The production of antibodies against the conserved cysteine region of steroid receptors and their use in characterizing the avian progesterone receptor.
The most highly conserved feature of steroid receptor primary structure, the conserved cysteine region, corresponds to the DNA-binding domain of steroid receptors. This domain of chick progesterone receptor (PR) and other receptors was immunochemically characterized using site-directed antibodies. Eight peptides were synthesized corresponding to portion of the conserved cysteine region from PR, glucocorticoid, or estrogen receptor proteins. Polyclonal antibodies were obtained by immunizing rabbits with peptide conjugated to a protein carrier. The cross-reactivity of each antiserum was tested by Western blotting against chick and human PR, human glucocorticoid receptor, and human estrogen receptor. Of the several antisera positive by Western blots, only one cross-reacted with native chick PR. It was found that antibody bound to 4S transformed receptor, but not to 8S nontransformed receptor. At 50 mM KCl, antibody bound preferentially to transformed receptor form A, but at 400 mM KCl antibody bound equally well to either receptor form A or B. Binding of PR to DNA-cellulose was partially inhibited by the presence of cross-reacting peptide antibody. Thus, we have obtained at least one site-directed antibody against steroid receptors which is a useful structural probe to the DNA-binding functional domain of these gene regulatory proteins.
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity; D002482:Cellulose / Q000031:analogs & derivatives / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D002645:Chickens; D003545:Cysteine; D004247:DNA / Q000031:analogs & derivatives / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000276:immunology; D005260:Female; D006367:HeLa Cells / Q000032:analysis; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D007118:Immunoassay; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D011817:Rabbits; D011960:Receptors, Estrogen / Q000276:immunology*; D011965:Receptors, Glucocorticoid / Q000276:immunology*; D011980:Receptors, Progesterone / Q000276:immunology*; D014599:Uterus / Q000032:analysis
D000906:Antibodies; C033190:DNA-cellulose; D000939:Epitopes; D007106:Immune Sera; D011960:Receptors, Estrogen; D011965:Receptors, Glucocorticoid; D011980:Receptors, Progesterone; D002482:Cellulose; D004247:DNA; D003545:Cysteine