Thesis (Ph. D.)--University of Rhode Island, 2006. / Typescript. Includes bibliographical references (leaves 104-121).

The 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. / Dissertation

Although a long standing convention maintained that G Protein Coupled Receptors (GPCRs) exist in the plasma membrane solely as monomers, substantial work over the last two decades has demonstrated that these ubiquitous receptors can and in many cases, preferentially, exist as homodimers, heterodimers, or higher order oligomers. Often, two GPCRs of the same class heterodimerize; it is less common for two GPCRs of different signaling pathways to interact. The work presented here studied the physical and functional interaction of two GPCRs from discrete classes, the Beta 2 Adrenergic Receptor (β2AR), a Gαs-coupled receptor, and Bradykinin type 2 Receptor (Bk2R), a Gαq coupled receptor. These data show that Bk2R and β2AR are physically coupled when heterologously expressed in Xenopus oocytes, and in pheochromocytoma (PC12) cells and in freshly isolated murine ventricular myocytes, two systems that endogenously express these receptors. This physical coupling led to functional consequences in heterologous and endogenous expression systems, as Bk2R was able to transactivate β2AR signaling via its direct interaction with the receptor. Furthermore, coexpression of Bk2R shifted the dose response curve of β2AR for its selective agonist rightward in Xenopus oocyte electrophysiology experiments, suggesting the presence of Bk2R negatively affected β2AR native pharmacology. Up to thirty minutes of either bradykinin (BK) or isoproterenol exposure did not change the relative amount of Bk2R/β2AR heterodimer in PC12 cells, a rat adrenal medulla tumor cell line that endogenously expresses these receptors. Despite the obvious signaling consequences, the Bk2R/β2AR heterodimer accounted for only 10% of the total β2AR protein detected and 20% of the total Bk2R protein detected. When other Bk2R-specific ligands were also tested to examine the extent of β2AR transactivation, our data showed that both Lys-des-Arg-Bradykinin, a Bk2R partial agonist and NPC 567, a Bk2R antagonist, transactivated β2AR to the same extent as BK. Taken together, our data provide a novel mode of receptor regulation and signaling via Bk2R/β2AR heterodimerization. Because a large percentage of therapeutics target GPCRs, a greater understanding of how a GPCR heterodimer functions could be beneficial for targeting new drugs and refining existing drugs. Understanding the Bk2R/β2AR heterodimer provides a new perspective on the myriad of fucntional consequences that occur when a GPCR is activated.

Thesis (Ph. D.)--University of Texas at Austin, 2003. / Vita. Includes bibliographical references. Available also from UMI Company.

Thesis (M.S.)--Ball State University, 2009. / Title from PDF t.p. (viewed on Nov. 30, 2009). Includes bibliographical references (p. [59]-63).

Thesis (Ph. D.)--University of Queensland, 2002. / Includes bibliographical references.

Not available / text

No description available.

The excitotoxin domoic acid (DOM) acts at both kainic acid (KA)- and α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-sensitive glutamate receptors. Clinical reports suggest that elderly people are hypersensitive to the neurological effects of DOM intoxication. Young, but not aged hippocampal slices which have been preconditioned with low concentrations of DOM or KA exhibit an acute �tolerance� to subsequent high doses of DOM or KA; application of the selective AMPA agonist fluorowillardiine (FW) fails to induce tolerance to excitotoxins. The aim of this study was to further investigate the molecular mechanism of tolerance induction in vitro, and to examine the ability of compounds to cross-condition against excitotoxic insult. In addition, in vivo techniques were used to explore the age-related susceptibility to the neurological effects of DOM and acute in vivo tolerance induction. Here we show that low doses of �classical� ionotropic kainate receptor agonists and AMPA/kainate receptor antagonists act as net inverse agonists at G-protein coupled receptors, reducing constitutive GTPase activity by up to 73% in the young hippocampus. Further evidence that inverse agonist activity at G-protein coupled receptors is responsible for acute in vitro tolerance induction by kainate receptor agonists and antagonists was also identified because preconditioning with the AMPA receptor antagonist GYKI-52466 significantly inhibited KA-induced population spike suppression in in vitro hippocampal brain slices from both Sprague-Dawley and Wistar rats. The broad-spectrum protein kinase inhibitor H-7 partially blocked tolerance induction when preconditioning occurs in the presence of suggesting that protein kinases are one of the downstream effectors of this phenomenon. Tolerance-inducing compounds are also capable of cross-conditioning against the effects of other excitotoxins; with 250 nM FW suppressed population spike area by only 62.8 � 10.0% at 30 minutes following a 500 nM KA preconditioning dose, compared to almost complete spike suppression within twenty minutes in naive hippocampal brain slices. In vivo experiments indicated that despite aged animals exhibiting significantly higher cumulative behavioural scores in response to i.p. DOM (1 mg kg⁻�; young = 102 � 9, aged = 179 � 19; P < 0.01) in response to DOM after two hours), and that this age-related supersensitivity is due to impaired renal clearance (young serum DOM = 41.5 � 30.3 ng ml⁻�, aged = 813.3 � 804.4 ng ml⁻� following administration of 1 mg kg⁻� DOM after 2.5 hours earlier). Tolerance to high doses of DOM was induced within a matter of minutes following i.p. preconditioning by low dose DOM in vivo. This was evidenced by severe seizure manifestations being almost absent in both young and aged animals, despite occurring frequently in naive animals. Therefore, this study concludes that tolerance is induced by kainate receptor ligands in vitro and in vivo within a matter of minutes, and is the result of a reduction in the turnover of G-protein coupled receptors and protein kinase activation. In addition, the increased sensitivity of aged rats to in vivo DOM is a result of elevated serum DOM concentrations most likely resulting from impaired renal clearance.

Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2007. / Includes bibliographical references (leaves 140-161). Also available in electronic version.