Biochemical\nAnalysis of the Regulatory Role of Gα<sub>o</sub> in the Conformational\nTransitions of Drosophila Pins

Drosophila Pins (and its\nmammalian\nhomologue LGN) play a crucial role in the process of asymmetric cell\ndivision (ACD). Extensive research has established that Pins/LGN functions\nas a conformational switch primarily through intramolecular interactions\ninvolving the N-terminal TPR repeats and the C-terminal GoLoco (GL)\nmotifs. The GL motifs served as binding sites for the α subunit\nof the trimeric G protein (Gα), which facilitates the release\nof the autoinhibited conformation of Pins/LGN. While LGN has been\nobserved to specifically bind to Gα<sub>i</sub>·GDP, Pins\nhas been found to associate with both Drosophila Gα<sub>i</sub> (<i>d</i>Gα<sub>i</sub>) and\nGα<sub>o</sub> (<i>d</i>Gα<sub>o</sub>) isoforms.\nMoreover, <i>d</i>Gα<sub>o</sub> was reported to be\nable to bind Pins in both the GDP- and GTP-bound forms. However, the\nprecise mechanism underlying the influence of <i>d</i>Gα<sub>o</sub> on the conformational states of Pins remains unclear, despite\nextensive investigations into the Gα<sub>i</sub>·GDP-mediated\nregulatory conformational changes in LGN/Pins. In this study, we conducted\na comprehensive characterization of the interactions between Pins-GL\nmotifs and <i>d</i>Gα<sub>o</sub> in both GDP- and\nGTP-loaded forms. Our findings reveal that Pins-GL specifically binds\nto GDP-loaded <i>d</i>Gα<sub>o</sub>. Through biochemical\ncharacterization, we determined that the intramolecular interactions\nof Pins primarily involve the entire TPR domain and the GL23 motifs.\nAdditionally, we observed that Pins can simultaneously bind three\nmolecules of <i>d</i>Gα<sub>o</sub>·GDP, leading\nto a partial opening of the autoinhibited conformation. Furthermore,\nour study presents evidence contrasting with previous observations\nindicating the absence of binding between <i>d</i>Gα<sub>i</sub> and Pins-GLs, thus implying the pivotal role of <i>d</i>Gα<sub>o</sub> as the principal participant in the ACD pathway\nassociated with Pins.