INVOLVEMENT OF THE N-TERMINAL REGION OF THE HUMAN MINERALOCORTICOID RECEPTOR HORMONE-BINDING DOMAIN IN AGONIST AND ANTAGONIST BINDING AS REVEALED BY A NEW MONOCLONAL-ANTIBODY

To gain a better understanding of the mechanism of binding to the huma n mineralocorticoid receptor (hMR), we developed a new monoclonal anti body (mAb) raised against the hormone-binding domain (HBD). For this p urpose, mice were immunized with a fusion protein including the sequen ce Thr(729)-Lys(984) of hMR. After ELISA screening, mAb 18C7 was selec ted for its specificity towards the HBD. This antibody recognized both the denatured and native MR forms, as well as the hetero-oligomeric M R form and the transformed MR state. By using several HBD subfragments , the mAb 18C7 epitope was located in the N-terminal region of the HBD from Thr(729) to Leu(765). We then studied the effect of the antibody on aldosterone and progesterone binding to the hMR. When 18C7 was inc ubated with liganded MR, it was able to partly displace (20%) the horm one from its binding site. When 18C7 was incubated with MR before aldo sterone or progesterone, the antibody inhibited 75-80% of the binding. The effect of 18C7 on the binding was similar with both hormones. A s ucrose gradient analysis indicated the simultaneous presence of two ki nds of receptor complexes: the steroid-MR complex and the antibody-MR complex. After its associated proteins, especially the heat-shock prot ein hsp90, had been crosslinked with the hMR by dimethylpimelimidate, 18C7 was still able to react with the receptor. Our results indicated that the epitope recognized by 18C7 was directly implicated in hormone binding. The lack of steroid binding of HBD mutants with the Thr(729) -Leu(765) sequence deleted [Jalaguier, Mesnier, Leger and Auzou (1996) J. Steroid Biochem. Mel. Biol. 57, 43-50] supports this hypothesis. B ecause of the similar behaviours of aldosterone and progesterone, we c onclude that the N-terminal Thr(729)-Leu(765) region of the HBD is sim ilarly involved in the binding of both hormones.