Receptor-proximal phosphoform organization precedes β2 integrin activation

Abstract

Receptor signaling is often framed as protein recruitment after stimulation, but whether receptor-proximal environments are organized in composition and phosphorylation state beforehand remains unclear. We used CD18-APEX2 proximity labeling and a spatial membrane reference to quantify proteins and phosphosites near leukocyte {beta}2 integrins. At rest, receptors, adaptors and Rap GTPases were preferentially represented relative to several catalytic classes. Parent-protein correction revealed spatial phosphoform organization, with 32% of 4,564 phosphosites deviating significantly from the distribution center and 56% of proteins with multiple quantified sites showing divergence beyond measurement error. Sustained activation altered 31% of phosphosites while 95.9% of proteins remained equivalent within {+/-}0.5 log2 and spatial phosphoform organization largely persisted. Most phosphorylation responses were shared across both labeling environments, whereas receptor-preferential changes were dominated by phosphosites. These findings suggest that receptor signaling can regulate pre-existing molecular states without extensive protein redistribution.