs by arrestins.” 2017. Doctoral Dissertation, ETH Zürich. Accessed April 22, 2019. http://hdl.handle.net/20.500.11850/215427.
Mayer, Daniel. “Molecular basis for the recognition of the phosphorylation pattern in the C-terminal tail of GPCRs by arrestins.” 2017. Web. 22 Apr 2019.
Mayer D. Molecular basis for the recognition of the phosphorylation pattern in the C-terminal tail of GPCRs by arrestins. [Internet] [Doctoral dissertation]. ETH Zürich; 2017. [cited 2019 Apr 22]. Available from: http://hdl.handle.net/20.500.11850/215427.
7. Masannat, Jude Naser. Beta Arrestin 2 Mediates Renal Cell Carcinoma Tumor Growth and Metastasis.
▼ Renal Cell Carcinoma (RCC) is one of the most lethal urological cancers worldwide, with incidence and mortality rates increasing in the past two decades. The disease does not present early clinical symptoms and is commonly diagnosed at the metastatic stage, leaving the 5-year survival rate at ~10-20%. For a wide array of neoplasms, many key molecular determinants involved in mediating the process of tumor cell growth, invasion, and colonization at a secondary site (i.e. metastasis) have been reported. However, few molecular predictors have been identified for RCC, rendering the mechanism(s) underlying RCC metastases poorly understood. This results in the lack of effective treatment for patients with advanced RCC. Advisors/Committee Members: DAAKA,YEHIA (committee chair), LAW,BRIAN K (committee member), SCOTT,EDWARD W (committee member), COGLE,CHRISTOPHER RAMIN (committee member).
Masannat, Jude Naser. “Beta Arrestin 2 Mediates Renal Cell Carcinoma Tumor Growth and Metastasis.” 2017. Doctoral Dissertation, University of Florida. Accessed April 22, 2019. http://ufdc.ufl.edu/UFE0051319.
Masannat, Jude Naser. “Beta Arrestin 2 Mediates Renal Cell Carcinoma Tumor Growth and Metastasis.” 2017. Web. 22 Apr 2019.
Masannat JN. Beta Arrestin 2 Mediates Renal Cell Carcinoma Tumor Growth and Metastasis. [Internet] [Doctoral dissertation]. University of Florida; 2017. [cited 2019 Apr 22]. Available from: http://ufdc.ufl.edu/UFE0051319.
8. Nobles, Kelly Nicole. Phosphorylation Bar Codes Induce Distinct Conformations and Functionalities of beta-Arrestin .
▼ Seven transmembrane spanning receptors (7TMRs), or G-protein coupled receptors (GPCRs), represent the largest and most ubiquitous of the several families of plasma membrane receptors and regulate virtually all known physiological processes in humans. The classical paradigm of signal transduction in response to 7TMR stimulation involves an agonist-induced conformational change of the receptor which leads to interaction with and dissociation of the heterotrimeric G-protein into independent Galpha and Gbeta;gamma signaling subunits. Following their activation, 7TMRs are phosphorylated by G-protein coupled receptor kinases (GRKs) and subsequently recruit beta-arrestins. beta-arrestins are multifunctional adaptor proteins which not only desensitize G-protein signals, but also facilitate receptor internalization and mediate numerous signaling pathways on their own. As beta-arrestins universally interact with members of the 7TMR superfamily, we (1) developed an in vitro model system to assess conformational changes that occur in beta-arrestins in response to phosphorylation and (2) to map the sites of phosphorylation on the beta2 adrenergic receptor by different GRKs which would determine the conformation(s) assumed by beta-arrestin and thereby, in turn, instruct its functional capabilities. We determined conformational changes in beta-arrestin1 in vitro using limited tryptic proteolysis and MALDI-TOF MS analysis in the presence of a phosphopeptides derived from the C-terminus of the V2 vasopressin receptor (V2Rpp or V2R4p) or the corresponding unphosphorylated peptide (V2Rnp). Upon V2Rpp binding, we show that the previously shielded R393 becomes accessible, which indicates release of the C-terminus. Moreover, we have shown that R285 becomes more accessible and this residue is located in a region of β-arrestin1 responsible for stabilization of its polar core. These two findings demonstrate "activation" of beta-arrestin1. We also show a functional consequence of the release of beta-arrestin1's C-terminus by enhanced clathrin binding. In addition, we have shown marked protection of beta-arrestin1's N-domain in the presence of V2Rpp; consistent with previous studies suggesting the N-domain is responsible for recognizing phosphates in 7TMRs. Using a differentially phsophorylated V2R peptide (V2R4p), we show that beta-arrestin1 is able to adopt distinct conformations in response to different phosphorylation patterns. Futhermore, a striking difference is observed in the conformation of V2Rpp-bound beta-arrestin1 when compared to beta-arrestin2, namely the flexibility of the inter-domain hinge region. These data represent the first direct evidence that the beta-arrestin1 conformation is differentially instructed by phosphorylation patterns and that the "receptor-bound" conformations of beta-arrestins1 and 2 are different. Phosphorylation of 7TMRs by GRKs plays essential roles in regulation of receptor function by promoting interactions of the receptors with beta-arrestins. We hypothesized that… Advisors/Committee Members: Lefkowitz, Robert J (advisor).
Nobles, Kelly Nicole. “Phosphorylation Bar Codes Induce Distinct Conformations and Functionalities of beta-Arrestin .” 2010. Thesis, Duke University. Accessed April 22, 2019. http://hdl.handle.net/10161/3117.
Nobles, Kelly Nicole. “Phosphorylation Bar Codes Induce Distinct Conformations and Functionalities of beta-Arrestin .” 2010. Web. 22 Apr 2019.
Nobles KN. Phosphorylation Bar Codes Induce Distinct Conformations and Functionalities of beta-Arrestin . [Internet] [Thesis]. Duke University; 2010. [cited 2019 Apr 22]. Available from: http://hdl.handle.net/10161/3117.
9. Wagener, Brant. Regulation of N-formyl peptide receptor trafficking and signaling by arrestins.
▼ This work focuses on how arrestin regulates trafficking and signaling of the N-formyl peptide receptor (FPR), a G protein-coupled receptor (GPCR). GPCRs are involved in almost all physiologic processes and numerous pathologic processes. There is an intimate relationship between GPCR trafficking and signaling that controls many cellular processes. However, the protein-protein interactions that control post-endocytic trafficking and signaling of GPCRs are poorly understood. Our previous reports demonstrated that three events take place upon FPR activation in the absence of arrestins: accumulation of FPR in the perinuclear recycling endosome, lack of FPR recycling and apoptosis. All of these phenotypes were rescued by reintroduction of arrestin-2 cDNA. We therefore hypothesized that 1) FPR trafficking and signaling defects were linked and causal and 2) specific regions of arrestin-2 regulate normal FPR trafficking and signaling. To address these hypotheses, we generated mutants of arrestin-2 that were previously described or changed regions of similar amino acids to alanine. We then screened these mutants for the ability to rescue FPR-mediated apoptosis. Subsequently, we examined the role of these arrestin mutants in FPR trafficking. We found that two arrestin-2 mutants demonstrated altered binding to adaptor protein (AP)-2. Furthermore, FPR recycling was inhibited in the presence of either arrestin-2 mutant or the absence of AP-2. We also examined the role of Src kinase in FPR trafficking and signaling and determined that Src kinase has two independent roles in FPR-arrestin-2 regulation: one that controls FPR trafficking and one that mediated FPR signaling. Finally, we found that different SH3-binding domains of arrestin-2 regulate FPR trafficking and signaling independently. An arrestin-2 mutant did not rescue FPR-mediated apoptosis, but did mediate normal FPR trafficking. These results indicate that FPR trafficking and signaling are coordinated processes, but may also be regulated independently. These studies have revealed novel aspects of arrestin-2 that regulate FPR signaling and trafficking. We hope they will serve as a model for the regulation of other GPCRs. Furthermore, we hope these data are used to create small molecule inhibitors to serve as experimental tools and chemotherapeutics to better understand and treat diseases caused by defects in GPCR trafficking and signaling. Advisors/Committee Members: Prossnitz, Eric, Sklar, Larry, Hudson, Luarie, Hathaway, Helen.
