Distribution of Activator of G-Protein Signaling 3 within the Aggresomal Pathway: Role of Specific Residues in the Tetratricopeptide Repeat Domain and Differential Regulation by the AGS3 Binding Partners Giα and Mammalian Inscuteable

Abstract

AGS3, a receptor-independent activator of G-protein signaling, is involved in unexpected functional diversity for G-protein signaling systems. AGS3 has seven tetratricopeptide (TPR) motifs upstream of four G-protein regulatory (GPR) motifs that serve as docking sites for Gi␣-GDP. The positioning of AGS3 within the cell and the intramolecular dynamics between different domains of the proteins are likely key determinants of their ability to influence G-protein signaling. We report that AGS3 enters into the aggresome pathway and that distribution of the protein is regulated by the AGS3 binding partners Gi␣ and mammalian Inscuteable (mInsc). Gi␣ rescues AGS3 from the aggresome, whereas mInsc augments the aggresome-like distribution of AGS3. The distribution of AGS3 to the aggresome is dependent upon the TPR domain, and it is accelerated by disruption of the TPR organizational structure or introduction of a nonsynonymous single-nucleotide polymorphism. These data present AGS3, G-proteins, and mInsc as candidate proteins involved in regulating cellular stress associated with protein-processing pathologies.The discovery of AGS3 (GPSM1) and related accessory proteins revealed unexpected functional diversity for G-protein signaling systems (8,36). AGS3 is involved in a number of different cellular activities, including asymmetric cell division during neuronal development (30), neuronal plasticity and addiction (9, 10, 12, 38, 39), autophagy (27), membrane protein trafficking (17), cardiovascular function (7), and metabolism (7). AGS3 is a multidomain protein consisting of seven tetratricopeptide repeats (TPR) in the amino-terminal portion of the protein and four G-protein regulatory (GPR) motifs in the carboxyl region of the protein. Each of the GPR motifs binds and stabilizes the GDP-bound conformation of G␣ (Gi␣, Gt␣, and Gi/o␣), essentially behaving as a guanine nucleotide dissociation inhibitor. As such, AGS3 may be complexed with up to four G␣ and function as an alternative binding partner for G␣ independently of the classical heterotrimeric G␣␤␥. Despite the clearly demonstrated function of AGS3 and the related protein LGN (GPSM2 or AGS5) in various model organisms and a fairly solid, basic biochemical understanding of the interaction of a GPR motif with G␣, the signals that operate "upstream" and/or "downstream" of AGS3 or an AGS3-Gi/o␣ complex are not well defined.AGS3 and other GPR proteins may regulate G-protein signaling directly by influencing the interaction of G␣ with G␤␥ or another G␣ binding partner. In addition, a portion of G␣ in the cell is complexed with GPR proteins to various degrees, and this interaction is regulated. Ric-8A interacts with an AGS3-Gi␣ complex in a manner somewhat analogous to the interaction of a G-protein-coupled receptor with heterotrimeric G␣␤␥, promoting nucleotide exchange and the apparent dissociation of AGS3 and Gi␣-GDP (37). The specific impact of AGS3 and other GPR proteins on signaling events is likely dependent upon where the individual protein is positioned within the c...

Direct Coupling of a Seven-Transmembrane-Span Receptor to a Gαi G-Protein Regulatory Motif Complex

“…B. Blumer, unpublished data). Additional signals regulating the interaction of Ga with GPR proteins and the subcellular distribution of GPR proteins may also be involved and may provide more rapid and dynamic control of cellular responses (Blumer et al, 2003;An et al, 2008;Nadella et al, 2010;Oner et al, 2010aOner et al, ,b, 2013cVural et al, 2010;Giguere et al, 2012). Finally, of particular interest, the coupling of a receptor to the GaGPR complex or the Gabg heterotrimer may be differentially regulated by hormones, neurotransmitters, and small molecules.…”

Translocation of Activator of G-protein Signaling 3 to the Golgi Apparatus in Response to Receptor Activation and Its Effect on the trans-Golgi Network

“…Cells were then processed for imaging 24 h later as described elsewhere (38). Primary antibodies were used at a dilution of 1:200 in cell washing solution (137 mM NaCl, 2.6 mM KCl, 1.8 mM KH 2 PO 4 , 10 mM Na 2 HPO 4 , pH 7.4), and secondary antibodies (goat anti-rabbit AlexaFluor488 or mouse AlexaFluor594, highly cross-adsorbed, Molecular Probes) were used at a dilution of 1:2000 in cell washing solution.…”

“…The nucleus was stained with 1 g/ml DAPI at the last washing step. Slides were then mounted with glass coverslips and analyzed with a Leica CTR5500 deconvolution fluorescence microscope using a 40ϫ or 63ϫ oil immersion objective as described elsewhere (38). All images were obtained from approximately the middle plane of the cells.…”

Activators of G Protein Signaling Exhibit Broad Functionality and Define a Distinct Core Signaling Triad

“…1). In mammalian systems, AGS3 is implicated in asymmetric cell division, neuronal plasticity and addiction, autophagy, membrane protein trafficking, polycystic kidney disease, renal response to ischemia, immune cell chemotaxis, insulin-like growth factor-1 mediated ciliary resorption, cardiovascular regulation, and metabolism (Blumer et al, 2002(Blumer et al, , 2006(Blumer et al, , 2008Ghosh et al, 2003;Gotta et al, 2003;Pattingre et al, 2003;Bowers et al, 2004Bowers et al, , 2008Sato et al, 2004;Yao et al, 2005Yao et al, , 2006Groves et al, 2007;Willard et al, 2008;Groves et al, 2010;Nadella et al, 2010;Vural et al, 2010;Hofler and Koelle, 2011;Regner et al, 2011;Chauhan et al, 2012;Kwon et al, 2012;Conley and Watts, 2013;Kamakura et al, 2013;Yeh et al, 2013). AGS3 single nucleotide polymorphisms are associated with diabetes and glucose handling (Scott et al, 2012;Hara et al, 2014;Huyghe et al, 2013).…”

“…2A), function as part of a distinct signaling pathway (e.g., GaGPR signaling module) (Fig. 2B), serve as a chaperone for Ga, and/or impact basic cellular events such as autophagy (Pattingre et al, 2003;Groves et al, 2010;Vural et al, 2010;Garcia-Marcos et al, 2011a) and secretory pathway dynamics (Groves et al, 2007;Oner et al, 2013b).…”