TABLE 2

Mutational analysis of residues potentially involved in metal site or as second site interaction

The effect of CuBip and ITAC were measured by the phosphatidyl inositol turnover experiments in transiently transfected COS-7 cells. n refers to the number of experiments. ± values are S.E.M.




Cu(II)-Bipyridine

ITAC


Log EC50
EC50
n
Fdec
Emax
Emax
Log EC50
EC50
Fdec
n
Emax
Emax
μM -fold fmol / 105 cells % μM -fold fmol / 105 cells %
III:05 G128H -4.7 ± 0.11 22 18 1.0 347 ± 32 162 -7.8 ± 0.09 15 1.0 16 215 ± 31 36
Background
III:08 F131A -2.8 ± 0.09 1415a 4 64 NE NE -7.5 ± 0.39 35 2.4 3 72 ± 10 8
III:12 F135L -4.3 ± 0.23 55 3 2.5 247 ± 29 172 -8.1 ± 0.14 8.3 0.57 3 144 ± 28 22
IV:20 D186N -3.1 ± 0.43 885a 8 40 NE NE -8.0 ± 0.20 9 0.6 8 67 ± 6.9 7
EC2 H202A -4.2 ± 0.27 58 3 2.6 153 ± 4.4 180 -7.8 ± 0.13 17 1.2 3 85 ± 9.6 11
V:01; R208A; -4.9 ± 0.05 12 4 0.22 236 ± 60 259 -7.8 ± 0.36 15 1.1 4 91 ± 14 12
V:05 R212A
V:05 R212H -4.7 ± 0.05 19 3 0.85 526 ± 122 227 -7.8 ± 0.03 16 1.1 2 232 ± 33 39
VI:13 W268A -4.3 ± 0.08 50 5 2.3 313 ± 68 460 -7.6 ± 0.18 27 1.9 6 68 ± 11 7
VI:16 Y271A -3.1 ± 0.21 794a 4 37 NE NE -8.8 ± 0.20 1.7 0.12 3 43 ± 1.8 2.5
VI:16 Y271L -2.9 ± 0.36 1278a 4 58 NE NE -8.9 ± 0.18 1.3 0.09 4 50 ± 3.0 3.9
VI:16 Y271N -3.2 ± 0.06 693a 3 31 NE NE -8.3 ± 0.38 5.6 0.39 3 39 ± 2.9 1.8
VI:16 Y271F -4.3 ± 0.05 51 3 2.3 263 ± 34 163 -8.1 ± 0.15 7.6 0.52 3 161 ± 37 25
VI:16 Y271H -4.0 ± 0.03 108 5 4.9 196 ± 29 190 -8.0 ± 0.28 9.6 0.66 5 103 ± 22 14
VI:23 D278N -4.3 ± 0.12 55 6 2.5 195 ± 31 267 -7.9 ± 0.02 13 0.88 6 73 ± 14 8
VII:02 K300A -4.7 ± 0.09 20 4 0.90 151 ± 17 150 -8.4 ± 0.05 4.2 0.29 3 101 ± 12 14
VII:10
Y308A
-3.2 ± 0.45
631
3
29
NE
NE
-8.1 ± 0.22
7.4
0.21
3
83 ± 40
10
  • Fdec, decrease in potency for CuBip and ITAC on a given mutation compared with His III:05-CXCR3 (background); Emax, maximal stimulation of each ligand; Emax (%), for Cu(II)-bipyridine refers to the efficacy of Cu(II)-bipyridine compared with ITAC. Emax (%), for ITAC refers to the efficacy on each mutation compared with wild-type CXCR3; NE, no detectable Emax.

  • a The EC50 was estimated by a nonlinear regression using the sigmoidal dose-response algorithm in GraphPad Prism with a constant Hill coefficient of 1.5 corresponding to the Hill coefficient of Cu(II)-bipyridine on His III:05 CXCR3.