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Title	Structural and functional characterization of the activator of G-protein signaling 3 (AGS3)
Author	Bernard, Michael LaBranche
Date	2001
Affiliation/College	College of Graduate Studies
Department	Molecular and Cellular Biology and Pathobiology
Extent	xiv, 187 leaves : ill.; 29 cm
Source	QU 55 B521s 2001
Subject.MeSH	Academic Dissertations Caenorhabditis elegans G-Proteins--analysis
Collection	MUSC Theses and Dissertations
Contributing Institution	Waring Historical Library (MUSC)
Rights	Copyright is held by the author.
Language	English
Type	Text
Format	application/pdf
Media Type	Manuscripts
Resource Identifier	mtd2001_bernard_ml
Abstract	AGS3 (Activator of G-protein Signaling 3) was isolated in yeast-based functional screen for receptor-independent activators of heterotrimeric G-proteins. To examine the role of AGS3 in mammalian signal processing, we defined the AGS3 sub domains involved in G-protein interaction, its selectivity for G-proteins and its influence on the activation state of G-protein. AGS3 co-immunoprecipitated with Gia3 from tissue lysates in a nucleotide dependent manner (GDP»GTPyS). The regions of AGS3 that bound Gin were localized to 4 thirty amino acid repeats (GPR - G-protein regulatory motif) in the carboxyl terminus (P463-S650), each of which were capable of binding Gin. AGS3-GPR domains selectively interacted with Gin in tissue lysates and with purified Gin/Gtn. The GPR domain of AGS3 containing four GPR motifs simultaneously bound more than one Gia. The AGS3-GPR domains competed with Gf3y for binding to GtaGDP and blocked GTPyS binding to Gial. When added to reconstituted receptor/Gprotein complex, AGS3-GPR disrupted the high affinity state of the receptor. From N-terminus to C-terminus, AGS3 contains a region of seven Tetratricopeptide repeats (TPRs), a one hundred amino acid linker region and the GPR domain. Using the TPR and linker region as bait, screening a mouse II-day old embryonic library using the yeast two-hybrid method yielded the C-terminal 107 aminoacids (D330-Q436) of a serine/theronine kinase, S/TKII. A GST-S/TKII (D330-Q436) fusion protein interacted with AGS3 from rat brain lysate. Ga subunits were also detected in this complex in a nucleotide dependent manner. To uncover the role of AGS3 in the intact organism we analyzed the function and expression profile of the AGS3 homolog in Caenorhabditus elegans. The pattern of LacZ staining indicated a strictly neuronal expression pattern for AGS3-CE. The expression in sub-adults appeared more widespread compared to that in adults. Using double stranded RNA corresponding to the coding region of the predicted AGS3-CE we performed RNA interference (RNAi). Injection of double stranded RNA into sub-adult worms generated a phenotype with nearly 85% penetrance. Offspring of dsRNA injected worms arrested in an early stage of embryogenesis.

Structural and functional characterization of the activator of G-protein signaling 3 (AGS3)

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