Extracellular cysteines of CCR5 are required for chemokine binding, but dispensable for HIV-1 coreceptor activity.
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 Cys20, which is thought to form a disulfide bond with Cys269, 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 Cys101 or Cys178, 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 degrees C. All cysteine mutants were unable to bind detectable levels of MIP-1beta, 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.
Blanpain
C
C
Institut de Recherche Interdisciplinaire, Université Libre de Bruxelles, Campus Erasme, 808 route de Lennik, B-1070 Bruxelles, Belgium.
Lee
B
B
Vakili
J
J
Doranz
B J
BJ
Govaerts
C
C
Migeotte
I
I
Sharron
M
M
Dupriez
V
V
Vassart
G
G
Doms
R W
RW
Parmentier
M
M
D019943:Amino Acid Substitution; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D054407:Chemokine CCL4; D018925:Chemokines / Q000378:metabolism*; D006224:Cricetinae; D003545:Cysteine / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D019402:Macrophage Inflammatory Proteins / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D019713:Receptors, CCR5 / Q000235:genetics / Q000378:metabolism*
D054407:Chemokine CCL4; D018925:Chemokines; D004220:Disulfides; D008024:Ligands; D019402:Macrophage Inflammatory Proteins; D019713:Receptors, CCR5; D003545:Cysteine