0 8 Analysis Analysis NNP 9 11 of of IN 12 15 the the DT 16 30 ligand-binding ligand-binding JJ 31 37 domain domain NN 38 40 of of IN 41 46 human human JJ 47 55 retinoic retinoic JJ 56 60 acid acid NN 61 69 receptor receptor NN 70 75 alpha alpha NN 76 78 by by IN 79 92 site-directed site-directed JJ 93 104 mutagenesis mutagenesis NN 104 105 . . . 107 112 Three three CD 113 121 subtypes subtype NNS 122 124 of of IN 125 133 retinoic retinoic JJ 134 138 acid acid NN 139 148 receptors receptor NNS 149 150 ( ( ( 150 153 RAR RAR NNP 153 154 ) ) ) 154 155 , , , 156 162 termed term VBN 163 166 RAR RAR NNP 167 172 alpha alpha NN 172 173 , , , 174 177 RAR RAR NNP 178 182 beta beta NN 182 183 , , , 184 187 and and CC 188 191 RAR rar NN 192 197 gamma gamma NN 197 198 , , , 199 203 have have VBP 204 208 been be VBN 209 218 described describe VBN 218 219 . . . 220 224 They they PRP 225 228 are be VBP 229 237 composed compose VBN 238 240 of of IN 241 250 different different JJ 251 261 structural structural JJ 262 269 domains domain NNS 269 270 , , , 271 280 including include VBG 281 289 distinct distinct JJ 290 297 domains domain NNS 298 301 for for IN 302 305 DNA DNA NNP 306 309 and and CC 310 316 ligand ligand NN 317 324 binding binding NN 324 325 . . . 326 330 RARs RAR NNP 331 343 specifically specifically RB 344 348 bind bind VBP 349 367 all-trans-retinoic all-trans-retinoic JJ 368 372 acid acid NN 373 374 ( ( ( 374 376 RA RA NNP 376 377 ) ) ) 377 378 , , , 379 387 9-cis-RA 9-cis-RA NNP 387 388 , , , 389 392 and and CC 393 401 retinoid retinoid NN 402 409 analogs analog NNS 409 410 . . . 411 413 In in IN 414 418 this this DT 419 424 study study NN 424 425 , , , 426 428 we we PRP 429 437 examined examine VBD 438 441 the the DT 442 452 functional functional JJ 453 457 role role NN 458 460 of of IN 461 469 cysteine cysteine NN 470 473 and and CC 474 482 arginine arginine NN 483 491 residues residue NNS 492 494 in in IN 495 498 the the DT 499 513 ligand-binding ligand-binding JJ 514 520 domain domain NN 521 523 of of IN 524 528 hRAR hRAR NNP 529 534 alpha alpha NN 535 536 ( ( ( 536 540 hRAR hRAR NNP 541 550 alpha-LBD alpha-lbd NN 550 551 , , , 552 557 amino amino NN 558 563 acids acid NNS 564 567 154 154 CD 568 570 to to TO 571 574 462 462 CD 574 575 ) ) ) 575 576 . . . 577 580 All all DT 581 590 conserved conserve VBN 591 599 cysteine cysteine NN 600 603 and and CC 604 612 arginine arginine NN 613 621 residues residue NNS 622 624 in in IN 625 629 this this DT 630 636 domain domain NN 637 641 were be VBD 642 649 mutated mutate VBN 650 652 by by IN 653 666 site-directed site-directed JJ 667 678 mutagenesis mutagenesis NN 678 679 , , , 680 683 and and CC 684 687 the the DT 688 694 mutant mutant JJ 695 703 proteins protein NNS 704 708 were be VBD 709 722 characterized characterize VBN 723 725 by by IN 726 734 blocking block VBG 735 744 reactions reaction NNS 744 745 , , , 746 760 ligand-binding ligand-binding JJ 761 772 experiments experiment NNS 772 773 , , , 774 789 transactivation transactivation NN 790 796 assays assay NNS 796 797 , , , 798 801 and and CC 802 810 protease protease NN 811 818 mapping mapping NN 818 819 . . . 820 827 Changes change NNS 828 830 of of IN 831 834 any any DT 835 843 cysteine cysteine NN 844 851 residue residue NN 852 854 of of IN 855 858 the the DT 859 863 hRAR hRAR NNP 864 873 alpha-LBD alpha-lbd NN 874 877 had have VBD 878 880 no no DT 881 892 significant significant JJ 893 902 influence influence NN 903 905 on on IN 906 909 the the DT 910 917 binding binding NN 918 920 of of IN 921 930 all-trans all-trans JJ 931 933 RA RA NNP 934 936 or or CC 937 942 9-cis 9-cis JJ 943 945 RA RA NNP 945 946 . . . 