Nuclear receptors engage a wide array of protein partners across cellular compartments. The canonical androgen receptor (AR) interactome had been cataloged at roughly 1,000 AR-interacting proteins (AR-IPs), but how a single receptor engages such a large and compartmentally distributed set of partners remained unclear. This study used proximity-labeling quantitative mass spectrometry to define an expanded, compartment-resolved AR-proximal interactome and to test whether short linear motifs organize receptor-proximal engagement.
The authors applied proximity-labeling coupled with quantitative mass spectrometry to LNCaP prostate cancer cells. Labeling experiments were resolved across three subcellular compartments: cytosolic, microsomal, and nuclear fractions. This compartmental approach was used to capture AR-proximal interacting proteins (AR-PIPs) that are present near the receptor in different cellular locations, producing a high-resolution proximal interaction landscape.
Across compartments, the authors resolved 4,751 AR-proximal interacting proteins (AR-PIPs)—a set more than four times larger than the previously described AR-interactome. This expanded proximal dataset includes many proteins beyond the canonical coactivators and core components previously associated with AR, reflecting the broad and compartment-specific environment surrounding AR in LNCaP cells.
To probe organizing principles within the AR-proximal cloud, the study anchored analyses on the LXXLL motif, a well-known nuclear receptor coactivator recognition sequence. After controlling for protein length, the authors observed a systematic depletion of LXXLL motifs among AR-PIPs. The depletion after length control is interpreted as consistent with low-affinity, transient engagement of LXXLL-containing sequences at the AR AF-2 charge clamp, rather than stable, high-affinity recruitment of many long LXXLL-bearing proteins.
A subset of AR-PIPs does contain LXXLL motifs; these include AR itself and canonical AR coactivators. The authors note that several of these LXXLL-bearing coactivators are altered in advanced disease—by amplification, deletion, or motif-spanning mutations—in metastatic and castration-resistant prostate cancer.
Based on motif distributions and compartmental patterns, the authors propose a two-mode engagement framework for nuclear receptor-proximal interactomes. In this model, one engagement mode comprises partners that are LXXLL-depleted (Mode 1), consistent with transient, low-affinity proximity, and a second mode (Mode 2) is LXXLL-enriched, reflecting interactions that depend on the AF-2 coactivator surface.
To test the model, the authors compared full-length AR to the splice variant AR-V7, which lacks the AF-2 surface. AR-V7 retained the Mode 1, LXXLL-depleted partners, but lost the LXXLL-enriched Mode 2 cloud. This differential retention and loss of partner sets in AR-V7 is presented as validation of the two-mode engagement framework for nuclear receptor proximal interactomes.
The expanded, compartment-resolved AR-proximal interactome and the motif-based interpretation have several implications:
This report is a bioRxiv preprint and has not been certified by peer review. The source does not provide peer-reviewed validation details or exhaustive methodological parameters in the abstract-level content presented here; readers should consult the full manuscript (and subsequent peer-reviewed versions) for detailed methods, statistical analyses, and validation experiments.
The work was funded in part by the National Institutes of Health (R01GM143399). Authors are affiliated with the University of Washington, National Jewish Health, and the University of Iowa. The authors declared no competing interests in the source report.