Extracellular Cysteines of CCR5 Are Required for Chemokine Binding, but Dispensable for HIV-1 Coreceptor Activity

• Published in: The Journal of biological chemistry Vol. 274; no. 27; pp. 18902 - 18908
• Main Authors: Blanpain, C, Lee, B, Vakili, J, Doranz, B J, Govaerts, C, Migeotte, I, Sharron, M, Dupriez, V, Vassart, G, Doms, R W, Parmentier, M
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 02-07-1999
• Subjects: Amino Acid Substitution; Animals; Cell Line; Chemokine CCL4; Chemokines - metabolism; CHO Cells; Cricetinae; Cysteine - metabolism; Disulfides - metabolism; HIV-1 - metabolism; Human immunodeficiency virus 1; Humans; Ligands; Macrophage Inflammatory Proteins - metabolism; Models, Molecular; Mutagenesis, Site-Directed; Protein Conformation; Receptors, CCR5 - genetics; Receptors, CCR5 - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.274.27.18902

CCR5 is the major coreceptor for macrophage-tropic human immunodeficiency virus type I (HIV-1). For most G-protein-coupled
receptors that have been tested so far, the disulfide bonds linking together the extracellular loops (ECL) are required for
maintaining the structural integrity necessary for ligand binding and receptor activation. A natural mutation affecting Cys 20 , which is thought to form a disulfide bond with Cys 269 , has been described in various human populations, although the consequences of this mutation for CCR5 function are not known.
Using site-directed mutagenesis, we mutated the four extracellular cysteines of CCR5 singly or in combination to investigate
their role in maintaining the structural conformation of the receptor, its ligand binding and signal transduction properties,
and its ability to function as a viral coreceptor. Alanine substitution of any single Cys residue reduced surface expression
levels by 40–70%. However, mutation of Cys 101 or Cys 178 , predicted to link ECL1 and ECL2 of the receptor, abolished recognition of CCR5 by a panel of conformation sensitive anti-CCR5
antibodies. The effects of the mutations on receptor expression and conformation were partially temperature-sensitive, with
partial restoration of receptor expression and conformation achieved by incubating cells at 32 °C. All cysteine mutants were
unable to bind detectable levels of MIP-1β, and did not respond functionally to CCR5 agonists. Surprisingly, all cysteine
mutants did support infection by R5 strains of HIV, though at reduced levels. These results indicate that both disulfide bonds
of CCR5 are necessary for maintaining the structural integrity of the receptor necessary for ligand binding and signaling.
Env binding and the mechanisms of HIV entry appear much less sensitive to alterations of CCR5 conformation.