Characterization of the ligand binding functionality of the extracellular domain of activin receptor type IIb.
The single transmembrane domain serine/threonine kinase activin receptor type IIB (ActRIIB) has been proposed to bind key regulators of skeletal muscle mass development, including the ligands GDF-8 (myostatin) and GDF-11 (BMP-11). Here we provide a detailed kinetic characterization of ActRIIB binding to several low and high affinity ligands using a soluble activin receptor type IIB-Fc chimera (ActRIIB.Fc). We show that both GDF-8 and GDF-11 bind the extracellular domain of ActRIIB with affinities comparable with those of activin A, a known high affinity ActRIIB ligand, whereas BMP-2 and BMP-7 affinities for ActRIIB are at least 100-fold lower. Using site-directed mutagenesis, we demonstrate that ActRIIB binds GDF-11 and activin A in different ways such as, for example, substitutions in ActRIIB Leu(79) effectively abolish ActRIIB binding to activin A yet not to GDF-11. Native ActRIIB has four isoforms that differ in the length of the C-terminal portion of their extracellular domains. We demonstrate that the C terminus of the ActRIIB extracellular domain is crucial for maintaining biological activity of the ActRIIB.Fc receptor chimera. In addition, we show that glycosylation of ActRIIB is not required for binding to activin A or GDF-11. Together, our findings reveal binding specificity and activity determinants of the ActRIIB receptor that combine to effect specificity in the activation of distinct signaling pathways.
Sako
Dianne
D
Acceleron Pharma, Cambridge, MA 02139, USA.
Grinberg
Asya V
AV
Liu
June
J
Davies
Monique V
MV
Castonguay
Roselyne
R
Maniatis
Silas
S
Andreucci
Amy J
AJ
Pobre
Eileen G
EG
Tomkinson
Kathleen N
KN
Monnell
Travis E
TE
Ucran
Jeffrey A
JA
Martinez-Hackert
Erik
E
Pearsall
R Scott
RS
Underwood
Kathryn W
KW
Seehra
Jasbir
J
Kumar
Ravindra
R
D030301:Activin Receptors, Type II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D018076:DNA, Complementary / Q000235:genetics; D017930:Genes, Reporter; D006801:Humans; D008024:Ligands; D016296:Mutagenesis; D050506:Mutant Chimeric Proteins / Q000737:chemistry / Q000378:metabolism; D055435:Myostatin / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D010960:Plasminogen Activators / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
D018076:DNA, Complementary; D008024:Ligands; D050506:Mutant Chimeric Proteins; D055435:Myostatin; D011993:Recombinant Fusion Proteins; D030301:Activin Receptors, Type II; C425584:activin receptor type II-B; D010960:Plasminogen Activators
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