Constitutively active CCR5 chemokine receptors differ in mediating HIV envelope-dependent fusion

The CCR5 chemokine receptor is a rhodopsin-like G protein-coupled receptor that mediates the effects of pro-inflammatory β-chemokines. CCR5 is also the major co-receptor for entry of human immunodeficiency virus (HIV) into human cells. G protein-coupled receptors exist in ensembles of active and inactive conformations. Active receptor conformations can be stabilized by mutations. Although binding of the HIV envelope protein to CCR5 stimulates cellular signaling, the CCR5 conformation that induces fusion of the viral membrane with cellular membranes is not known. We mutated conserved amino acids to generate constitutively active CCR5 receptors, which are stabilized in active conformations, and tested the ability of constitutively active CCR5 receptors to mediate HIV envelope-directed membrane fusion. Mutation of the Asp 3.49(125) and Arg 6.32(225) residues of CCR5 did not cause constitutive activity, but Lys or Pro substitutions for Thr 2.56(82) , in the TxP motif, caused high basal inositol phosphate signaling. Signaling did not increase in response to MIP-1β, suggesting that the Thr 2.56(82) mutants were fully stabilized in active conformations. The Thr 2.56(82) Lys mutation severely decreased cell surface CCR5 expression. Combining the Thr 2.56(82) Lys mutation with an Arg 6.32(225) Gln mutation partially reversed the decrease in expression. Mutants with Thr 2.56(82) Lys substitutions were poor mediators of HIV envelope-directed membrane fusion, but mutants with the Thr 2.65(82) Pro substitution exhibited full co-receptor function. Our results suggest that the Thr 2.65(82) Lys and Thr 2.65(82) Pro mutations stabilize distinct constitutively active CCR5 conformations. Lys in position 2.65(82) stabilizes activated receptor conformations that appear to be constitutively internalized and do not induce envelope-dependent membrane fusion, whereas Pro stabilizes activated conformations that are not constitutively internalized and fully mediate envelope-directed membrane fusion.