Wagener, Brant. “Regulation of N-formyl peptide receptor trafficking and signaling by arrestins.” 2009. Doctoral Dissertation, University of New Mexico. Accessed April 22, 2019. https://digitalrepository.unm.edu/biom_etds/105.
Wagener, Brant. “Regulation of N-formyl peptide receptor trafficking and signaling by arrestins.” 2009. Web. 22 Apr 2019.
Wagener B. Regulation of N-formyl peptide receptor trafficking and signaling by arrestins. [Internet] [Doctoral dissertation]. University of New Mexico; 2009. [cited 2019 Apr 22]. Available from: https://digitalrepository.unm.edu/biom_etds/105.
10. Jones, Brian William. Phosphorylation of the Thyrotropin-releasing Hormone Receptor.
Jones, Brian William. “Phosphorylation of the Thyrotropin-releasing Hormone Receptor.” 2009. Doctoral Dissertation, University of Rochester. Accessed April 22, 2019. http://hdl.handle.net/1802/6618.
Jones, Brian William. “Phosphorylation of the Thyrotropin-releasing Hormone Receptor.” 2009. Web. 22 Apr 2019.
Jones BW. Phosphorylation of the Thyrotropin-releasing Hormone Receptor. [Internet] [Doctoral dissertation]. University of Rochester; 2009. [cited 2019 Apr 22]. Available from: http://hdl.handle.net/1802/6618.
11. Jaeger, Werner Christian. Biophysical insights into orexin receptor-β-arrestin complexes and their role in cellular signalling.
Jaeger, Werner Christian. “Biophysical insights into orexin receptor-β-arrestin complexes and their role in cellular signalling.” 2011. Doctoral Dissertation, University of Western Australia. Accessed April 22, 2019. http://repository.uwa.edu.au:80/R/?func=dbin-jump-full&object_id=32908&local_base=GEN01-INS01.
Jaeger, Werner Christian. “Biophysical insights into orexin receptor-β-arrestin complexes and their role in cellular signalling.” 2011. Web. 22 Apr 2019.
Jaeger WC. Biophysical insights into orexin receptor-β-arrestin complexes and their role in cellular signalling. [Internet] [Doctoral dissertation]. University of Western Australia; 2011. [cited 2019 Apr 22]. Available from: http://repository.uwa.edu.au:80/R/?func=dbin-jump-full&object_id=32908&local_base=GEN01-INS01.
12. Hoeppner, Crystal Z. Identification of a Nuclear Localization Sequence in Beta-Arrestin1:Implications in NF- kB Activation.
▼ A mounting body of evidence suggests that beta-arrestin1 plays important roles in the nucleus, but how beta-arrestin1 enters the nucleus remains unclear since no nuclear import signal has been identified in the -arrestins. We sought to characterize the cellular localization of wild type beta-arrestin1 and a series of N domain mutants to determine the structural basis and functional implications of beta-arrestin1 nuclear localization. A seven-residue candidate nuclear localization sequence (NLS) was identified based on sequence analysis. Mutation of the NLS led to a loss of beta-arrestin1 nuclear localization in transfected cells. Exogenous expression of wild type beta-arrestin1 enhanced the transcriptional activity of nuclear factor kappaB (NF-kappaB) induced by bradykinin, while mutation of the NLS reduced this effect by two thirds relative to wild type controls. Loss of beta-arrestin1 nuclear localization was accompanied by reduced recruitment of the CREB binding protein and altered post-translational modification profile of p65/RelA. Further mutational analysis identified Lys157 within the putative NLS as being critical to nuclear localization of beta-arrestin1. Substitution of Lys157 to Ala led to reduced nuclear localization, decreased promoter binding by p65/RelA and decreased IL-1beta gene transcription. These results demonstrate a critical role for beta-arrestin1 nuclear localization in scaffolding and transcriptional regulation. Advisors/Committee Members: Ye, Richard D (advisor).
Hoeppner, Crystal Z. “Identification of a Nuclear Localization Sequence in Beta-Arrestin1:Implications in NF- kB Activation.” 2012. Thesis, University of Illinois – Chicago. Accessed April 22, 2019. http://hdl.handle.net/10027/9243.
Hoeppner, Crystal Z. “Identification of a Nuclear Localization Sequence in Beta-Arrestin1:Implications in NF- kB Activation.” 2012. Web. 22 Apr 2019.
Hoeppner CZ. Identification of a Nuclear Localization Sequence in Beta-Arrestin1:Implications in NF- kB Activation. [Internet] [Thesis]. University of Illinois – Chicago; 2012. [cited 2019 Apr 22]. Available from: http://hdl.handle.net/10027/9243.
13. Chen, Qiuyan. The structure basis of arrestin mediated GPCR signaling.
▼ Arrestin selectively binds the phosphorylated active receptor to either terminate the G protein dependent signaling or initiate G protein independent signaling. Receptor binding induces global conformational changes in arrestin and allosterically activates the region for downstream effectors binding. My research is focused on elucidating the structural basis of arrestin signaling. In particular, I am trying to answer the following questions: 1. What is the conformation of arrestin in the active form? 2. How does arrestin activate downstream signaling? 3. How is the receptor activation allosterically linked to the effector binding in arrestin? I have used NMR, EPR, crystallography and various techniques to look at arrestin dynamics and have determined the structure of active arrestin-3. Collectively, my results revealed the high dynamic of arrestin and identified key elements for receptor and downstream effector binding. Advisors/Committee Members: Charles R. Sanders (committee member), Heidi Hamm (committee member), Tina M. Iverson (committee member), Vsevolod V. Gurevich (committee member), Brian Wadzinski (chair).
Chen, Qiuyan. “The structure basis of arrestin mediated GPCR signaling.” 2015. Doctoral Dissertation, Vanderbilt University. Accessed April 22, 2019. http://etd.library.vanderbilt.edu//available/etd-11162015-201134/ ;.
Chen, Qiuyan. “The structure basis of arrestin mediated GPCR signaling.” 2015. Web. 22 Apr 2019.
Chen Q. The structure basis of arrestin mediated GPCR signaling. [Internet] [Doctoral dissertation]. Vanderbilt University; 2015. [cited 2019 Apr 22]. Available from: http://etd.library.vanderbilt.edu//available/etd-11162015-201134/ ;.
