The identification and characterization of three distinct estrogen receptor subtypes in a teleost fish, the Atlantic croaker (Micropogonias undulatus)

This dissertation identifies and characterizes three distinct estrogen receptors in a teleost fish, the Atlantic croaker, Micropogonias undulatus. Atlantic croaker possess ERα, ERβ, and a previously unrecognized form we call ERγ. This is the first description of three ER subtypes in any vertebrate. Phylogenetic analysis shows that ERγ arose via gene duplication from ERβ early in the teleost lineage, and indicates that ERγ is present in other teleosts, but has not been recognized. The three ER subtypes are genetically distinct and have different distribution patterns in Atlantic croaker tissues. The Atlantic croaker (ac)ERγ shows amino acid differences in regions important for ligand-binding and receptor activation that are conserved in all other ERγs. Bacterially expressed fusion proteins for acERα, β, and γ show specific, high affinity binding to [3 H] estradiol (E2) with Kds of 0.61± 0.013, 0.40 ± 0.006, and 0.38 ± 0.059 nM respectively. The rank orders of binding to the acER fusion proteins are DES >> ICI182 > TOH > ICI164 > E2 ≥ ZEAR > MOX E >TAM > E1 ≥ 17αE2

E3 > 2OH E = GEN >> RU 486 for acERα; ICI182 > DES > TOH > E2 > ICI164 > GEN > MOX E > TAM > ZEAR = E1 > E3 = 17αE2 > 2OH E >> RU 486 for acERβ; and E2 ≥ DES > TOH > ICI182 > ICI164 > E3 ≥ GEN > MOX E > ZEAR > E1 > 17αE2 > RU 486 ≥ TAM > 2OH E for acERγ. The acER subtypes are expressed in expected areas of the forebrain, but their distributions within these regions differ. acERα is found in all brain regions previously demonstrated to concentrate E2 or possess ERs. acERγ, but not acERβ, is detected in the nucleus suprachiasmaticus and dorsal preopticus parvocellularis anterior (PPa). Conversely, acERβ, but not acERγ was found in the ventral PPa, preopticus magnocellularis pars magnocellularis, and cerebellar Purkinje cells. The presence of three functional ERs in one species expands the role of estrogen receptor multiplicity in estrogen signaling systems and clarifies the dynamics and mechanisms of estrogen receptor evolution.