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.