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.

Crystal structure of an HSA/FcRn complex reveals recycling by competitive mimicry of HSA ligands at a pH-dependent hydrophobic interface.

The long circulating half-life of serum albumin, the most abundant protein in mammalian plasma, derives from pH-dependent endosomal salvage from degradation, mediated by the neonatal Fc receptor (FcRn). Using yeast display, we identified human serum albumin (HSA) variants with increased affinity for human FcRn at endosomal pH, enabling us to solve the crystal structure of a variant HSA/FcRn complex. We find an extensive, primarily hydrophobic interface stabilized by hydrogen-bonding networks involving protonated histidines internal to each protein. The interface features two key FcRn tryptophan side chains inserting into deep hydrophobic pockets on HSA that overlap albumin ligand binding sites. We find that fatty acids (FAs) compete with FcRn, revealing a clash between ligand binding and recycling, and that our high-affinity HSA variants have significantly increased circulating half-lives in mice and monkeys. These observations open the way for the creation of biotherapeutics with significantly improved pharmacokinetics.