Structural survey of HIF-2α reveals regulation of its subcellular localization and protein interactome

Hypoxia‑inducible factor 2α (HIF‑2α) is a central regulator of cellular homeostasis and a known oncogenic driver in multiple cancers. Although HIF‑2α is canonically defined as a nuclear transcription factor, its cytoplasmic presence and potential non‑transcriptional functions remain poorly understood. Here, we dissect the structural determinants of HIF‑2α localization, stability, and activity using a deletion‑construct library, transcriptional assays, and in vivo xenograft models. We show that the oxygen‑dependent degradation domain (ODD), and N‑terminal intrinsically disordered region (IDR) and transactivation domain (NTAD) promote cytoplasmic localization, whereas the C‑terminal IDR drives nuclear accumulation. Notably, we demonstrate that HIF‑2α nuclear localization occurs independently of ARNT dimerization, resolving a long-standing question in the field. While both NTAD and C‑terminal (C)TAD contribute to transcriptional activation, only CTAD mediates hypoxia responsive element (HRE) transactivation, and neither domain is required for tumor growth in vivo. These data suggest a dominant role for non‑canonical cytoplasmic mechanisms in HIF‑2α-driven tumorigenesis. Proteomic analyses reveal HIF‑2α interactions with regulators of mitochondrial function, translation initiation, RNA splicing, vesicular transport, and DNA replication. Together, these findings uncover previously unrecognized structural and functional complexity of HIF‑2α and expand its role beyond canonical transcriptional regulation