Characterization of the extracellular ligand-binding domain of the type II activin receptor.
The binding of a ligand to cell surface receptors initiates a cascade of intracellular 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 biological processes. To study the structural basis that underlies the transmembrane 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 large quantity of ActRII-ECD. Expression was carried out in a fermentor with a typical yield of 10 mg of pure ActRII-ECD from a liter of growth media. Biological function was confirmed by the ability to decrease the activin-stimulated release of FSH from cultured rat pituitary cells in addition to several activin-binding assays, including native gel shift and chemical cross-linking. The glycosylation on ActRII-ECD was shown to be dispensable for high-affinity activin binding, and nonnatural sugars from the yeast expression host did not interfere with binding, indicating that the binding of activin is not sensitive to the environment near the two positions of N-linked glycosylation. Analytical ultracentrifugation of the complex between activin A and ActRII-ECD reveals that two receptors associate with one activin A dimer, consistent with results from chemical cross-linking experiments.
Greenwald
J
J
Structural Biology Laboratory, The Salk Institute, La Jolla, California 92037, USA.
Le
V
V
Corrigan
A
A
Fischer
W
W
Komives
E
E
Vale
W
W
Choe
S
S
D029404:Activin Receptors; D000818:Animals; D002850:Chromatography, Gel; D003460:Crystallization; D004789:Enzyme Activation; D005110:Extracellular Space / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D008024:Ligands; D008297:Male; D051379:Mice; D010843:Pichia / Q000201:enzymology; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D017207:Rats, Sprague-Dawley; D017978:Receptors, Growth Factor / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D008024:Ligands; D017978:Receptors, Growth Factor; D017346:Protein Serine-Threonine Kinases; D029404:Activin Receptors