0 7 Limited limit VBN 8 19 proteolysis proteolysis NN 20 23 for for IN 24 32 assaying assay VBG 33 39 ligand ligand NN 40 47 binding binding NN 48 58 affinities affinity NNS 59 61 of of IN 62 69 nuclear nuclear JJ 70 79 receptors receptor NNS 79 80 . . . 82 85 The the DT 86 93 binding binding NN 94 96 of of IN 97 104 natural natural JJ 105 107 or or CC 108 117 synthetic synthetic JJ 118 125 ligands ligand NNS 126 128 to to TO 129 136 nuclear nuclear JJ 137 146 receptors receptor NNS 147 149 is be VBZ 150 153 the the DT 154 164 triggering trigger VBG 165 170 event event NN 171 178 leading lead VBG 179 181 to to TO 182 186 gene gene NN 187 200 transcription transcription NN 201 211 activation activation NN 212 214 or or CC 215 225 repression repression NN 225 226 . . . 227 233 Ligand Ligand NNP 234 241 binding binding NN 242 244 to to TO 245 248 the the DT 249 255 ligand ligand NN 256 263 binding binding NN 264 270 domain domain NN 271 273 of of IN 274 279 these these DT 280 289 receptors receptor NNS 290 297 induces induce VBZ 298 312 conformational conformational JJ 313 320 changes change NNS 321 325 that that WDT 326 329 are be VBP 330 339 evidenced evidence VBN 340 342 by by IN 343 345 an an DT 346 355 increased increase VBN 356 366 resistance resistance NN 367 369 of of IN 370 374 this this DT 375 381 domain domain NN 382 384 to to TO 385 394 proteases protease NNS 394 395 . . . 396 398 In in FW 399 404 vitro vitro FW 405 412 labeled label VBN 413 422 receptors receptor NNS 423 427 were be VBD 428 437 incubated incubate VBN 438 442 with with IN 443 450 various various JJ 451 460 synthetic synthetic JJ 461 463 or or CC 464 471 natural natural JJ 472 480 agonists agonist NNS 481 483 or or CC 484 495 antagonists antagonist NNS 496 499 and and CC 500 509 submitted submit VBN 510 512 to to TO 513 520 trypsin trypsin NN 521 530 digestion digestion NN 530 531 . . . 532 543 Proteolysis Proteolysis NNP 544 552 products product NNS 553 557 were be VBD 558 567 separated separate VBN 568 570 by by IN 571 579 SDS-PAGE sds-page NN 580 583 and and CC 584 594 quantified quantify VBN 594 595 . . . 596 599 The the DT 600 606 amount amount NN 607 609 of of IN 610 627 trypsin-resistant trypsin-resistant JJ 628 637 fragments fragment NNS 638 641 was be VBD 642 654 proportional proportional JJ 655 657 to to TO 658 666 receptor receptor NN 667 676 occupancy occupancy NN 677 679 by by IN 680 683 the the DT 684 690 ligand ligand NN 690 691 , , , 692 695 and and CC 696 703 allowed allow VBD 704 707 the the DT 708 721 determination determination NN 722 724 of of IN 725 737 dissociation dissociation NN 738 747 constants constant NNS 748 749 ( ( ( 749 752 kDa kda NN 752 753 ) ) ) 753 754 . . . 755 760 Using use VBG 761 764 the the DT 765 774 wild-type wild-type JJ 775 777 or or CC 778 785 mutated mutate VBN 786 791 human human JJ 792 800 retinoic retinoic JJ 801 805 acid acid NN 806 814 receptor receptor NN 815 820 alpha alpha NN 821 823 as as IN 824 825 a a DT 826 831 model model NN 831 832 , , , 833 836 kDa kda NN 837 843 values value NNS 844 854 determined determine VBN 855 857 by by IN 858 867 classical classical JJ 868 879 competition competition NN 880 887 binding binding NN 888 894 assays assay NNS 895 900 using use VBG 901 910 tritiated tritiate VBN 911 918 ligands ligand NNS 919 922 are be VBP 923 925 in in IN 926 935 agreement agreement NN 936 940 with with IN 941 946 those those DT 947 955 measured measure VBN 956 958 by by IN 959 962 the the DT 963 974 proteolytic proteolytic JJ 975 980 assay assay NN 980 981 . . . 982 986 This this DT 987 993 method method NN 994 997 was be VBD 998 1010 successfully successfully RB 1011 1019 extended extend VBN 1020 1022 to to TO 1023 1028 human human JJ 1029 1037 retinoic retinoic JJ 1038 1039 X X NNP 1040 1048 receptor receptor NN 1049 1054 alpha alpha NN 1054 1055 , , , 1056 1070 glucocorticoid glucocorticoid NN 1071 1079 receptor receptor NN 1079 1080 , , , 1081 1084 and and CC 1085 1097 progesterone progesterone NN 1098 1106 receptor receptor NN 1106 1107 , , , 1108 1112 thus thus RB 1113 1122 providing provide VBG 1123 1124 a a DT 1125 1130 basis basis NN 1131 1134 for for IN 1135 1136 a a DT 1137 1140 new new JJ 1140 1141 , , , 1142 1148 faster fast JJR 1149 1154 assay assay NN 1155 1157 to to TO 1158 1167 determine determine VB 1168 1182 simultaneously simultaneously RB 1183 1186 the the DT 1187 1195 affinity affinity NN 1196 1199 and and CC 1200 1212 conformation conformation NN 1213 1215 of of IN 1216 1225 receptors receptor NNS 1226 1230 when when WRB 1231 1236 bound bind VBN 1237 1239 to to TO 1240 1241 a a DT 1242 1247 given give VBN 1248 1254 ligand ligand NN 1254 1255 . . .