Biomolecular Condensates defined by Receptor Independent Activator of G protein Signaling: Properties and Regulation

Abstract

Activator of G-protein Signaling 3 (AGS3), a receptor independent activator of G-protein signaling, oscillates among different subcellular compartments in a regulated manner including punctate entities referred to as biomolecular condensates (BMCs). The dynamics of the AGS3 oscillation and the specific subcompartment within the cell is intimately related to the functional diversity of the protein. 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 (oxidative, pHi, thermal) induced the formation of AGS3 BMCs in two cell lines (Hela, COS7) as determined by fluorescent microscopy. The AGS3-BMCs generated in response to oxidative stress were distinct from stress granules (SG) as defined by the SG marker protein G3BP1 and RNA processing BMCs defined by the P-body protein Dcp1a. Immunoblots of fractionated cell lysates indicated that cellular stress shifted AGS3 to the membrane pellet fraction, whereas the protein markers for stress granules (G3BP1) SG- BMCs remained in the supernatant. We next asked if the formation of the stress-induced AGS3 BMCs was regulated by protein binding partners involved with signal processing. The stress-induced generation of AGS3 BMCs was regulated by the signaling protein Gi3, but not by the AGS3 binding partner DVL2. Finally, we addressed the fluidity or rigidity of the stress-induced AGS3-BMCs using fluorescent recovery following photobleaching of individual AGS3-BMCs. The AGS3-BMCs indicated distinct diffusion kinetics that were consistent with restricted mobility of AGS3 within the stress-induced AGS3-BMCs. These data suggest that AGS3 BMCs represents a distinct class of stress granules that define a new type of BMC that may serve as previously unappreciated signal processing nodes. Summary statementAGS3 assembles into distinct biomolecular condensates in response to cell stress and this assembly is selectively regulated by AGS3 binding partners involved in signal transduction within the cell.