Biomolecular condensates defined by Activator of G protein Signaling 3 exhibit distinct properties and regulation

Abstract

<b>Abstract ID 52172</b> <b>Poster Board 528</b> Activator of G-protein Signaling 3 (AGS3), a receptor independent activator of G-protein signaling, consists of 7 tetratricopeptide repeats (TPR) upstream from 4 G-protein regulatory motifs (GPR), each of which interact with GalphaGDP. AGS3 oscillates among different subcellular compartments in a regulated manner, which is intimately related to the functional diversity of the protein. AGS3 also exhibits an inherent and apparently regulated propensity to form biomolecular condensates (BMCs). BMCs are non-membranous, micron-scale compartments involving liquid-liquid phase separation within the cell in a regulatable manner. BMCs provide a platform to coordinate the assembly of a specific subset of molecules sequestered from the rest of the cytoplasm and are engaged in a wide range of biological and chemical events within the cell. To further address the properties and regulation of AGS3 BMCs, we asked initial questions regarding a) the distribution of AGS3 across the broader BMC landscape with and without cellular stress, and b) the core material properties of these punctate structures. Cellular stress (arsenate treatment) induced the formation of distinct stress granule BMCs and AGS3 BMCs as determined by fluorescent microscopy in two cell lines (Hela, COS7). In contrast, AGS3 colocalized with processing (P) body BMCs and this colocalization was disrupted by cellular stress with the generation of distinct AGS3 BMCs. Immunoblots of fractionated cell lysates indicated that cellular stress shifted AGS3 to the membrane pellet fraction, whereas the protein markers for stress granule and P-body BMCs remained in the supernatant. These data suggest that AGS3 BMCs may define a distinct type of BMC. To further address this hypothesis, we characterized the AGS3 BMCs by a) analysis of cell lysates under non-reducing gel electrophoresis and b) through examination of AGS3 diffusion dynamics in specific BMCs using fluorescence recovery after photobleaching (FRAP). Stress-induced AGS3 BMCs migrated as higher order structures as determined by immunoblots following non-reducing gel electrophoresis and the stress-induced properties of AGS3 BMCs were reversed by co-expression of the AGS3 binding partner Gialpha3. Results from a series of FRAP experiments indicated differences in the diffusion dynamics of AGS3 in AGS3-DVL2 BMCs versus stress-induced AGS3 BMCs as well as AGS3 BMCs generated by changes in phosphorylation status. These data indicate that AGS3 BMCs, the formation of which is regulated by both cellular stress and the signaling proteins Gialpha3 and DVL2, define a new type of BMC that may serve as previously unappreciated signal processing nodes.