947 960 Interestingly interestingly RB 960 961 , , , 962 969 residue residue NN 970 975 C-235 c-235 NN 976 978 is be VBZ 979 991 specifically specifically RB 992 1001 important important JJ 1002 1004 in in IN 1005 1015 antagonist antagonist NN 1016 1023 binding binding NN 1023 1024 . . . 1025 1029 With with IN 1030 1037 respect respect NN 1038 1040 to to TO 1041 1049 arginine arginine NN 1050 1058 residues residue NNS 1058 1059 , , , 1060 1064 only only RB 1065 1068 the the DT 1069 1072 two two CD 1073 1079 single single JJ 1080 1089 mutations mutation NNS 1090 1092 of of IN 1093 1098 R-276 R-276 NNP 1099 1102 and and CC 1103 1108 R-394 r-394 NN 1109 1111 to to TO 1112 1119 alanine alanine NN 1120 1126 showed show VBD 1127 1128 a a DT 1129 1137 dramatic dramatic JJ 1138 1146 decrease decrease NN 1147 1149 of of IN 1150 1157 agonist agonist NN 1158 1161 and and CC 1162 1172 antagonist antagonist NN 1173 1180 binding binding NN 1181 1188 whereas whereas IN 1189 1192 the the DT 1193 1198 R272A R272A NNP 1199 1207 mutation mutation NN 1208 1214 showed show VBD 1215 1219 only only RB 1220 1221 a a DT 1222 1228 slight slight JJ 1229 1235 effect effect NN 1235 1236 . . . 1237 1240 For for IN 1241 1244 all all DT 1245 1250 other other JJ 1251 1259 arginine arginine NN 1260 1269 mutations mutation NNS 1269 1270 , , , 1271 1273 no no DT 1274 1285 differences difference NNS 1286 1288 in in IN 1289 1297 affinity affinity NN 1298 1302 were be VBD 1303 1313 detectable detectable JJ 1313 1314 . . . 1315 1318 The the DT 1319 1322 two two CD 1323 1332 mutations mutation NNS 1333 1338 R217A R217A NNP 1339 1342 and and CC 1343 1348 R294A R294A NNP 1349 1355 caused cause VBD 1356 1358 an an DT 1359 1368 increased increase VBN 1369 1376 binding binding NN 1377 1387 efficiency efficiency NN 1388 1391 for for IN 1392 1403 antagonists antagonist NNS 1404 1407 but but CC 1408 1410 no no DT 1411 1417 change change NN 1418 1420 in in IN 1421 1428 agonist agonist NN 1429 1436 binding binding NN 1436 1437 . . . 1438 1442 From from IN 1443 1448 these these DT 1449 1456 results result NNS 1456 1457 , , , 1458 1460 we we PRP 1461 1464 can can MD 1465 1473 conclude conclude VB 1474 1478 that that IN 1479 1492 electrostatic electrostatic JJ 1493 1505 interactions interaction NNS 1506 1508 of of IN 1509 1518 retinoids retinoid NNS 1519 1523 with with IN 1524 1527 the the DT 1528 1531 RAR RAR NNP 1532 1541 alpha-LBD alpha-lbd NN 1542 1546 play play VBP 1547 1548 a a DT 1549 1560 significant significant JJ 1561 1565 role role NN 1566 1568 in in IN 1569 1575 ligand ligand NN 1576 1583 binding binding NN 1583 1584 . . . 1585 1587 In in IN 1588 1596 addition addition NN 1596 1597 , , , 1598 1609 antagonists antagonist NNS 1610 1614 show show VBP 1615 1625 distinctly distinctly RB 1626 1635 different different JJ 1636 1648 requirements requirement NNS 1649 1652 for for IN 1653 1662 efficient efficient JJ 1663 1670 binding binding NN 1670 1671 , , , 1672 1677 which which WDT 1678 1681 may may MD 1682 1692 contribute contribute VB 1693 1695 to to TO 1696 1701 their their PRP$ 1702 1714 interference interference NN 1715 1717 in in IN 1718 1721 the the DT 1722 1738 ligand-inducible ligand-inducible JJ 1739 1754 transactivation transactivation NN 1755 1763 function function NN 1764 1766 of of IN 1767 1770 RAR RAR NNP 1771 1776 alpha alpha NN 1776 1777 . . .