Alpha 2-macroglobulin conformation determines binding affinity for activin A and plasma clearance of activin A/alpha 2-macroglobulin complex

Abstract

Activin A is a member of the transforming growth factor-beta family of growth factors and a potent regulator of cellular activity. A number of binding proteins for activin A have been identified, including alpha 2-macroglobulin (alpha 2M). Alpha 2M has several conformational states that are known to have different growth factor-binding properties. The effect of alpha 2M conformation on activin A binding has not been characterized. The aims of this study were to determine 1) whether activin A binds preferentially to the native (alpha 2M-N) or "activated" (alpha 2M*) conformation of alpha 2M, 2) the affinity of different alpha 2M conformations for activin A, and 3) the fate of activin A complexed with alpha 2M-N or alpha 2M* in vivo. [125I]Activin A associated with alpha 2M in plasma and follicular fluid and with purified alpha 2Ms. In this qualitative assay, more activin A was associated with alpha 2M* than with alpha 2M-N. The affinity of the activin A-alpha 2M interaction was determined. The Kd values for activin A-alpha 2M* and activin A-alpha 2M-N were 190 +/- 30 and 510 +/- 60 nM, respectively. The plasma clearance profiles and tissue distribution of uncomplexed activin A and purified alpha 2M*-activin A complex were determined. Radiolabeled activin A cleared in a biphasic manner, with rapid clearance over the initial 10 min and substantially slower clearance over the subsequent 20 min. During the slow phase of clearance, activin A formed a complex with circulating alpha 2M-N. In contrast, radiolabeled activin A-alpha 2M* complexes were rapidly cleared from plasma with a half-life of approximately 5 min and were specifically targeted to alpha 2M receptors in vivo. These studies reveal that alpha 2M can maintain activin A in the circulation or rapidly target the hormone for plasma clearance depending on the conformational state of the carrier protein in vivo.

Hepcidin Bound to α2-Macroglobulin Reduces Ferroportin-1 Expression and Enhances Its Activity at Reducing Serum Iron Levels

“…complexed at a 4:1 ratio) was injected into the tail vein. Within 10 s of injection, an aliquot (5 l) of blood was withdrawn from a tail snip, and this measurement was taken to represent 100% of total circulating radiation (8,36,37). Blood samples (5 l) were collected at predetermined times (1-60 min) in Terumo Capiject Micro-Collection tubes (Somerset, NJ), and the radioactivity was determined using the gamma counter above.…”

“…The transport of hepcidin within the blood is of interest as: 1) many peptide hormones are bound by carriers that influence their function and distribution (7)(8)(9)(10)(11)(12)(13); 2) a greater understanding of the mechanism of hepcidin delivery to the cell could provide further information on its regulatory role; and 3) understanding the binding of hepcidin to a carrier protein would facilitate development of quantitative hepcidin assays.…”

“…defensin (HNP1) (17), transforming growth factor-␤1/2, etc. (7)(8)(9)(10)(11)(12)(13)24). The consequence of these interactions with ␣ 2 M/␣ 2 M-MA can be passive (24) or stimulatory (10).…”

Expression of Panza, an α2-macroglobulin, in a restricted dorsal domain of the primitive gut in Xenopus laevis

“…The conformational change that occurs with α 2 M activation also exposes binding sites for nonprotease ligands, including PDGF (Huang et al, 1984), FGF (Mathew et al, 2003), TGFβ1 (Huang et al, 1988;Stouffer et al, 1993;Feige et al, 1996;Arandjelovic et al, 2003), Activin A (Niemuller et al, 1995;Mather, 1996;Phillips, 2000), NGF (Ronne et al, 1979), TNF (James et al, 1992) and multiple Interleukins (Borth and Luger, 1989;Borth et al, 1990;Garber et al, 2000;Kurdowska et al, 2000). In complex with these ligands, α 2 M can serve either as a carrier that stabilizes ligand in circulation, a clearance factor for ligand degradation, or as a targeting factor for cellular uptake and subcellular localization of ligand.…”

“…In complex with these ligands, α 2 M can serve either as a carrier that stabilizes ligand in circulation, a clearance factor for ligand degradation, or as a targeting factor for cellular uptake and subcellular localization of ligand. For example, native α 2 M is an Activin A carrier that stabilizes circulating levels, while activated α 2 M clears circulating Activin A via LRP-mediated internalization (Niemuller et al, 1995). The ability of α 2 M to bind and regulate such a diverse group of ligands suggests that α 2 M may modulate multiple signaling and morphogenetic processes in the embryo and adult.…”