Biochemical Analysis of the Regulatory Role of Gαo in the Conformational Transitions of Drosophila Pins

Abstract

Drosophila Pins (and its mammalian homologue LGN) play a crucial role in the process of asymmetric cell division (ACD). Extensive research has established that Pins/LGN functions as a conformational switch primarily through intramolecular interactions involving the N-terminal TPR repeats and the C-terminal GoLoco (GL) motifs. The GL motifs served as binding sites for the α subunit of the trimeric G protein (Gα), which facilitates the release of the autoinhibited conformation of Pins/LGN. While LGN has been observed to specifically bind to Gα_i·GDP, Pins has been found to associate with both Drosophila Gα_i (dGα_i) and Gα_o (dGα_o) isoforms. Moreover, dGα_o was reported to be able to bind Pins in both the GDP- and GTP-bound forms. However, the precise mechanism underlying the influence of dGα_o on the conformational states of Pins remains unclear, despite extensive investigations into the Gα_i·GDP-mediated regulatory conformational changes in LGN/Pins. In this study, we conducted a comprehensive characterization of the interactions between Pins-GL motifs and dGα_o in both GDP- and GTP-loaded forms. Our findings reveal that Pins-GL specifically binds to GDP-loaded dGα_o. Through biochemical characterization, we determined that the intramolecular interactions of Pins primarily involve the entire TPR domain and the GL23 motifs. Additionally, we observed that Pins can simultaneously bind three molecules of dGα_o·GDP, leading to a partial opening of the autoinhibited conformation. Furthermore, our study presents evidence contrasting with previous observations indicating the absence of binding between dGα_i and Pins-GLs, thus implying the pivotal role of dGα_o as the principal participant in the ACD pathway associated with Pins.