14. Cleghorn, Whitney Marie. Arrestins regulate cell spreading and motility via focal adhesion dynamics.
▼ Arrestins bind G protein-coupled receptors and more than 100 non-receptor partners, regulating various signaling pathways and cellular functions. The interactions of many proteins (e.g., Src, JNK3, ERKÂ½, Mdm2, etc.) with receptor-bound arrestin localize these molecules to receptor-rich membranes. Our recent finding that arrestins bind microtubules and recruit signaling proteins to the cytoskeleton prompted us to investigate whether arrestins affect cell motility and morphology. Here we describe a novel function of arrestins, their direct effect on focal adhesion dynamics. We demonstrate excessive spreading of cells lacking both non-visual arrestins, which is substrate-independent, evident on both fibronectin and poly-D-lysine. Reduced activity of small GTPases RhoA and Rac1 in arrestin-deficient cells is only partially responsible for the cell spreading phenotype. Increased adhesion, reflected by elevated activity of focal adhesion proteins paxillin and focal adhesion kinase, underlies the exaggerated spreading of arrestin-null cells and their reduced motility. The absence of arrestins greatly increases the size and lifespan of focal adhesions, indicating that arrestins are necessary for rapid focal adhesion turnover. Focal adhesions in arrestin-deficient cells are insensitive to microtubules, suggesting that arrestins likely mediate the induction of focal adhesion disassembly upon microtubule regrowth. Overexpression of WT arrestins and their receptor binding-deficient mutants in arrestin-null cells rescues the phenotype, demonstrating that regulation of focal adhesion dynamics by arrestins is receptor-independent. This is the first demonstration that arrestins play a direct role in focal adhesion dynamics. Advisors/Committee Members: Roy Zent (committee member), Heidi E. Hamm (committee member), Brian E. Wadzinski (chair), Vsevolod V. Gurevich (committee member), Alissa M. Weaver (committee member).
Cleghorn, Whitney Marie. “Arrestins regulate cell spreading and motility via focal adhesion dynamics.” 2012. Doctoral Dissertation, Vanderbilt University. Accessed April 22, 2019. http://etd.library.vanderbilt.edu/available/etd-06222012-142651/ ;.
Cleghorn, Whitney Marie. “Arrestins regulate cell spreading and motility via focal adhesion dynamics.” 2012. Web. 22 Apr 2019.
Cleghorn WM. Arrestins regulate cell spreading and motility via focal adhesion dynamics. [Internet] [Doctoral dissertation]. Vanderbilt University; 2012. [cited 2019 Apr 22]. Available from: http://etd.library.vanderbilt.edu/available/etd-06222012-142651/ ;.
15. Sethi, Shalini. Effect of Melatonin on Differentiation of Human Mesenchymal Stem Cells and a Study on C-Terminal Domains of MT1 and MT2 Melatonin Receptors.
▼ Melatonin has been reported to enhance the differentiation of osteoblasts. The purpose of this study was to determine the melatonin treatment that would differentiate human mesenchymal stem cells (hAMSCs) into osteoblasts. A 21 d continuous melatonin treatment significantly increased the alkaline phosphatase (ALP) activity and the deposition of calcium in hAMSCs. These effects were inhibited by MT2 specific antagonist- 4P-PDOT. The time periods of melatonin treatment that increased the expression of osteogenic genes indicated both a sensitized and desensitized receptors with respect to cAMP signaling, signifying two distinct mechanisms of melatonin's action. Unlike the parathyroid hormone which is administered in intermittent doses to increase bone mass, a continuous melatonin treatment may be effective in having an anabolic effect on bone. Advisors/Committee Members: Paula Witt-Enderby, Vicki Davis, Jane Cavanaugh, John Pollock, Rehana Leak.
Sethi, Shalini. “Effect of Melatonin on Differentiation of Human Mesenchymal Stem Cells and a Study on C-Terminal Domains of MT1 and MT2 Melatonin Receptors.” 2010. Doctoral Dissertation, Duquesne University. Accessed April 22, 2019. https://dsc.duq.edu/etd/1173.
Sethi, Shalini. “Effect of Melatonin on Differentiation of Human Mesenchymal Stem Cells and a Study on C-Terminal Domains of MT1 and MT2 Melatonin Receptors.” 2010. Web. 22 Apr 2019.
Sethi S. Effect of Melatonin on Differentiation of Human Mesenchymal Stem Cells and a Study on C-Terminal Domains of MT1 and MT2 Melatonin Receptors. [Internet] [Doctoral dissertation]. Duquesne University; 2010. [cited 2019 Apr 22]. Available from: https://dsc.duq.edu/etd/1173.
16. Brust Fernandes, Tarsis. FUNCTIONAL SELECTIVITY DOWNSTREAM OF Gαi/o-COUPLED RECEPTORS.
▼ G protein-coupled receptors (GPCRs) are drug targets that often activate multiple signaling pathways. The multiple GPCR responses provide opportunities for biased or functionally selective ligands to preferentially modulate one signaling pathway over another. Studies with several GPCRs have suggested that selective activation of signaling pathways downstream of a GPCR may lead to safer and more effective drug therapies. The dopamine D2 receptor is the main target in therapies for Parkinson’s disease and schizophrenia. First and second generation antipsychotic drugs antagonize dopamine D2 receptor. Notably, both these classes of drugs may cause side effects associated with D2 receptor antagonism (e.g. hyperprolactemia and extrapyramidal symptoms). The novel, “third generation” antipsychotic drug, aripiprazole is also used to treat schizophrenia, with the remarkable advantage that its tendency to cause extrapyramidal symptoms is minimal. In this work we studied the molecular pharmacology of aripiprazole and showed that the compound displays ligand bias for modulation of G proteins, being a partial agonist for Gαi/o and a robust antagonist for Gβγ signaling. We have also examined the activation of immediate effectors of the dopamine D2 receptor (i.e. Gαi/o, Gβγ, β-arrestin recruitment) and more complex signaling pathways (i.e., extracellular signal-regulated kinase phosphorylation, heterologous sensitization, and dynamic mass redistribution) in response to a series of D2 receptor ligands. The most commonly used methods to measure ligand bias were employed and compared. Functional selectivity analyses were also employed as tools to explore the relative contribution of immediate dopamine D2 receptor effectors for the activation of more complex signaling pathways. We have further identified novel classes of AC1 inhibitors through both chemical library screening and structure-activity relationship studies. The effects of our best inhibitor (W001) on acute and chronic signaling through the μ-opioid receptor were also examined, revealing an alternative method to induce functional selectivity (i.e. by targeting signaling components that are downstream of GPCRs). Lastly, we showed that W001, which is the most potent selective small molecule AC1 inhibitor described to date, has analgesic properties in a mouse model of inflammatory pain. Advisors/Committee Members: Val J Watts, Jean-Christophe Rochet, Gregory H Hockerman, Donald F Ready.
Brust Fernandes, Tarsis. “FUNCTIONAL SELECTIVITY DOWNSTREAM OF Gαi/o-COUPLED RECEPTORS.” 2015. Doctoral Dissertation, Purdue University. Accessed April 22, 2019. https://docs.lib.purdue.edu/open_access_dissertations/1448.
Brust Fernandes, Tarsis. “FUNCTIONAL SELECTIVITY DOWNSTREAM OF Gαi/o-COUPLED RECEPTORS.” 2015. Web. 22 Apr 2019.
