Multiple charged and aromatic residues in CCR5 amino-terminal domain are involved in high affinity binding of both chemokines and HIV-1 Env protein

CCR5 is a functional receptor for MIP-1 alpha, MIP-1 beta, RANTES (regulate d on activation normal T cell expressed), MCP-2, and MCP-4 and constitutes the main coreceptor for macrophage tropic human and simian immunodeficiency viruses. By using CCR5-CCR2b chimeras, we have shown previously that the s econd extracellular loop of CCR5 is the major determinant for chemokine bin ding specificity, whereas the amino-terminal domain plays a major role for human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency vir us coreceptor function. In the present work, by using a panel of truncation and alanine-scanning mutants, we investigated the role of specific residue s in the CCR5 amino-terminal domain for chemokine binding, functional respo nse to chemokines, HIV-1 gp120 binding, and coreceptor function. Truncation of the amino-terminal domain resulted in a progressive decrease of the bin ding affinity for chemokines, which correlated with a similar drop in funct ional responsiveness. Mutants lacking residues 2-13 exhibited fairly weak r esponses to high concentrations (500 nM) of RANTES or MIP-1 beta. Truncated mutants also exhibited a reduction in the binding affinity for R5 Env prot eins and coreceptor activity. Deletion of 4 or 12 residues resulted in a 50 or 80% decrease in coreceptor function, respectively. Alanine-scanning mut agenesis identified several charged and aromatic residues (Asp-2, Tyr-3, Ty r-10, Asp-11, and Glu-18) that played an important role in both chemokine a nd Env high affinity binding. The overlapping binding site of chemokines an d gp120 on the CCR5 amino terminus, as well as the involvement of these res idues in the epitopes of monoclonal antibodies, suggests that these regions are particularly exposed at the receptor surface.