Date of Award

2001

Embargo Period

8-1-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular and Cellular Biology and Pathobiology

College

College of Graduate Studies

First Advisor

Steven A. Rosenzweig

Second Advisor

Kathryn E. Meier

Third Advisor

Stephen M. Lanier

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»GTPγS). The regions of AGS3 that bound Giα 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 Giα. AGS3-GPR domains selectively interacted with Giα in tissue lysates and with purified Giα/Gtα. The GPR domain of AGS3 containing four GPR motifs simultaneously bound more than one Giα. The AGS3-GPR domains competed with Gβy for binding to GtαGDP and blocked GTPγS binding to Giα. 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. Gα 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.

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