TABLE 1

Molecular interaction of CCL1 and LMD-009 with CCR8

The whole panel of CCR8 mutations was screened for the ability of the endogenous chemokine CCL1 (left) and the nonpeptide LMD-009 (right) to activate every single mutation. The CCR8 receptors were expressed in transiently transfected COS-7 cells, and the activation was measured by means of accumulated inositol phosphates (see Materials and Methods). The table shows the potency (both as Log EC50 and as EC50) and the level of activation [basal and agonist stimulated levels (efficacy)]. The differences between the potency of CCL1 or LMD-009 on a given mutation and the potency of each ligand on wt CCR8 are shown in the columns named “-Fold.” The number of experiments is given as n. The receptor residues are given according to the Schwartz (S) and Ballesteros/Weinstein (B-W) nomenclatures (see footnote 1 in the text.)

Residue CCL1 LMD-009
Potency Potency
Position Number EC50 EC50 -Fold n Efficacy (Emax) Basal Activity EC50 EC50 -Fold n Efficacy (Emax)
S B-W
Log nM cpm cpm Log nM cpm
wt wt wt −8.1 ± 0.03 8.7 1.0 65 4000 ± 314 712 ± 40 −8.0 ± 0.17 11 1.0 11 3774 ± 448
I:07 1.39 Y42A −7.9 ± 0.13 11 1.3 9 2238 ± 370 786 ± 120 −6.8 ± 0.09 158 15 3 1471 ± 302
I:11 1.43 F46A
II:13 2.53 F84A −8.4 ± 0.13 4.0 0.46 8 2098 ± 278 1009 ± 87 −7.9 ± 0.09 14 1.3 3 1441 ± 158
II:17 2.57 F88A −8.0 ± 0.05 10 1.2 9 2445 ± 419 799 ± 118 −7.0 ± 0.05 108 10 4 1107 ± 450
II:20 2.60 Q91W −7.2 ± 0.14 69 7.9 6 1232 ± 101 665 ± 58 −7.0 ± 0.18 108 10 3 2916 ± 92
III:04 3.28 V109A −8.1 ± 0.07 8.2 0.95 3 2100 ± 356 940 ± 269 −8.1 ± 0.21 7.7 0.73 3 2193 ± 297
III:05 3.29 S110A −8.8 ± 0.10 1.6 0.18 8 2804 ± 723 861 ± 214 −8.8 ± 0.41 1.5 0.14 4 1066 ± 596
III:05 3.29 S110H <−6 >1000 >137 7 N.E. 556 ± 850 <−5 >10,000 >950 2 N.E.
III:05 3.29 S110W <−6 >1000 >137 5 N.E. 400 ± 108 <−5 >10,000 >950 2 N.E.
III:07 3.31 F112A −8.3 ± 0.07 4.6 0.53 9 3706 ± 552 651 ± 88 −7.9 ± 0.04 11 1.1 4 2394 ± 702
III:08 3.32 Y113A −6.5 ± 0.12 323 37 10 1047 ± 185 570 ± 57 <−5 ± 0.00 >10,000 >950 5 N.E.
III:09 3.33 Y114A −6.9 ± 0.13 131 15 4 2692 ± 784 927 ± 200 −7.4 ± 0.09 42 3.9 6 3652 ± 710
III:18 3.42 F123A −8.4 ± 0.15 4.4 0.51 6 1466 ± 371 426 ± 64 −8.2 ± 0.14 7.0 0.66 3 1430
IV:20 4.60 L168D −7.9 ± 0.18 12 1.4 11 3549 ± 537 948 ± 122 −7.8 ± 0.17 18 1.7 2 4410 ± 1576
IV:24 4.64 Y172A −8.0 ± 0.11 11 1.3 8 2000 ± 155 1315 ± 110 −7.7 ± 0.15 21 2.0 4 1557 ± 329
V:−02 5.33 K193A −7.7 ± 0.17 22 2.6 6 2956 ± 352 659 ± 89 −8.1 ± 0.37 8.5 0.80 2 3414 ± 298
V:−01 5.34 W194A −6.9 ± 0.29 128 15 5 2500 ± 142 673 ± 44 −8.1 ± 0.23 7.6 0.72 3 1273 ± 207
V:01 5.35 K195A −8.1 ± 0.09 7.5 0.87 7 1947 ± 314 666 ± 97 −8.2 ± 0.30 6.2 0.59 3 2370 ± 485
V:12 5.46 G206A −7.2 ± 0.13 67 7.7 9 904 ± 63 508 ± 54 −7.7 ± 0.08 19 1.4 3 744 ± 59
V:12 5.46 G206W N.A. 8 501 ± 75 N.A. N.A. 3
VI:13 6.48 W251Q 8.5 ± 0.12 3.4 0.39 10 2245 ± 347 1146 ± 152 −7.8 ± 0.34 44 4.1 3 1112 ± 468
VI:13 6.48 W251Q −8.2 ± 0.14 6.6 0.76 6 2528 ± 320 1097 ± 166 −7.4 ± 0.34 44 4.1 3 1112 ± 468
VI:16 6.51 F254A −7.0 ± 0.09 109 13 10 3131 ± 609 835 ± 74 −9.2 ± 0.24 0.57 0.05 5 6640 ± 1484
VI:20 6.55 L258A −7.6 ± 0.11 27 3.2 9 2699 ± 262 591 ± 54 −7.9 ± 0.20 13 1.2 3 3162 ± 568
VI:24 6.59 S262A −8.3 ± 0.10 5.2 0.60 10 2949 ± 420 621 ± 67 −7.7 ± 0.16 21 2.0 4 1854 ± 815
VII:03 7.36 H283A −7.9 ± 0.09 12 1.4 8 4003 ± 444 705 ± 58 −7.6 ± 0.14 24 2.2 3 3644 ± 1595
VII:06 7.39 E286A −7.9 ± 0.11 12 1.4 11 3732 ± 578 930 ± 96 <−5 >10000 >950 5 N.E.
VII:09 7.42 S289A −7.7 ± 0.14 18 2.1 3 9229 ± 3007 842 ± 293 −7.8 ± 0.03 16 1.5 3 10833 ± 2827
VII:10 7.43 F290A −8.2 ± 0.20 6 0.68 5 4325 ± 940 2497 ± 517 −7.7 ± 0.08 20 1.9 5 3700 ± 789

  • N.E., not estimated due to no plateau; N.A., no activation at all.