Abstract
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 Asp3.49(125) and Arg6.32(225) residues of CCR5 did not cause constitutive activity, but Lys or Pro substitutions for Thr2.56(82), in the TxP motif, caused high basal inositol phosphate signaling. Signaling did not increase in response to MIP-1β, suggesting that the Thr2.56(82) mutants were fully stabilized in active conformations. The Thr2.56(82)Lys mutation severely decreased cell surface CCR5 expression. Combining the Thr2.56(82)Lys mutation with an Arg6.32(225)Gln mutation partially reversed the decrease in expression. Mutants with Thr2.56(82)Lys substitutions were poor mediators of HIV envelope-directed membrane fusion, but mutants with the Thr2.65(82)Pro substitution exhibited full co-receptor function. Our results suggest that the Thr2.65(82)Lys and Thr2.65(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.
Highlights
The CCR5 chemokine receptor is a G protein-coupled receptor (GPCR) that mediates leukocyte chemotaxis and recruitment to sites of inflammation in response to pro-inflammatory bchemokines, including macrophage inflammatory protein 1b (MIP-1b, CCL4) [1,2]
GPCR proteins exist in ensembles of inactive conformations, which are stabilized by inverse agonists and do not support intracellular signaling, and active receptor conformations, which are stabilized by agonists and activate corresponding ensembles of cellular signaling pathways
Our results suggest that Pro and Lys substitutions in position 2.56(82) stabilize distinct activated CCR5 conformations that differ in their localization at the cell surface and in their ability to induce human immunodeficiency virus (HIV) envelope glycoprotein (Env)-dependent membrane fusion
Summary
The CCR5 chemokine receptor is a G protein-coupled receptor (GPCR) that mediates leukocyte chemotaxis and recruitment to sites of inflammation in response to pro-inflammatory bchemokines, including macrophage inflammatory protein 1b (MIP-1b, CCL4) [1,2]. GPCR proteins exist in ensembles of inactive conformations, which are stabilized by inverse agonists and do not support intracellular signaling, and active receptor conformations, which are stabilized by agonists and activate corresponding ensembles of cellular signaling pathways. Ligands may selectively stabilize ensembles of receptor conformations that activate subsets of cellular signaling pathways [3,4]. Chemokines stabilize CCR5 receptor conformations that activate G protein signaling and conformations that are recognized by G proteincoupled receptor kinases and arrestins, which promote receptor internalization. Some chemokine ligands have distinct efficacies for stimulating intracellular signaling and internalization of CCR5 [5]. HIV binding to CCR5 must stabilize a receptor conformation that induces the fusion conformation of Env. HIV stimulates CCR5-dependent cellular signaling [6,7,8]
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