Ed-receptor conformations that differ in their interactions with HIV Env protein. In summary, we generated four CCR5 mutants that constitutively activate IP signaling. The Thr2.56(82)Pro and Thr2.56(82)Pro/Arg6.32(225)Gln mutants, which were expressed at levels similar to the wild type receptor in HEK 293 cells, the Thr2.56(82)Lys mutant, which was poorly expressed, and the double mutant, Thr2.56(82)Lys/Arg6.32(225)Gln, which showed enhanced expression relative to the Thr2.56(82)Lys mutant. Constitutively active mutants with Lys in position 82 showed very low fusion efficiency, but mutants with Pro in position 82 showed good fusion efficiency that was comparable to the wild type receptor.DiscussionWe have investigated the ability of get Gracillin activated CCR5 conformations to mediate HIV Env-directed membrane fusion by generating constitutively active mutant CCR5 receptors. Chargeneutralizing substitutions for Asp3.49(125) in the DRY motif and substitutions of the naturally occurring Arg6.32(225)Gln mutation of CCR5 did not increase constitutive activation of IP signaling. However, substitution of the Thr2.56(82) residue of the TxP motif caused high levels of ligand-independent cellular signaling. The Thr2.56(82)Lys mutation also decreased cell surface CCR5 protein. Severely decreased expression of mutants with Lys, but not Pro, in position 82 suggests that the conformations of the constitutively active mutant receptors differ. Mutant CCR5 receptors with Lys 18325633 in position 82, which constitutively activated IP signaling, were poor mediators of Env-directed membrane fusion, suggesting that HIV might not enter cells via the activated receptor conformation. However, constitutively active receptors with Pro substituted into the TxP motif mediated Env-directed membrane fusion very efficiently. The differential effects on receptor expression and membrane fusion suggest that Lys and Pro substitutions in position 82 stabilize distinct activated conformations of CCR5 that vary in their ability to mediate Env-dependent membrane fusion. Constitutively active GPCR mutants are defined by increased ligand-independent (basal) signaling activity. The increased signaling results from an increased population of activated receptor conformations by mutant receptors. Many constitutively active mutants exhibit decreased cell surface expression, whichConstitutively Active CCR5 Receptor ConformationsFigure 2. IP production, expression and competition binding of CCR5 receptors with mutations of Thr2.56(82) and Arg6.32(225). A, HEK-Gqi cells were transfected with the wild type ( ) or mutant CCR5 receptors Thr 2.56(82) Lys ( ), Thr 2.56(82) Pro (m), Thr 2.56(82) Lys/ Arg6.32(225)Gln (#) or Thr2.56(82)Pro/Arg6.32(225)Gln (D). Untransfected cells ( ) were used as a negative control. Cells pre-labeled with [3H]myo-inositol were incubated with increasing 16402044 Tramiprosate concentrations of MIP1b. Data are from a single experiment that is representative of at least three independent experiments performed in duplicate. B, HEK cells were transfected with wild type or mutant CCR5 receptors and stained with PE-2D7 for FACS analysis. Results are mean values 6 SEM from at least three independent experiments performed in duplicate. C, HEK 293 cells were transiently transfected with wild type ( ) or mutant CCR5 receptors, Thr2.56(82)Lys ( ), Thr2.56(82)Pro (m), Thr2.56(82)Lys/ Arg6.32(225)Gln (#) or Thr2.56(82)Pro/Arg6.32(225)Gln (D) and incubated with 125I-MIP-1b and various concentrations of unlabelled MIP.Ed-receptor conformations that differ in their interactions with HIV Env protein. In summary, we generated four CCR5 mutants that constitutively activate IP signaling. The Thr2.56(82)Pro and Thr2.56(82)Pro/Arg6.32(225)Gln mutants, which were expressed at levels similar to the wild type receptor in HEK 293 cells, the Thr2.56(82)Lys mutant, which was poorly expressed, and the double mutant, Thr2.56(82)Lys/Arg6.32(225)Gln, which showed enhanced expression relative to the Thr2.56(82)Lys mutant. Constitutively active mutants with Lys in position 82 showed very low fusion efficiency, but mutants with Pro in position 82 showed good fusion efficiency that was comparable to the wild type receptor.DiscussionWe have investigated the ability of activated CCR5 conformations to mediate HIV Env-directed membrane fusion by generating constitutively active mutant CCR5 receptors. Chargeneutralizing substitutions for Asp3.49(125) in the DRY motif and substitutions of the naturally occurring Arg6.32(225)Gln mutation of CCR5 did not increase constitutive activation of IP signaling. However, substitution of the Thr2.56(82) residue of the TxP motif caused high levels of ligand-independent cellular signaling. The Thr2.56(82)Lys mutation also decreased cell surface CCR5 protein. Severely decreased expression of mutants with Lys, but not Pro, in position 82 suggests that the conformations of the constitutively active mutant receptors differ. Mutant CCR5 receptors with Lys 18325633 in position 82, which constitutively activated IP signaling, were poor mediators of Env-directed membrane fusion, suggesting that HIV might not enter cells via the activated receptor conformation. However, constitutively active receptors with Pro substituted into the TxP motif mediated Env-directed membrane fusion very efficiently. The differential effects on receptor expression and membrane fusion suggest that Lys and Pro substitutions in position 82 stabilize distinct activated conformations of CCR5 that vary in their ability to mediate Env-dependent membrane fusion. Constitutively active GPCR mutants are defined by increased ligand-independent (basal) signaling activity. The increased signaling results from an increased population of activated receptor conformations by mutant receptors. Many constitutively active mutants exhibit decreased cell surface expression, whichConstitutively Active CCR5 Receptor ConformationsFigure 2. IP production, expression and competition binding of CCR5 receptors with mutations of Thr2.56(82) and Arg6.32(225). A, HEK-Gqi cells were transfected with the wild type ( ) or mutant CCR5 receptors Thr 2.56(82) Lys ( ), Thr 2.56(82) Pro (m), Thr 2.56(82) Lys/ Arg6.32(225)Gln (#) or Thr2.56(82)Pro/Arg6.32(225)Gln (D). Untransfected cells ( ) were used as a negative control. Cells pre-labeled with [3H]myo-inositol were incubated with increasing 16402044 concentrations of MIP1b. Data are from a single experiment that is representative of at least three independent experiments performed in duplicate. B, HEK cells were transfected with wild type or mutant CCR5 receptors and stained with PE-2D7 for FACS analysis. Results are mean values 6 SEM from at least three independent experiments performed in duplicate. C, HEK 293 cells were transiently transfected with wild type ( ) or mutant CCR5 receptors, Thr2.56(82)Lys ( ), Thr2.56(82)Pro (m), Thr2.56(82)Lys/ Arg6.32(225)Gln (#) or Thr2.56(82)Pro/Arg6.32(225)Gln (D) and incubated with 125I-MIP-1b and various concentrations of unlabelled MIP.

Ed-receptor conformations that differ in their interactions with HIV Env protein.

About author

Leave a reply

You must be logged in to post a comment.