Brust Fernandes T. FUNCTIONAL SELECTIVITY DOWNSTREAM OF Gαi/o-COUPLED RECEPTORS. [Internet] [Doctoral dissertation]. Purdue University; 2015. [cited 2019 Apr 22]. Available from: https://docs.lib.purdue.edu/open_access_dissertations/1448.
17. Cervantes, David. Modulation of mitogenic signaling and growth by sympathetic adrenergic regulation.
▼ Heart disease and diabetes mellitus are growing epidemics, consistently ranking within the top ten causes of death in the United States. Both diseases are associated with cardiac remodeling, including cellular growth and fibrosis. Central to the progression of heart disease and diabetes are the adrenergic receptors (ARs) and insulin receptors (IR), respectively. Classically considered to elicit separate, and even opposing, cellular processes, increasing evidence suggests that ARs are capable of signaling cross-talk with other G-protein coupled receptors (GPCRs) as well as other receptor families, including receptor tyrosine kinases (RTKs). While the ARs have been implicated at numerous stages of heart failure, the adrenergic signaling mechanisms underlying this remodeling remain unknown. Further, studies investigating signaling cross-talk among different adrenergic receptors as well as between adrenergic receptors and other signaling pathways in myocardium remain in their infancy. In addition, few studies have implied functional interactions between IRs and βARs in cardiac tissues. Understanding how cells integrate information from a variety of chemically diverse signals into complex, orchestrated responses such as cell proliferation, differentiation and apoptosis is an overarching goal of cell biology. Thus, an understanding of the mechanisms and physiologic consequences of adrenergic receptor cross-talk within the heart is essential to develop novel treatments designed to prevent the cardiac remodeling observed in heart disease and diabetes. In concordance, the goals of this thesis research are two-fold. First, we aim to define mechanisms of AR cross-talk in cardiac cells. Secondly, we aim to determine the importance of these mechanisms in cardiac remodeling in response to pathological conditions. To begin to address the goals presented we first examined cross-talk within GPCRs. We elucidated a general mechanism in which non-traditional GPCR signaling is capable of regulating mitogen-activated protein kinase (MAPK) signaling originating from another GPCR. Interestingly, this cross-talk impaired GPCR-induced cellular proliferation. Next, we characterized a novel signaling mechanism in which type II RTK activation at high concentrations of mitogen can recruit non-traditional GPCR signaling components to fine-tune activation of MAPK signaling for cell proliferation. In sum, this work presents a significant expansion of our understanding of GPCR signaling cross-talk and its role in altering growth signaling originating from another receptor. Further, due to the complexities of hormonal signaling in vivo, this work highlights the need to further pursue a more in-depth understanding of how concomitant activation of one signaling pathway can alter the signaling and physiologic outcome of another receptor’s signaling. Advisors/Committee Members: Xiang, Yang (advisor), Nardulli, Ann M. (committee member), Chen, Jie (committee member), Raetzman, Lori T. (committee member).
Cervantes, David. “Modulation of mitogenic signaling and growth by sympathetic adrenergic regulation.” 2012. Doctoral Dissertation, University of Illinois – Urbana-Champaign. Accessed April 22, 2019. http://hdl.handle.net/2142/29837.
Cervantes, David. “Modulation of mitogenic signaling and growth by sympathetic adrenergic regulation.” 2012. Web. 22 Apr 2019.
Cervantes D. Modulation of mitogenic signaling and growth by sympathetic adrenergic regulation. [Internet] [Doctoral dissertation]. University of Illinois – Urbana-Champaign; 2012. [cited 2019 Apr 22]. Available from: http://hdl.handle.net/2142/29837.
18. Cahill, Thomas J. New Insights into GPCR–Transducer Coupling .
▼ β-arrestins (βarrs) interact with G protein-coupled receptors (GPCRs) to desensitize G protein signaling, initiate signaling on their own, and mediate receptor endocytosis. Using a panel of GPCRs believed to couple differently to βarrs we demonstrate how distinct conformations of GPCR–βarr complexes are specialized to perform different subsets of these cellular functions. Our results thus provide a new signaling paradigm for the understanding of GPCRs, whereby a specific GPCR–βarr conformation mediates receptor desensitization, while another drives internalization and some forms of signaling. In addition, some GPCRs activate G proteins from within internalized cellular compartments resulting in sustained signaling. We used a variety of biochemical, biophysical, and cell-based methods to demonstrate the existence, functionality, and architecture of internalized receptor “mega-complexes” composed of a single GPCR, βarr, and G protein. EM of purified ‘megaplexes’ reveals that a single receptor binds simultaneously through its core region with G protein and with βarr in the tail conformation. Thus, the two GPCR–βarr conformations carry out distinct cellular functions. Advisors/Committee Members: Lefkowitz, Robert J (advisor).
Cahill, Thomas J. “New Insights into GPCR–Transducer Coupling .” 2018. Thesis, Duke University. Accessed April 22, 2019. http://hdl.handle.net/10161/16774.
Cahill, Thomas J. “New Insights into GPCR–Transducer Coupling .” 2018. Web. 22 Apr 2019.
Cahill TJ. New Insights into GPCR–Transducer Coupling . [Internet] [Thesis]. Duke University; 2018. [cited 2019 Apr 22]. Available from: http://hdl.handle.net/10161/16774.
19. Groer, Chad E. Agonist-selective regulation of the mu opioid receptor by ßarrestins.
▼ Morphine and other opiates mediate their effects through activation of the mu opioid receptor (MOR). Activation of the MOR results in recruitment of regulatory proteins, ßarrestins, that can regulate how this receptor signals. In vivo studies suggest that disruption of ßarrestin-mediated MOR regulation may enhance opiate-induced antinociception and reduce tolerance and certain unwanted side effects. Therefore, by understanding the cellular mechanisms by which this receptor is regulated, the development of analgesics which preserve the beneficial effects of opiates while eliminating unwanted side effects may be possible. In this dissertation we test the hypothesis that MOR agonists can bias MOR-ßarrestin interactions, and that ßarrestin recruitment profiles, in turn, may determine cellular responses evoked by these agonists. In the first data portion of this dissertation, we characterize several novel MOR agonists that are unable to promote ßarrestin recruitment. Herkinorin is a moderately selective agonist at the MOR, based on the structure of a natural product, Salvinorin A. We find that herkinorin promotes very little MOR phosphorylation, does not recruit ßarrestins, and does not induce receptor internalization in transfected cells. Herkinorin is unable to induce ßarrestin recruitment or MOR internalization under conditions that facilitate receptor phosphorylation and subsequent ßarrestin recruitment with other agonists. We also evaluated several derivatives of herkinorin with similar ßarrestin recruitment and MOR internalization profiles. Therefore, herkinorin and its derivatives may be a promising step toward recapitulating morphine’s effects in ßarr2-KO mice, which have been used to demonstrate that MOR activation without recruiting ßarrestin2 may be therapeutically useful, by producing analgesia with reduced side effects. In the second data portion of this dissertation, we evaluate the interaction and functional consequences of MOR regulation by ßarrestin1 and ßarrestin2, in response to the classical agonists, DAMGO and morphine. Using both qualitative (microscopy) and quantitative (cell surface biotinylation and BRET) approaches, we have confirmed that DAMGO can induce robust interactions between the MOR and both ßarrestins. Morphine, however, selectively promotes interactions with ßarrestin2. Additionally, the agonist specific ßarrestin interactions are required for internalization of the MOR. Finally, we show that ßarrestin1 is required for agonist-induced MOR ubiquitination, such that only DAMGO, and not morphine, is able to promote MOR ubiquitination. Taken together, these data suggest that MOR regulation is highly dependent on the complement of proteins available to interact with the MOR, and that the nature of the ligand can determine how the MOR is regulated by the available proteins. Therefore, the development of biased ligands for the MOR should focus activation of the MOR, but circumventing ßarrestin-mediated regulation. These concepts may be critical to consider in the… Advisors/Committee Members: Sadee, Wolfgang (Committee Chair), Bohn, Laura (Advisor).
