Characterization of the extracellular ligand-binding domain of the type IIactivin receptor

The binding of a ligand to cell surface receptors initiates a cascade of in tracellular signals that generate responses to the external stimuli. Thus, this event plays a pivotal role in the mechanism of transmembrane signaling . Activin is a member of a cytokine family that is involved in diverse biol ogical processes. To study the structural basis that underlies the transmem brane signaling mechanism, we have overexpressed the soluble extracellular domain of the type II activin receptor from mouse (ActRII-ECD). We used the methylotrophic yeast Pichia pastoris as an expression host to produce a la rge quantity of ActRII-ECD. Expression was carried out in a fermenter with a typical yield of 10 mg of pure ActRII-ECD from a liter of growth media. B iological function was confirmed by the ability to decrease the activin-sti mulated release of FSH from cultured rat pituitary cells in addition to sev eral activin-binding assays, including native gel shift and chemical cross- linking. The glycosylation on ActRII-ECD was shown to be dispensable for hi gh-affinity activin binding, and nonnatural sugars from the yeast expressio n host did not interfere with binding, indicating that the binding of activ in is not sensitive to the environment near the two positions of N-linked g lycosylation. Analytical ultracentrifugation of the complex between activin A and ActRII-ECD reveals that two receptors associate with one activin A d imer, consistent with results from chemical cross-linking experiments.