A Spatiotemporal Atlas of Extranuclear Androgen Receptor Proximal Interaction Networks

Androgen receptor-interacting proteins (AR-IPs) comprise nearly 1,000 proteins, yet their organization across subcellular space and time remains uncharted. Proximity labeling captures direct binding partners along with neighboring proteins that populate a receptors local environment, thereby broadening AR-IPs into a broader population of AR-proximal interacting proteins (AR-PIPs). Here, we apply proximity labeling quantitative mass spectrometry (PL-qMS) to construct a spatiotemporal atlas of the extranuclear AR-proximal interactome in LNCaP prostate tumor cells. PL-qMS recovered 82.2% of the known AR-IPs and identified 3,947 AR-PIPs across cytosolic and membrane compartments, revealing dynamic remodeling across an androgen time course. Functional enrichment and network analyses identified the retromer complex as an androgen-sensitive AR-proximal interaction, which was verified by proximity ligation assays. Partial genetic disruption of VPS26A attenuated transcription of canonical androgen-regulated genes by mislocalizing the AR coactivator TMF1, establishing the retromer-AR-TMF1 axis as a functionally validated AR-proximal interaction network (AR-PIN). This work establishes subcellular proximal proteomes as a spatiotemporal framework for probing AR function and its dysregulation in disease. SynopsisProximity labeling constructs a spatiotemporal atlas of the extranuclear AR-proximal interactome and identifies the retromer complex as an androgen-sensitive regulator of AR transcription. O_LIPL-qMS constructs a spatiotemporal atlas of the extranuclear AR-proximal interactome C_LIO_LIAR-PIPs recover 3,947 proximal interactors across cytosolic and membrane fractions C_LIO_LIRetromer complex is an androgen-sensitive AR-proximal interaction C_LIO_LIPartial VPS26A disruption attenuates AR-dependent gene transcription via TMF1 mislocalization C_LI