Abstract
The Gi family of G protein‐coupled receptors (GPCRs) drives various cellular processes to regulate physiological functions that encompass neuronal, immune, and endocrine health. Agonist‐bound GPCRS result in the activation of the heterotrimeric G protein, leading to the dissociation of the Gα subunit from the Gβγ dimer. While Gβγ has been considered to be a main driver of signaling downstream of Gi‐coupled receptors, the lack of tools to inhibit Gαi subunits without disrupting Gβγ signaling has limited our appreciation of potential novel pathways downstream of Gαi. To identify new Gαi interaction partners, our laboratory adopted an unbiased proximity‐based approach where we fused BioID2, a biotin ligase, within a specific site of the helical domain of the inactive Gαi1(Gαi‐BioID2) and the constitutively active Gαi1 (Gαi1‐Q204L‐BioID2). Using mass spectrometry, we specifically looked for targets enriched in Gαi1‐Q204L‐BioID2 samples. Among 104 targets, the activity‐dependent neuroprotective protein (ADNP) was found to be the most enriched target enriched in active Gαi1 in two separate screens. There is no previously identified relationship between Gαi and ADNP.ADNP is a transcription factor and cytoskeletal binding protein involved in brain formation and maturation. To investigate the interaction of ADNP with active Gαi1, HEK293 cells were transfected with ADNP alone, ADNP with Gαi1‐Q204L‐BioID2, and ADNP with Gαi1‐BioID2 and supplemented with biotin for 24 hours. Cells were lysed, and streptavidin beads were used to capture biotinylated proteins. Using immunoblotting, we confirmed the selective enrichment of ADNP biotinylation by Gαi1‐Q204L‐BioID2.To further determine whether ADNP interacts with Gαi1, we used a nanoluciferase‐based complementation assay where we co‐transfected HEK cells with ADNP with Gαi1 WT and Gαi1 QL respectively. In this assay, significantly greater interaction between ADNP and Gαi1 QL was observed when compared to Gαi1 WT and ADNP.Next, we addressed the subcellular localization of ADNP and Gαi1. Using cell fractionation, we observed that ADNP is localized to the nucleus of HEK cells while Gαi1 immunoreactivity was found in a cytosolic and membrane fraction, and in the nuclear fraction. Gαi1 expression does not alter the localization of ADNP, but ADNP can be exported and imported through the nucleus. Current work is focused on manipulation of the cellular localization of ADNP to determine where in the cell Gαi1 interacts with ADNP. Overall, we have identified ADNP as a novel interactor of active Gαi. Considering the distinct localization and expression of ADNP at different neurodevelopmental stages, further characterization of this interaction may provide new insight into Gαi‐mediated cAMP‐independent signaling pathways in neurophysiological and pathological states.
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