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 v irus type I (HIV-1). For most G-protein-coupled receptors that have been te sted so far, the disulfide bonds linking together the extracellular loops ( ECL) are required for maintaining the structural integrity necessary for li gand binding and receptor activation, A natural mutation affecting Cys(20), which is thought to form a disulfide bond with Cys(269), has been describe d in various human populations, although the consequences of this mutation for CCR5 function are not known. Using site-directed mutagenesis, we mutate d the four extracellular cysteines of CCR5 singly or in combination to inve stigate their role in maintaining the structural conformation of the recept or, its ligand binding and signal transduction properties, and its ability 60 function as a viral coreceptor. Alanine substitution of any single Cys r esidue reduced surface expression levels by 40-70%. However, mutation of Cy s(101) or Cys(178), predicted to link ECL1 and ECL2 of the receptor, abolis hed recognition of CCR5 by a panel of conformation sensitive anti-CCR5 anti bodies. The effects of the mutations on receptor expression and conformatio n were partially temperature-sensitive, with partial restoration of recepto r expression and conformation achieved by incubating cells at 32 degrees C. All cysteine mutants were unable 60 bind detectable levels of MIP-1 beta, and did not respond functionally to CCR5 agonists. Surprisingly, all cystei ne mutants did support infection by R5 strains of HIV, though at reduced le vels. These results indicate that both disulfide bonds of CCR5 are necessar y for maintaining the structural integrity of the receptor necessary for li gand binding and signaling. Env binding and the mechanisms of HIV entry app ear much less sensitive to alterations of CCR5 conformation.