Groer, Chad E. “Agonist-selective regulation of the mu opioid receptor by ßarrestins.” 2010. Doctoral Dissertation, The Ohio State University. Accessed April 22, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1284396146.
Groer, Chad E. “Agonist-selective regulation of the mu opioid receptor by ßarrestins.” 2010. Web. 22 Apr 2019.
Groer CE. Agonist-selective regulation of the mu opioid receptor by ßarrestins. [Internet] [Doctoral dissertation]. The Ohio State University; 2010. [cited 2019 Apr 22]. Available from: http://rave.ohiolink.edu/etdc/view?acc_num=osu1284396146.
20. Raehal, Kirsten Michele. Opioid-Induced Side Effects in Beta-arrestin2 adn G Protein-Coupled Receptor Kinase Knockout Mice.
▼ Opioid drugs are potent analgesics; however, they also produce several adverse side effects including constipation, antinociceptive tolerance, and physical dependence by activating the mu opioid receptor, a G protein-coupled receptor (GPCR). There is a substantial literature that suggests that the GPCR regulatory proteins G protein-coupled receptor kinases (GRKs) and beta-arrestins play a key role in regulating mu opioid receptor signaling and responsiveness. In vivo, the loss of beta-arrestin2 significantly alters morphine-induced analgesia, antinociceptive tolerance, respiratory suppression, and reward. Moreover, distinct opioid agonists have been shown to differ in their propensity to promote interactions between the mu opioid receptor and beta-arrestins and such agonist directed events may ultimately determine the functional response of the receptor to a particular drug. Therefore, we hypothesize that GRK and beta-arrestin2-mediated mu opioid receptor regulation may determine the extent of opioid-induced side effects including constipation, antinociceptive tolerance, and physical dependence, in a manner that is specifically influenced by different opioid agonists and cellular environments. Using mice genetically lacking individual GRKs and beta-arrestin2, we evaluated distinct opioid agonists (morphine, methadone, and fentanyl) for their ability to elicit constipation, antinociceptive tolerance, and physical dependence. We find that beta-arrestin2 is important in determining the expression of morphine-induced constipation, antinociceptive tolerance, and physical dependence, even more so than GRKs. However, while all responses evaluated in response to morphine were affected by the loss of beta-arrestin2, only methadone-induced physical dependence was altered in the beta-arrestin2-knockout mice, suggesting that beta-arrestin2 differentially affects these opioid-mediated responses in an agonist-dependent manner. Collectively, these results provide evidence that distinct opioid agonists can influence mu opioid receptor regulation and responsiveness and that the contribution of a particular regulatory factor to receptor function can differ based upon the specific cell composition and physiology assessed. Advisors/Committee Members: Bohn, Laura (Advisor).
Raehal, Kirsten Michele. “Opioid-Induced Side Effects in Beta-arrestin2 adn G Protein-Coupled Receptor Kinase Knockout Mice.” 2009. Doctoral Dissertation, The Ohio State University. Accessed April 22, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1236884585.
Raehal, Kirsten Michele. “Opioid-Induced Side Effects in Beta-arrestin2 adn G Protein-Coupled Receptor Kinase Knockout Mice.” 2009. Web. 22 Apr 2019.
Raehal KM. Opioid-Induced Side Effects in Beta-arrestin2 adn G Protein-Coupled Receptor Kinase Knockout Mice. [Internet] [Doctoral dissertation]. The Ohio State University; 2009. [cited 2019 Apr 22]. Available from: http://rave.ohiolink.edu/etdc/view?acc_num=osu1236884585.
21. Chan, Sanny Kai-Wai. Signaling cascades: a functional characterization of cone arrestin and a differential gene expression analysis of developing retinal ganglion cells.
▼ The mature retina is a specialized tissue critical for vision. Individual cell-types that comprise the retina have specialized functional roles mediated by differential gene expression. To identify the contribution of specific genes in defining a cell-type, the function of the cone arrestin protein was analyzed in vivo in photoreceptors and a differential transcript expression profile generated for perinatal developing retinal ganglion cells.; Photoreceptors are responsible for converting photon inputs into neural signals through a process called phototransduction. Rods and cones express unique proteins that enable them to execute this function. The mechanism of rod phototransduction has been well characterized, but little is known about cone phototransduction. Since many homologous components of the rod phototransduction cascade have been identified in cones, it has been suggested that cone phototransduction is similar to rod phototransduction. We hypothesize that CAR functions in cone photoreceptors similar to rod arrestin's role in terminating rod phototransduction.; Using a genetic approach, we expressed cone arrestin (CAR) in rod photoreceptors without rod arrestin to directly compare the functional role of CAR and rod arrestin in terminating the photoresponse. We provide light damage, translocation and membrane association, and single cell electrophysiology evidence that show CAR functions similarly to rod arrestin in the termination of the rod photoresponse in rod photoreceptors in vivo although not as effectively as rod arrestin. These data suggest that the role of CAR in cone phototransduction is akin to rod arrestin's role in quenching rod phototransduction.; In addition to analyzing the specific functional capabilities of CAR, transcripts were screened from perinatal developing retinal ganglion cells (RGCs) for differential expression in order to identify genes that may play a role in developing functional RGCs. We hypothesize that there is uniform coordinated expression of specific transcripts during perinatal RGC maturation that mediate neurite growth loss. High-density microarray analysis of the differentially expressed transcripts from CD90+ RGCs was confirmed by semi-quantitative PCR and in situ hybridization for temporal and spatial expression. Seventy-one transcripts out of ~26,000 genes were identified as differentially expressed (ANOVA corrected p Advisors/Committee Members: Chen, Jeannie (Committee Chair), Hinton, David R. (Committee Member), Garner, Judy A. (Committee Member), Craft, Cheryl (Committee Member).
Chan, Sanny Kai-Wai. “Signaling cascades: a functional characterization of cone arrestin and a differential gene expression analysis of developing retinal ganglion cells.” 2007. Doctoral Dissertation, University of Southern California. Accessed April 22, 2019. http://digitallibrary.usc.edu/cdm/compoundobject/collection/p15799coll127/id/583837/rec/5846.
Chan, Sanny Kai-Wai. “Signaling cascades: a functional characterization of cone arrestin and a differential gene expression analysis of developing retinal ganglion cells.” 2007. Web. 22 Apr 2019.
Chan SK. Signaling cascades: a functional characterization of cone arrestin and a differential gene expression analysis of developing retinal ganglion cells. [Internet] [Doctoral dissertation]. University of Southern California; 2007. [cited 2019 Apr 22]. Available from: http://digitallibrary.usc.edu/cdm/compoundobject/collection/p15799coll127/id/583837/rec/5846.
