Short Linear Motifs as a General Organizing Principle of the Nuclear-Receptor Proximal Interactome

The AR-interactome comprises ∼1,000 androgen receptor-interacting proteins (AR-IPs), yet how a single receptor engages so many partners across compartments remains mechanistically unclear. We integrate proximity-labeling quantitative mass spectrometry across cytosolic, microsomal, and nuclear compartments of LNCaP prostate cancer cells and resolve 4,751 AR-proximal interacting proteins (AR-PIPs), more than four times the size of the AR-interactome. Anchoring on the LXXLL coactivator recognition motif, LXXLL motifs are systematically depleted in AR-PIPs after length control, consistent with low-affinity, transient engagement at the AR AF-2 charge clamp. LXXLL-bearing AR-PIPs include AR itself and canonical AR coactivators altered by amplification, deletion, or motif-spanning mutations in metastatic and castration-resistant prostate cancers. AR-V7, which lacks AF-2, retains LXXLL-depleted Mode 1 partners and loses the LXXLL-enriched Mode 2 cloud, thereby validating a two-mode engagement framework for nuclear receptor-proximal interactomes.