Characterization of the Extracellular Ligand-Binding Domain of the Type II Activin Receptor

Abstract

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.

Mutations of the TGF-β type II receptorBMPR2 in pulmonary arterial hypertension

“…Importantly, in these two exons, amino acid substitutions represent the most common type of mutation, indicating conserved structural and functional requirements of this region for BMPR-II signaling activity. In vitro mutagenesis and X-ray crystallography studies identified 10 cysteine residues conserved across the related type II BMP, MIS, and activin receptors, each of which is necessary for receptor ligand protein folding of the ligand domain [Greenwald et al, 1999]. FPAH-causing missense mutations have now been identified for eight of the 10 cysteine residues, and of those studied by transient transfection of GFP-or myc-tagged BMPR2 constructs, each appears to lead to significant protein misfolding and/or cytoplasmic retention [Nishihara et Frequency refers to the number of times recurrent mutation has been identi¢ed by each investigation.…”

Structural and functional characterizations of activin type 2B receptor (acvr2b) ortholog from the marine fish, gilthead sea bream, Sparus aurata: evidence for gene duplication of acvr2b in fish

“…Yet another possibility is that differences exist between the affinity of Acvr2b-1 and -2 to MSTN1 and MSTN2. Interestingly, a 1000-fold excess of mouse ACVR2B-ECD to activin was needed in order to achieve about 50% inhibition of activin-induced FSH secretion from pituitary cells (Greenwald et al 1998). This later study showed binding between the two by cross-linking experiments, using radiolabeled activin and mouse ACVR2-ECD expressed in P. pastoris.…”

Molecular cloning, characterization and tissue distribution of six splice variants of activin type IIA receptor (ActRIIA) from grass carp (Ctenopharyngodon idellus)

“…Alignment of deduced amino acid sequences was performed by using DNAMAN software (Lynnon Biosoft, Canada). In order to understand the secondary structures of the extracellular domain and kinase domain of grass carp ActRIIA, secondary structure-based alignment was conducted with the known structures of mouse ActRII and human ActRIIB (Greenwald et al, 1999;Han et al, 2007). Furthermore, modeling of tertiary structure for grass carp ActRIIA was achieved by using the automated compara-tive protein modeling server SWISS-MODEL (http:// swissmodel.expasy.org) with the first approach mode.…”

“…Furthermore, modeling of tertiary structure for grass carp ActRIIA was achieved by using the automated compara-tive protein modeling server SWISS-MODEL (http:// swissmodel.expasy.org) with the first approach mode. In this case, the crystal structures of the extracellular ligand-binding domain of mouse ActRII (PDB 1bteB) (Greenwald et al, 1999) and human ActRIIB kinase domain (PDB 2qluA) (Han et al, 2007) were utilized as the templates to create the ligand-binding domain and the intracellular kinase domain of grass carp ActRIIA, respectively. The three-dimensional (3D) models were visualized with Molsoft ICM software (Molsoft L.L.C., USA).…”

“…Upon ligand binding, type II receptors (ActRIIA and ActRIIB) recruit and phosphorylate type I receptors at the serine residues within the highly conserved GS domain (a glycine/serine-rich region). The activated type I receptors in turn phosphorylate receptor-regulated Smads (R-Smad, Smad2 and Smad3), allowing R-Smad to form a heterocomplex with co-mediator Smad (Smad4), and the Smad complex is translocated into the nucleus to regulate target gene transcription (Greenwald et al, 1999;Shi and Massague, 2003). Therefore, type II receptors are responsible for ligand recognition and binding, as well as signal transduction in the activin signaling pathway.…”