Cooperative actions of Tbc1d1 and AS160/Tbc1d4 in GLUT4-trafficking activities

AS160 and Tbc1d1 are key Rab GTPase-activating proteins (RabGAPs) that mediate release of static GLUT4 in response to insulin or exercise-mimetic stimuli, respectively, but their cooperative regulation and its underlying mechanisms remain unclear. By employing GLUT4 nanometry with cell-based reconstitution models, we herein analyzed the functional cooperative activities of the RabGAPs. When both RabGAPs are present, Tbc1d1 functionally dominates AS160, and stimuli-inducible GLUT4 release relies on Tbc1d1-evoking proximal stimuli, such as AICAR and intracellular Ca²⁺ Detailed functional assessments with varying expression ratios revealed that AS160 modulates sensitivity to external stimuli in Tbc1d1-mediated GLUT4 release. For example, Tbc1d1-governed GLUT4 release triggered by Ca²⁺ plus insulin occurred more efficiently than that in cells with little or no AS160. Series of mutational analyses revealed that these synergizing actions rely on the phosphotyrosine-binding 1 (PTB1) and calmodulin-binding domains of Tbc1d1 as well as key phosphorylation sites of both AS160 (Thr⁶⁴²) and Tbc1d1 (Ser²³⁷ and Thr⁵⁹⁶). Thus, the emerging cooperative governance relying on the multiple regulatory nodes of both Tbc1d1 and AS160, functioning together, plays a key role in properly deciphering biochemical signals into a physical GLUT4 release process in response to insulin, exercise, and the two in combination.