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 degenerate primers followed by 3' rapid amplification of cDNA ends PCR (3'Race-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, human, African toad: 68-70% amino acid identity), and encompasses the second part 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 sequence. COS-1 cells transfected with a pCMV5 expression vector containing the whole 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 detected in untransfected cells. Aldosterone displaced (3)H-cortisol binding, though was less effective by than unlabeled cortisol (P<0.05). Competition experiments with other steroids gave the following hierarchy for the displacement of the (3) dexamethasone, whereas 17, 20beta-dihydroxy-4-pregnen-3-one and 17,20beta,21beta-trihydroxy-4 pregnen-3-one (two fish specific progestins) did not show any specific binding. These results strongly suggest that this cDNA sequence encodes a rainbow trout mineralocorticoid-like receptor, and represent the first description of such a receptor in teleost fish where aldosterone, the classic mineralocorticoid, is believed to be absent.
Colombe
L
L
INRA SCRIBE, Campus de Beaulieu, 35042, Rennes, France.
Fostier
A
A
Bury
N
N
Pakdel
F
F
Guiguen
Y
Y
D019326:17-alpha-Hydroxyprogesterone / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D019556:COS Cells / Q000378:metabolism; D003001:Cloning, Molecular; D003600:Cytosol / Q000378:metabolism; D006801:Humans; D006854:Hydrocortisone / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D017686:Oncorhynchus mykiss / Q000235:genetics*; D018161:Receptors, Mineralocorticoid / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013256:Steroids / Q000378:metabolism*; D013739:Testosterone / Q000031:analogs & derivatives / Q000378:metabolism
D018161:Receptors, Mineralocorticoid; D011994:Recombinant Proteins; D013256:Steroids; D013739:Testosterone; D019326:17-alpha-Hydroxyprogesterone; C003600:11-ketotestosterone; D006854:Hydrocortisone