22. Murphy-Marshman, Hannah E. Identification Of A Novel Interaction Between Integrin-Linked Kinase And Beta-Arrestin 1.
▼ Integrin-linked kinase (ILK) is a ubiquitous scaffold protein essential for the development of front-rear polarity and directional migration of epidermal keratinocytes. β-arrestin 1 is another adaptor protein which has recently emerged as a key factor in modulating proliferation and migration of various cell types. Previous studies have demonstrated an association between β-arrestin 2 and ILK in cerebellar granule precursor cells. I have now identified a novel interaction between ILK and β-arrestin 1 in primary keratinocytes that occurs directly and without post-translational modifications. The N-terminal 67 residues of ILK and multiple regions in β-arrestin 1 are important for this interaction. This association occurs regardless of the conformational state of β-arrestin 1. Furthermore, ILK and β-arrestin 1 colocalize at cell extensions, suggesting a possible role for this complex in the development of front-rear cell polarity and migration. My analysis of this novel interaction provides new insight into mechanisms of keratinocyte regulation.
Murphy-Marshman, Hannah E. “Identification Of A Novel Interaction Between Integrin-Linked Kinase And Beta-Arrestin 1.” 2016. Thesis, University of Western Ontario. Accessed April 22, 2019. https://ir.lib.uwo.ca/etd/4182.
Murphy-Marshman, Hannah E. “Identification Of A Novel Interaction Between Integrin-Linked Kinase And Beta-Arrestin 1.” 2016. Web. 22 Apr 2019.
Murphy-Marshman HE. Identification Of A Novel Interaction Between Integrin-Linked Kinase And Beta-Arrestin 1. [Internet] [Thesis]. University of Western Ontario; 2016. [cited 2019 Apr 22]. Available from: https://ir.lib.uwo.ca/etd/4182.
23. McLean, Sarah E.A. Transforming Growth Factor Beta Receptor Partitioning: Molecular Mechanisms and Functional Consequences.
▼ The transforming growth factor beta (TGFbeta) pathway has been conserved throughout evolution and plays important roles in tissue homeostasis. Dysregulation of the TGFbeta pathway has been implicated in a number of disorders, including cancer, fibrosis, and vascular conditions. The signalling potential of the TGFbeta pathway is regulated by the route of internalization of its cell-surface receptors: Receptors internalized by clathrin-mediated endocytosis propagate signal transduction while those internalized by membrane rafts are targeted for degradation. Given the importance of trafficking of the TGFbeta receptors to signal propagation, this thesis focuses on evaluating proteins which direct TGFb receptor internalization and trafficking. Initial work in this thesis shows that the extracellular domain of the type II TGFbeta receptor (TbRII) and the glycosylation state of the cell are important factors in permitting membrane-raft localization of TbRII. Using this information I assessed the ability of TbRIII, a glycosylated cell surface protein, to direct TbRII internalization. I found that TbRIII increases membrane-raft independent internalization of TbRII, increases TbRII/TbRI complex half-life, and basal TGFbeta signalling. I next assessed the role of beta arrestin2, a protein which interacts with TbRIII, in regulating TbRII trafficking and signalling. I show that beta arrestin2 interacts with TbRII and traffics with TbRII to the early endosome to increase Smad-dependent signalling. Also, I show that depletion of beta arrestin2 increases Smad-independent signal transduction. In the last data chapter of this thesis, I evaluate the role of TGFbeta1 and TGFbeta3, to direct TGFbeta trafficking and signalling. I found that TGFbeta3 is less potent than TGFbeta1 at propagating TGFbeta signalling. I also show that TGFbeta3 induces a different binding ratio of TbRII/TbRI cell-surface complexes, which could explain its decreased potency. Overall my studies highlight the role of receptor-interacting proteins in directing TGFbeta receptor trafficking and signal transduction. Since this pathway is dysregulated in a number of pathologies, my studies suggest that TGFbeta receptor trafficking is an important avenue to modifying TGFbeta signal transduction.
McLean, Sarah E A. “Transforming Growth Factor Beta Receptor Partitioning: Molecular Mechanisms and Functional Consequences.” 2012. Thesis, University of Western Ontario. Accessed April 22, 2019. https://ir.lib.uwo.ca/etd/752.
McLean, Sarah E A. “Transforming Growth Factor Beta Receptor Partitioning: Molecular Mechanisms and Functional Consequences.” 2012. Web. 22 Apr 2019.
McLean SEA. Transforming Growth Factor Beta Receptor Partitioning: Molecular Mechanisms and Functional Consequences. [Internet] [Thesis]. University of Western Ontario; 2012. [cited 2019 Apr 22]. Available from: https://ir.lib.uwo.ca/etd/752.
24. C. Cancellieri. BETA-ARRESTIN DEPENDENT REGULATION OF CYTOSKELETON DYNAMICS AND SIGNALLING OF CHEMOKINE RECEPTOR ACKR2.
▼ Chemokines promote leukocyte migration through the activation of dedicated G-protein coupled receptors. Beyond conventional chemokine receptors, which directly induce cell migration through heterotrimeric Gαi-mediated signalling events, a set of atypical chemokine receptors (ACKRs) have been described. ACKRs do not activate Gαi-mediated signalling activity, but they are mainly involved in shaping the chemokine gradient. The best characterized member of this family is ACKR2. ACKR2, previously referred to as D6, is a scavenger receptor that binds with high affinity to 13 inflammatory CC chemokines. The scavenging activity of ACKR2 relies on its intracellular traffic properties. Under homeostatic conditions, ACKR2 is mainly localized in intracellular stores associated with both early Rab4/5-positive and recycling Rab11-positive endosomes. At increasing levels of chemokines, ACKR2 increases plasma membrane abundance through an acceleration in the rate of Rab11-depedent recycling pathway, in order to optimize its chemokine scavenging activity. Here, I demonstrated that the intracellular distribution of ACKR2 is maintained by cytoskeletal dynamics. After chemokine engagement, ACKR2 activate a G-protein-independent and β-arrestin-dependent Rac1-PAK1-LIMK1 signalling cascade to finely regulate the actin cytoskeletal and the microtubules network reorganization, to promote receptor up-regulation and scavenging function. ACKR2 is able to recruit and associates both β-arrestins in basal condition, at membrane and intracellular levels, but only β-arrestin1 is recruited after active ligand stimulation, in order to promote a β-arrestin1-dependent signalling pathway, required for supporting the myosin Vb-dependent ACKR2 up-regulation and scavenging properties. Advisors/Committee Members: tutor: M. Locati, supervisore: E.M Borroni, coordinatore del corso di dottorato: M. Locati, LOCATI, MASSIMO, LOCATI, MASSIMO.
Cancellieri, C.. “BETA-ARRESTIN DEPENDENT REGULATION OF CYTOSKELETON DYNAMICS AND SIGNALLING OF CHEMOKINE RECEPTOR ACKR2.” 2014. Thesis, Università degli Studi di Milano. Accessed April 22, 2019. http://hdl.handle.net/2434/229565.
Cancellieri, C.. “BETA-ARRESTIN DEPENDENT REGULATION OF CYTOSKELETON DYNAMICS AND SIGNALLING OF CHEMOKINE RECEPTOR ACKR2.” 2014. Web. 22 Apr 2019.
