Characterization of in vitro translated human mineralocorticoid receptor. Structure and activation.
The structures of the unactivated and activated mineralocorticoid receptors have been difficult to characterize because of receptor lability and steroid dissociation. Therefore, human mineralocorticoid receptor mRNA was translated in rabbit reticulocyte lysate in the presence and absence of [35S]methionine to compare the structure of [3H]aldosterone-bound and [35S]labeled receptor. In vitro synthesized receptor was able to specifically bind [3H]aldosterone. Unactivated [3H]aldosterone-bound and 60% of unactivated [35S]labeled receptor eluted from DEAE-cellulose with 250 mM salt and had a Rs of 72A. Forty percent of unactivated [35S]labeled receptor eluted from DEAE-cellulose with 100 mM salt and had a Rs of 54A. SDSPAGE showed intact hMR was present in both DEAE-cellulose eluates as three bands between M(r) 110,000-120,000. However, the low salt eluate contained less intact receptor and more lower MW bands. Neither [3H]aldosterone-bound nor [35S]labeled receptor was activated by incubation at 25 degrees C as readily as glucocorticoid receptor studied under identical conditions. Activated [3H]aldosterone-bound receptor eluted from DEAE-cellulose at 100 mM salt and had a Rs of 37A. After activation, 60% of [35S]labeled receptor eluted from DEAE-cellulose with 100 mM salt and had a Rs of 91A. SDS-PAGE of the high and low salt DEAE-cellulose eluates showed that 50% of intact receptor eluted in the low salt peak after activation. These data indicate that: 1. Some in vitro synthesized mineralocorticoid receptor assembles into the heteromeric unactivated form; 2. The remaining intact receptor remains monomeric and unable to bind steroid; 3. Activation causes dissociation of intact receptor from a larger complex; and 4. Activated receptor tends to aggregate.
Schulman
G
G
Department of Medicine, Temple University School of Medicine, Philadelphia, PA.
Daniel
V
V
Cooper
M
M
Alnemri
E S
ES
Maksymowych
A B
AB
Litwack
G
G
D000450:Aldosterone / Q000378:metabolism; D000818:Animals; D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D066298:In Vitro Techniques; D008901:Mineralocorticoids / Q000378:metabolism*; D015394:Molecular Structure; D008970:Molecular Weight; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D011817:Rabbits; D018161:Receptors, Mineralocorticoid; D011987:Receptors, Steroid / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D008901:Mineralocorticoids; D012333:RNA, Messenger; D018161:Receptors, Mineralocorticoid; D011987:Receptors, Steroid; D000450:Aldosterone