Cellular context and ligand class shape CXCR4-CCR5 heteromerization in live cells.

G protein-coupled receptors (GPCRs) can form heteromeric assemblies, yet whether class A GPCR heteromerization is dictated by cellular context remains unclear. Here we identify CXCR4-CCR5 heteromerization as a cell-dependent, cholesterol-sensitive, and ligand-regulated feature of live-cell membrane organization. In cancer-derived MDA-MB-231 cells, CXCR4 and CCR5 formed stable, slow-diffusing higher-order assemblies, whereas in COS7, HEK293, and MCF-10A cells the receptors were detected mainly as weaker monomer-dimer mixtures. Cholesterol depletion selectively reduced CXCR4-CCR5 heteromerization in MDA-MB-231 cells, implicating membrane composition as a major determinant of receptor assembly. Agonists induced transient heteromerization coupled to receptor internalization, while antagonists, especially plerixafor together with maraviroc, stabilized persistent surface-associated complexes. Molecular dynamics simulations in asymmetric bilayers resembling MDA-MB-231 and MCF-10A membranes identified cholesterol-enriched receptor interfaces that prolong CXCR4-CCR5 dimer lifetimes in MDA-like membranes. These results show that GPCR heteromerization is not an intrinsic fixed property of receptor pairs, but an emergent behavior shaped by cell state, lipid environment, and ligand input.