Constitutively active CCR5 chemokine receptors differ in mediating HIV envelope-dependent fusion.
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.
Medical Research Council Receptor Biology Research Unit, Division of Medical Biochemistry, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa.
Alex
A
de Voux
Mei-Chi
MC
Chan
Asongna T
AT
Folefoc
Michael T
MT
Madziva
Colleen A
CA
Flanagan
D019943:Amino Acid Substitution; D057809:HEK293 Cells; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D020125:Mutation, Missense; D019713:Receptors, CCR5 / Q000235:genetics / Q000378:metabolism*; D053586:Virus Internalization; D054299:env Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism*
D019713:Receptors, CCR5; D054299:env Gene Products, Human Immunodeficiency Virus