Carp Activation Factors ¢ñ And ¢ò Receptor Cdna Clones, Structural Analysis And Its Regulation Of Expression In Pituitary Cells

Since the initial discovery activin, cumulative evidence further has established that activin and its two functional modulators, the activin binding protein follistatin and the activin antagonist inhibin, regulated reproductive function by exerting effects at all levels of the reproductive axis, in particular at the ovary and pituitary. However, the intracellular mechanisms for activin actions, e.g. receptor expression levels, have not been examined. In the present study,two cDNAs encoding the activin type IIB receptor (ActRIIB) and activin type IB receptor (ActRIB) were cloned and characterized from grass carp. The deduced ActRIIB protein of 510 amino acids shared 79-90% identity with those in other vertebrates, while the predicted ActRIB protein of 505 amino acids exhibited high sequence identity (80-96%) to its counterparts in human, rat, mouse, frog and zebrafish. Phylogenetic analysis revealed that two receptors were highly conserved during vertebrate evolution. Furthermore, comparative analysis showed that each of these two receptors contained the conserved amino acid residues required for activin binding, and comprised the characteristic regions of an extracellular ligand binding domain, a single transmembrane region, and an intracellular serine/threonine kinase domain. The high degree of conservation of receptor functional domains between human and grass carp supports the idea that human activin-A used in the present study could interact with grass carp ActRIIB and ActRIB. Real-time PCR analysis revealed that both ActRIIB and ActRIB transcripts were ubiquitously expressed in all of the tissues examined, including pituitary. Using a static incubation approach, the direct actions of exogenous activin on its own receptors, ActRIIB and ActRIB, mRNA expression were examined at the pituitary level. Activin significantly stimulated mRNA expression of grass carp ActRIB in a time- and dose-dependent manner, but didn't alter the expression level of ActRIIB. These findings support the notion that activin receptors may serve as a local regulatory point involving pituitary function of activin in grass carp. In addition, expression plasmids for kinase deficient form and constitutively active form of ActRIB, and the ligand-binding inefficient form of ActRIIB were constructed using site-directed mutagenesis, respectively, providing the molecular tool for further study on the functional roles of these two receptors.