Cancellieri C. BETA-ARRESTIN DEPENDENT REGULATION OF CYTOSKELETON DYNAMICS AND SIGNALLING OF CHEMOKINE RECEPTOR ACKR2. [Internet] [Thesis]. Università degli Studi di Milano; 2014. [cited 2019 Apr 22]. Available from: http://hdl.handle.net/2434/229565.
25. Kuhar, Jamie Rose. Mechanisms of Opioid Receptor Desensitization.
Kuhar, Jamie Rose. “Mechanisms of Opioid Receptor Desensitization.” 2015. Doctoral Dissertation, University of Washington. Accessed April 22, 2019. http://hdl.handle.net/1773/34122.
Kuhar, Jamie Rose. “Mechanisms of Opioid Receptor Desensitization.” 2015. Web. 22 Apr 2019.
Kuhar JR. Mechanisms of Opioid Receptor Desensitization. [Internet] [Doctoral dissertation]. University of Washington; 2015. [cited 2019 Apr 22]. Available from: http://hdl.handle.net/1773/34122.
26. Armando, Sylvain. Structure quaternaire des récepteurs de chimiokines CXCR4 et CCR2 et interaction avec leur effecteurs. : Quaternary arrangements of the CXCR4-CCR2 homo- and hetero-oligomers and of their complexes with their signaling effectors.
Les récepteurs couplés aux protéines G (RCPG) sont la famille de récepteurs membranaires la plus représentée chez les vertébrés, et la plus grande cible thérapeutique chez l'Homme. L'évolution du paradigme initial qui énonçait une stœchiométrie récepteur : protéine G : effecteur de 1 :1 :1 sera présentée sur le modèle des récepteurs aux chimiokines CXCR4 et CCR2. Grâce à la technique de transfert d'énergie par bioluminescence (BRET), les travaux réalisés durant cette thèse montrent (1) que c'est par un couplage alternatif de CXCR4 à Gα13 au lieu de la voie classique Gαi que les cellules de cancer du sein migrent pour former des métastases, (2) que la désensibilisation de CXCR4 implique le recrutement d'une combinaison définie de protéines (GRK et arrestines) permettant l'arrêt sélectif des multiples voies engagées en réponse à l'agoniste, et (3) que le protomère CXCR4 a un rôle déterminant dans l'engagement de la protéine Gαi et le recrutement de la β-arrestine par l'hétéro-oligomère CXCR4/CCR2 lorsque CCR2 est activé. Dans cette dernière et principale étude, les résultats montrent également que le dimère CCR2 peut s' assembler au dimère CXCR4 pour former un tétramère, et que l'activation de CCR2 influence la conformation du dimère CXCR4. Les phénomènes de coopérativité et d'activation asymétrique déjà rapportés pour cet hétérodimère pourraient donc impliquer l'interaction de quatre protomères. En conclusion les travaux effectués durant cette thèse démontrent une régulation supplémentaire de l'activité des récepteurs chimiokines au niveau de leur structure quaternaire, de leur signalisation, et de l'arrêt de cette signalisation.
Advisors/Committee Members: Prézeau, Laurent (thesis director), Bouvier, Michel (thesis director).
Armando, Sylvain. “Structure quaternaire des récepteurs de chimiokines CXCR4 et CCR2 et interaction avec leur effecteurs. : Quaternary arrangements of the CXCR4-CCR2 homo- and hetero-oligomers and of their complexes with their signaling effectors.” 2010. Doctoral Dissertation, Université Montpellier II. Accessed April 22, 2019. http://www.theses.fr/2010MON20208.
Armando, Sylvain. “Structure quaternaire des récepteurs de chimiokines CXCR4 et CCR2 et interaction avec leur effecteurs. : Quaternary arrangements of the CXCR4-CCR2 homo- and hetero-oligomers and of their complexes with their signaling effectors.” 2010. Web. 22 Apr 2019.
Armando S. Structure quaternaire des récepteurs de chimiokines CXCR4 et CCR2 et interaction avec leur effecteurs. : Quaternary arrangements of the CXCR4-CCR2 homo- and hetero-oligomers and of their complexes with their signaling effectors. [Internet] [Doctoral dissertation]. Université Montpellier II; 2010. [cited 2019 Apr 22]. Available from: http://www.theses.fr/2010MON20208.
27. Walker III, William Benjamin. On the nature of the sensory arrestins of the dipteran insects Anopheles gambiae and Drosophila melanogaster.
▼ This project is concerned with the functional roles of the sensory arrestin genes, arr1 and arr2, in the regulation of dipteran olfactory signal transduction. Specifically, I have examined the sensory arrestins of the fruit fly, Drosophila melanogaster, and the malaria vector mosquito, Anopheles gambiae (An. gambiae). I have demonstrated that transgenic expression of An. gambiae arr1 in the antennae of arr1 mutant fruit flies sufficiently rescues olfactory deficits in these mutant flies. This implies functional orthology between the fruit fly and mosquito arr1 homologues. I also sought to examine spatial and temporal characteristics of these genes in the antennae of D. melanogaster. I attempted to identify the spatial expression patterns of arr1 and arr2 mRNA in antennal neurons via fluorescence in situ hybridization. However, the expression levels of these genes lie below the threshold of detection for the applied methodology, as no signal corresponding to the expression of these genes was detected. I utilized time-controlled induction of wild-type arr1 transgene paradigms in arr1 mutant fruit flies to assess the role of this gene as it pertains to the temporal dynamics of the effects of this gene on the olfactory system of the fly. The results of these studies are inconclusive and require further experimentation. Advisors/Committee Members: Vsevolod Gurevich (chair), Laurence J Zwiebel (committee member), Todd R Graham (committee member), Kendal Scot Broadie (committee member).
Walker III, William Benjamin. “On the nature of the sensory arrestins of the dipteran insects Anopheles gambiae and Drosophila melanogaster.” 2009. Doctoral Dissertation, Vanderbilt University. Accessed April 22, 2019. http://etd.library.vanderbilt.edu/available/etd-03312008-162553/ ;.
Walker III, William Benjamin. “On the nature of the sensory arrestins of the dipteran insects Anopheles gambiae and Drosophila melanogaster.” 2009. Web. 22 Apr 2019.
Walker III WB. On the nature of the sensory arrestins of the dipteran insects Anopheles gambiae and Drosophila melanogaster. [Internet] [Doctoral dissertation]. Vanderbilt University; 2009. [cited 2019 Apr 22]. Available from: http://etd.library.vanderbilt.edu/available/etd-03312008-162553/ ;.
