Abstract
Acrp30/adiponectin is an adipocyte-derived serum protein with important roles in regulation of lipid and glucose metabolism, but which of its isoforms are biologically active remains controversial. We addressed this issue by first characterizing the structure of each individual Acrp30 oligomer and the determinants responsible for multimer formation. Freeze etch electron microscopy showed the trimer to exhibit a ball-and- stick-like structure containing a large globular sphere, an extended collagen stalk, and a smaller sphere on the opposite end of the stalk. The hexamer consists of two adjacent trimeric globular domains and a single stalk composed of collagen domains from two trimers. Although not necessary for trimer formation or stability, two of the three monomers in an Acrp30 trimer are covalently linked by a disulfide bond between cysteine residues at position 22. In contrast, assembly of hexameric and higher molecular weight (HMW) forms of Acrp30 depends upon formation of Cys22-mediated disulfide bonds because their reduction with dithiothreitol or substitution of Cys22 with alanine led exclusively to trimers. HMW and hexamer isoforms of Acrp30 activated NF-kappaB in C2C12 cells, but trimers, either natural, formed by reduction of Acrp30 hexamer, or formed by the C22A mutant, did not. In contrast, incubation of isolated rat extensor digitorum longus with naturally formed Acrp30 trimers or trimeric C22A Acrp30 led to increased phosphorylation of AMP-activated protein kinase-alpha at Thr172 and its activation. Hexameric and HMW Acrp30 could not activate AMP-activated protein kinase. Thus, trimeric and HMW/hexameric Acrp30 activate different signal transduction pathways, and Acrp30 represents a novel example of the control of ligand signaling via changes in its oligomerization state.
Highlights
Adipocyte complement related protein of 30 kDa (Acrp30)/adiponectin is an adipocyte-derived serum protein with important roles in regulation of lipid and glucose metabolism, but which of its isoforms are biologically active remains controversial
We performed freeze etch electron microscopy to visualize the architecture of human embryonic kidney (HEK) 293T cell-produced Acrp30 trimer, hexamer, and higher molecular weight (HMW) isoforms following their separation by gel filtration chromatography (Fig. 1)
We previously reported that globular domain of Acrp30 (gAcrp30) activated AMPK in isolated rat skeletal muscle but that full-length Acrp30 hexamer produced in E. coli did not [11]
Summary
Acrp30/adiponectin is an adipocyte-derived serum protein with important roles in regulation of lipid and glucose metabolism, but which of its isoforms are biologically active remains controversial. We addressed this issue by first characterizing the structure of each individual Acrp oligomer and the determinants responsible for multimer formation. How Acrp acts as a hormone to regulate these physiological processes remains unknown We addressed this issue by analyzing the structure of Acrp secreted from cells and in serum. Because the crystal structure of the Acrp globular domain shows a trimeric fold similar to tumor necrosis factor ␣, the collagen domain of Acrp may mediate multimerization into hexamer and HMW isoforms. Trimeric Acrp activates AMPK and hexameric/HMW Acrp activates NF-B, and we propose that the oligomerization state of Acrp determines the signaling pathway it can activate
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