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Characterization of beta-arrestin-mo...

Duke University.

Characterization of beta-arrestin-modulated lipid kinase activities for diacylglycerol and phosphatidylinositol 4-phosphate.

Record Type:	Electronic resources : Monograph/item
Title/Author:	Characterization of beta-arrestin-modulated lipid kinase activities for diacylglycerol and phosphatidylinositol 4-phosphate.
Author:	Nelson, Christopher David.
Description:	195 p.
Notes:	Adviser: Robert J. Lefkowitz.
Notes:	Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1621.
Contained By:	Dissertation Abstracts International68-03B.
Subject:	Biology, Cell.
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3255594

Characterization of beta-arrestin-modulated lipid kinase activities for diacylglycerol and phosphatidylinositol 4-phosphate.
Nelson, Christopher David.

Characterization of beta-arrestin-modulated lipid kinase activities for diacylglycerol and phosphatidylinositol 4-phosphate. - 195 p.

Adviser: Robert J. Lefkowitz.

Thesis (Ph.D.)--Duke University, 2007.

Phosphatidic acid is an effector for several enzymes, including the phosphatidylinositol 5-kinases (PIP5K), which phosphorylate PIP to make PIP2. Thus, we hypothesized beta-arrestin-targeted DGKs may regulate PIP5K activity. PIP5K Ialpha associated with beta-arrestin2 in an agonist-dependent manner in HEK293 cells, and a beta-arrestin2 mutant defective in receptor endocytosis (a PIP2-dependent function) was impaired. Furthermore, knockdown of beta-arrestin2 by RNAi significantly decreased the amount of PIP5K Ialpha detected in receptor immunoprecipitates. In TLC assays, overexpressing both beta-arrestin2 and PIP5K Ialpha enhanced agonist-stimulated PIP2 labeling, while either protein alone had no effect. These data support the concept of beta-arrestin binding to 7TMRs and enriching local membrane concentrations of PA, which then stimulates production of PIP2, promoting receptor internalization.Subjects--Topical Terms:

226967
Biology, Cell.

Characterization of beta-arrestin-modulated lipid kinase activities for diacylglycerol and phosphatidylinositol 4-phosphate.
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100 0 $a Nelson, Christopher David. $3 264358
245 1 0 $a Characterization of beta-arrestin-modulated lipid kinase activities for diacylglycerol and phosphatidylinositol 4-phosphate.
300 $a 195 p.
500 $a Adviser: Robert J. Lefkowitz.
500 $a Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1621.
502 $a Thesis (Ph.D.)--Duke University, 2007.
520 # $a Phosphatidic acid is an effector for several enzymes, including the phosphatidylinositol 5-kinases (PIP5K), which phosphorylate PIP to make PIP2. Thus, we hypothesized beta-arrestin-targeted DGKs may regulate PIP5K activity. PIP5K Ialpha associated with beta-arrestin2 in an agonist-dependent manner in HEK293 cells, and a beta-arrestin2 mutant defective in receptor endocytosis (a PIP2-dependent function) was impaired. Furthermore, knockdown of beta-arrestin2 by RNAi significantly decreased the amount of PIP5K Ialpha detected in receptor immunoprecipitates. In TLC assays, overexpressing both beta-arrestin2 and PIP5K Ialpha enhanced agonist-stimulated PIP2 labeling, while either protein alone had no effect. These data support the concept of beta-arrestin binding to 7TMRs and enriching local membrane concentrations of PA, which then stimulates production of PIP2, promoting receptor internalization.
520 # $a 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, beta-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 beta-arrestins recruiting PDE4 phosphodiesterase to Gs-coupled beta 2-adrenergic receptors, enhancing metabolism of the second messenger cAMP. As beta-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.
520 # $a We screened for beta-arrestin-binding proteins among modulators of diacylglycerol and IP3 (second messengers downstream of Gq-coupled 7TMRs). We observed beta-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 beta-arrestin enhanced phosphatidic acid (PA) production after M1 muscarinic receptor stimulation. Conversely, depletion of beta-arrestins by RNA interference showed significantly decreased agonist-stimulated PA accumulation. Additionally, overexpression of a beta-arrestin2 mutant that binds DGKs but not receptors served as a dominant negative for agonist-dependent DGK activity. These results demonstrate a requirement for beta-arrestins in DGK translocation to the membrane, and specifically to activated 7TMRs, where concentrations of second messengers are at their highest.
590 $a School code: 0066.
650 # 0 $a Biology, Cell. $3 226967
650 # 0 $a Chemistry, Biochemistry. $3 226900
690 $a 0379
690 $a 0487
710 0 # $a Duke University. $3 226880
773 0 # $g 68-03B. $t Dissertation Abstracts International
790 $a 0066
790 1 0 $a Lefkowitz, Robert J., $e advisor
791 $a Ph.D.
792 $a 2007
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