Cloning and identification of functional domains in quail Brain aromatase

Recent evidence indicates that aromatase activity (AA) in the hypothalamus is not only modulated by slow (hours to days) genomic actions but also through fast (seconds to minutes) non-genomic mechanisms. We recently showed that Calcium (Ca2+)-dependent phosphorylations catalyzed by multiple protein kinases including PKC, and possibly PKA and CAMK, rapidly down-regulate AA in quail hypothalamic homogenates. Western blotting experiments also indicated that phosphorylations affect the aromatase molecule itself but it was impossible to fully characterize the putative phosphorylation sites on the quail enzyme because its sequence was unknown. We therefore cloned and sequenced the quail brain aromatase. We identified a 1541-bp open reading frame that encodes a predicted 490-amino acid protein containing all functional domains previously described in mammalian and other avian aromatases. Multiple motifs match consensus sequences corresponding to several protein kinases including those that were shown to affect AA during pharmacological experiments with specific kinase inhibitors (e.g., PKC, PKA, MAPK, Myosine light chain kinase, Tyr. kinase). Another potential control pathway of AA, independent from phosphorylations, could involve a direct control by Ca2+-dependent calmodulin (CAM), as suggested by the identification in Western blots of CAM on purified aromatase from quail hypothalamic homogenates. Accordingly, two Ca2+-dependent calmodulin binding motifs (1-8-14b) as defined by Rhoads and Friedberg (FASEB, 1997, 11:331-340) are present and conserved in most vertebrates including quail aromatase. These results suggest that the phosphorylation of some residues as well as the direct binding of calmodulin on the aromatase protein represent part of the mechanism(s) underlying the rapid changes in AA.