Liu, S.; Chen, Y.; Xu, R.-G.; Zhang, H.; Mostafa, F.; Liu, L.

Integrins are heterodimeric transmembrane receptors that mediate cell-cell and cell-extracellular matrix interactions and play essential roles in development and disease. Within the PS2 alpha-integrin subfamily, four paralogs (alphaIIb, alpha5, alpha8, and alphaV) share a conserved RGD-binding motif yet exhibit diverse functional specializations. Integrins have been widely targeted therapeutically for various clinical conditions, though achieving subtype specificity remains a major challenge. Here, we performed an integrative evolutionary analysis of 114 PS2 alpha-integrin sequences across 28 vertebrate species, combining phylogenetic reconstruction, time calibration, ancestral sequence inference, and structural mapping. Our time-calibrated phylogeny indicates that the PS2 lineage originated ~862 Mya, with diversification of the four paralogs occurring prior to vertebrate radiation. Ancestral state reconstruction reveals that fibronectin and vitronectin binding are ancestral traits, whereas fibrinogen binding and beta3 pairing arose independently in the alphaIIb and alphaV lineage. Evolutionary rate analysis shows domain-specific divergence, with the beta-propeller acting as a hotspot of evolutionary change, likely driven by combined pressures from ligand binding and beta-subunit interaction. These pressures vary across paralogs: alphaIIb exhibits accelerated evolution in ligand-binding regions, while alphaV displays elevated rates in beta-subunit interaction domains. Mapping sequence variation onto structural interfaces identifies lineage-specific substitutions underlying functional divergence, including distinct molecular solutions for fibrinogen binding in alphaIIb and alphaV. These findings collectively demonstrate that PS2 alpha-integrins evolved from a generalist ancestor through neofunctionalization and lineage-specific specialization. This work provides an evolutionary framework for identifying subtype-specific functional sites and highlights the potential of evolution-informed strategies to guide the development of more selective integrin-targeting therapeutics.