A mineralocorticoid-like receptor in the rainbow trout, Oncorhynchus mykiss: cloning and characterization of its steroid binding domain

Using reverse transcriptase polymerase chain reaction (PCR) (RT-PCR) with d egenerate primers followed by 3' rapid amplification of cDNA ends PCR (3'Ra ce-PCR) we have isolated a new fish steroid receptor cDNA sequence of 1806 bp from rainbow trout (Oncorhynchus mykiss) testis. This sequence has clear homology with various mineralocorticoid receptor cDNA sequences (rat, huma n, African toad: 68-70% amino acid identity), and encompasses the second pa rt of DNA binding domain (C domain), the whole hinge region (D domain) and the steroid binding domain (E domain) plus 726 bp of 3' untranslated sequen ce. COS-I cells transfected with a pCMV5 expression vector containing the w hole E domain (pCMV5-rtMR) showed high affinity binding for cortisol (K-a = 0.53 +/- 0.03 nM, K-d = 1.9 nM) in the cytosol, which could not be detecte d in untransfected cells. Aldosterone displaced H-3-cortisol binding, thoug h was less effective by than unlabeled cortisol (P < 0.05). Competition exp eriments with other steroids gave the following hierarchy for the displacem ent of the H-3-cortisol from the receptor-ligand complex: cortisol = cortic osterone = 11-deoxycortisol = 21-deoxycortisol > Il-deoxycorticosterone = 1 1 beta-hydroxyprogesterone = 17-hydroxyprogesterone > dexamethasone, wherea s 17,20 beta-dihydroxy-4-pregnen-3-one and 17,20 beta,21 beta-trihydroxy-4- pregnen-3-one (two fish specific progestins) did not show any specific bind ing. These results strongly suggest that this cDNA sequence encodes a rainb ow trout mineralocorticoid-like receptor, and represent the first descripti on of such a receptor in teleost fish where aldosterone, the classic minera locorticoid, is believed to be absent. (C) 2000 Elsevier Science Inc. All r ights reserved.