28. Davidson, Reshma. Regulation of Septum Formation by Two Novel Proteins Art1 and Bga1 in Fission Yeast Cytokinesis.
▼ Cytokinesis is the final step in the cell cycle that partitions a mother cell into two daughter cells. The final step in cytokinesis involves extracellular matrix formation/ remodelling and midbody abscission in mammals. Many of the proteins and steps involved in cytokinesis are conserved from yeast to mammals. This makes genetically tractable unicellular fission yeast a good model system to study cytokinesis. In fission yeast extracellular matrix remodeling involves the deposition of a trilaminar polysaccharide septum and its subsequent digestion to separate the two daughter cells. For my thesis I worked on two novel proteins that are involved in cytokinesis and the regulation of septum synthesis. The septum and the outer cell wall in fission yeast are rigid structures that protect the cell from environmental stresses and give the cell its characteristic rod shape. The process of cell wall and septum formation are tightly regulated. Rho GTPases, activated by guanine nucleotide exchange factors (GEFs), are essential regulators of polarized cell growth, cytokinesis, and many other cellular processes. Amongst other things, they control the function of the cell wall synthesizing enzymes. However, the regulations of Rho GEFs themselves are not well understood. For my first project I studied the regulation of the Rho GEF Rgf3. Rgf3 is an essential GEF for Rho1 GTPase in fission yeast. The regulation of Rho GEF is a well-studied topic although much remains to be understood regarding its different modes of regulation. I have shown a novel mechanism of regulation of Rgf3 protein level and localization by an arrestin-related protein Art1. I found that art1¿ cells have a defective septum similar to rgf3 mutants. Art1 binds Rgf3 and plays a role in maintaining Rgf3 cellular levels. Finally, in art1¿ cells the amount of active Rho GTPase found at the division site was also reduced. This showed that Art1 played a role in septum formation by regulating the levels of active Rho1 found at the division site, through regulation of Rgf3. For my second project I explored how the localization and level of the cell wall synthesizing enzyme are controlled. Regulation of fungal cell wall synthesis is of great interest due to the diseases and economic damage caused by several pathogenic fungi. ß-Glucan synthase 1 (Bgs1), popular anti-fungal drug target, synthesizes the linear 1,3-ß-glucan which is the main component of primary septum. Rho1 GTPase and the F-BAR protein Cdc15 are known to play a role in Bgs1 regulation. However, they also regulate the function of several other proteins during cytokinesis. No specific regulator for Bgs1 function was known. In my second project I have characterized a novel protein Bga1 that regulates Bgs1 protein levels and localization to the plasma membrane. Bga1 is essential for contractile-ring constriction and septum formation during cytokinesis. Bga1 and Bgs1 physically interact and are interdependent for localization to the plasma membrane. Furthermore, Bga1 plays an earlier and more important role in… Advisors/Committee Members: Wu, Jian-Qiu (Advisor).
Davidson, Reshma. “Regulation of Septum Formation by Two Novel Proteins Art1 and Bga1 in Fission Yeast Cytokinesis.” 2016. Doctoral Dissertation, The Ohio State University. Accessed April 22, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1469185292.
Davidson, Reshma. “Regulation of Septum Formation by Two Novel Proteins Art1 and Bga1 in Fission Yeast Cytokinesis.” 2016. Web. 22 Apr 2019.
Davidson R. Regulation of Septum Formation by Two Novel Proteins Art1 and Bga1 in Fission Yeast Cytokinesis. [Internet] [Doctoral dissertation]. The Ohio State University; 2016. [cited 2019 Apr 22]. Available from: http://rave.ohiolink.edu/etdc/view?acc_num=osu1469185292.
29. Bonneterre, Julien. Impact de différentes modalités de recrutement de la β-arrestine au récepteur de chimiokine CXCR4 .
Bonneterre, Julien. “Impact de différentes modalités de recrutement de la β-arrestine au récepteur de chimiokine CXCR4 .” 2018. Thesis, Université de Montréal. Accessed April 22, 2019. http://hdl.handle.net/1866/20403.
Bonneterre, Julien. “Impact de différentes modalités de recrutement de la β-arrestine au récepteur de chimiokine CXCR4 .” 2018. Web. 22 Apr 2019.
Bonneterre J. Impact de différentes modalités de recrutement de la β-arrestine au récepteur de chimiokine CXCR4 . [Internet] [Thesis]. Université de Montréal; 2018. [cited 2019 Apr 22]. Available from: http://hdl.handle.net/1866/20403.
30. Nguyen, Peter. CANNABINOID RECEPTORS IN THE 3D RECONSTRUCTED MOUSE BRAIN: FUNCTION AND REGULATION.
▼ CB1 receptors (CB1R) mediate the psychoactive and therapeutic effects of cannabinoids including ∆9-tetrahydrocannabinol (THC), the main psychoactive constituent in marijuana. However, therapeutic use is limited by side effects and tolerance and dependence with chronic administration. Tolerance to cannabinoid-mediated effects is associated with CB1R adaptations, including desensitization (receptor-G-protein uncoupling) and downregulation (receptor degradation). The objectives of this thesis are to investigate the regional-specificity in CB1R function and regulation. Previous studies have investigated CB1Rs in a subset of regions involved in cannabinoid effects, but an inclusive regional comparison of the relative efficacies of different classes of cannabinoids to activate G-proteins has not been conducted. A novel unbiased whole-brain analysis was developed based on Statistical Parametric Mapping (SPM) for 3D-reconstructed mouse brain images derived from agonist-stimulated [35S]GTPgS autoradiography, which has not been described before. SPM demonstrated regional differences in the relative efficacies of cannabinoid agonists methanandamide (M-AEA), CP55,940 (CP), and WIN55,212-2 (WIN) in mouse brains. To assess potential contribution of novel sites, CB1R knockout (KO) mice were used. SPM analysis revealed that WIN, but not CP or M-AEA, stimulated [35S]GTPgS binding in regions that partially overlapped with the expression of CB1Rs. We then examined the role of the regulatory protein Beta-arrestin-2 (βarr2) in CB1R adaptations to chronic THC treatment. Deletion of βarr2 reduced CB1R desensitization/downregulation in the cerebellum, caudal periaqueductal gray (PAG), and spinal cord. However in hippocampus, amygdala and rostral PAG, similar desensitization was present in both genotypes. Interestingly, enhanced desensitization was found in the hypothalamus and cortex in βarr2 KO animals. Intra-regional differences in the magnitude of desensitization were noted in the caudal hippocampus, where βarr2 KO animals exhibited greater desensitization compared to WT. Regional differences in βarr2-mediated CB1R adaptation were associated with differential effects on tolerance, where THC-mediated antinociception, but not catalepsy or hypothermia, was attenuated in βarr2 KO mice. Overall, studies using SPM revealed intra- and inter-regional specificity in the function and regulation of CB1Rs and underscores an advantage of using a whole-brain unbiased approach. Understanding the regulation of CB1R signaling within different anatomical contexts represents an important fundamental prerequisite in the therapeutic exploitation of the cannabinoid system. Advisors/Committee Members: Laura Sim-Selley.
Nguyen, Peter. “CANNABINOID RECEPTORS IN THE 3D RECONSTRUCTED MOUSE BRAIN: FUNCTION AND REGULATION.” 2010. Doctoral Dissertation, Virginia Commonwealth University. Accessed April 22, 2019. https://scholarscompass.vcu.edu/etd/2274.
Nguyen, Peter. “CANNABINOID RECEPTORS IN THE 3D RECONSTRUCTED MOUSE BRAIN: FUNCTION AND REGULATION.” 2010. Web. 22 Apr 2019.
Nguyen P. CANNABINOID RECEPTORS IN THE 3D RECONSTRUCTED MOUSE BRAIN: FUNCTION AND REGULATION. [Internet] [Doctoral dissertation]. Virginia Commonwealth University; 2010. [cited 2019 Apr 22]. Available from: https://scholarscompass.vcu.edu/etd/2274.

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