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Signal."],"published-print":{"date-parts":[[2026,10,13]]},"abstract":"<jats:p>Chemokine receptors are G protein\u2013coupled receptors (GPCRs) that drive cell migration through interactions with chemokines. Even in the absence of ligand, chemokine receptors spend a portion of their time in active conformational states, rendering them constitutively active. Here, we identified and characterized two mutations in the third intracellular loop of atypical chemokine receptor 3 (ACKR3) that inhibited or promoted its constitutive activity as assessed by ligand-independent \u03b2-arrestin recruitment and receptor internalization. Single-molecule fluorescence resonance energy transfer experiments showed that these effects on activity corresponded to changes in the conformational equilibrium of the receptor, which also affected the kinetics of chemokine binding. The potencies of chemokines and small-molecule agonists for \u03b2-arrestin recruitment were differentially affected by the mutations, showing that the effects of an altered receptor conformational equilibrium on receptor activation were ligand dependent. 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