Two Activin Type 2B Receptors from Sea Bream Function Similarly in vitro

Myostatin (MSTN) and activin are members of the transforming growth factor-β superfamily. Both signal through the activin type II receptors (ActRII and ActRIIB). In a previous report, we demonstrated that fish possess at least 2 genes for ActRIIB: ActRIIB-1 and ActRIIB-2, which differ in their amino acid sequence. We also showed that affinity-purified, fish-soluble ActRIIB-1 (extracellular domain; ECD), produced in the yeast Pichia pastoris, inhibited recombinant mouse/rat/human mature MSTN activity in vitro using a reporter gene assay in the mammalian A204 cell line. In the present study, we produced soluble ActRIIB-2a in P. pastoris, and showed that it is N-glycosylated, similar to soluble ActRIIB-1. Inhibition of MSTN and activin A activities by affinity-purified ActRIIB-2a was compared with that of soluble ActRIIB-1 using the CAGA-luciferase assay in A204 cells. The findings of this study provide evidence that both paralogs, which probably resulted from gene duplication, did not diversify in their functionality (neofunctionalization), but rather retained a similar function. Both ActRIIB isoforms are equally potent in the mammalian system, and both exhibited an inhibitory effect on mammalian MSTN and activin A. Moreover--in spite of the amino acid differences in ECD between the two paralogs--it appears that the residues important for ligand binding are conserved, and that they recognize the mammalian ligands activin A and MSTN to the same extent.