Activin and inhibin binding to the soluble extracellular domain of activin receptor II.
Activins and inhibins belong to the transforming growth factor-beta-like superfamily of growth and differentiation factors that exert pleiotropic effects in many target tissues. Heteromeric association of activin with two structurally related receptor serine/threonine kinases, activin receptor types I and II, initiates downstream signaling events. The extracellular domain of type II mouse activin receptor (ActRII ECD) was expressed in the baculovirus system, purified in three steps by lectin affinity, anion exchange, and reverse phase chromatography, and further characterized by mass spectrometry. The reduction in the apparent size of the purified ActRII ECD on SDS-PAGE after treatment with glycosidases provided evidence for N- and O-linked oligosaccharides. Specific receptor/ligand complexes of [125I] activin A to ActRII ECD or [125I]ActRII ECD to activin A were analyzed by cross-linking and immunoprecipitation. Two major radiolabeled bands were observed on SDS-PAGE with mobilities consistent with the expected size of ActRII ECD/betaA or ActRII ECD/betaAbetaA. When inhibin A was cross-linked to [125I]ActRII ECD, a slower migrating complex corresponding to ActRII ECD/betaAalpha was also observed. The apparent dissociation constant (Kd) for activin A binding to ActRII ECD was 2-7 nM. This Kd value is approximately an order of magnitude greater than that of the full-length membrane-associated type II receptor. Treatment of cultured rat anterior pituitary cells with ActRII ECD attenuated FSH secretion in response to exogenous activin A or endogenous activin B. These data indicate that the soluble ActRII ECD has structural determinants that are sufficient for high affinity ligand binding.
Donaldson
C J
CJ
The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, California 92037, USA.
Vaughan
J M
JM
Corrigan
A Z
AZ
Fischer
W H
WH
Vale
W W
WW
D029404:Activin Receptors; D028341:Activins; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002478:Cells, Cultured; D003432:Cross-Linking Reagents; D005110:Extracellular Space / Q000737:chemistry; D005640:Follicle Stimulating Hormone / Q000378:metabolism; D015870:Gene Expression; D007265:Inhibins / Q000378:metabolism* / Q000494:pharmacology; D007457:Iodine Radioisotopes; D051379:Mice; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010903:Pituitary Gland, Anterior / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D017978:Receptors, Growth Factor / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
D003432:Cross-Linking Reagents; D007457:Iodine Radioisotopes; D010446:Peptide Fragments; D017978:Receptors, Growth Factor; D011994:Recombinant Proteins; D028341:Activins; D007265:Inhibins; D005640:Follicle Stimulating Hormone; D029404:Activin Receptors