Acrp30/adiponectin is an adipocyte-specific secretory protein that has recently been implicated as a mediator of systemic insulin sensitivity with liver and muscle as target organs. Acrp30 is found as two forms in serum, as a lower molecular weight trimer-dimer and a high molecular weight complex. Little is know about the regulation and significance of these Acrp30 complexes in serum and about the events that lead to the generation of the bioactive ligand. Here, we show that there is a profound sexual dimorphism of Acrp30 levels and complex distribution in serum. Female mice display significantly higher levels of the high molecular weight complex in serum than males. In both females and males, levels of the high molecular weight complex are significantly reduced in response to a systemic increase of insulin. The ratio of the two complexes is restored upon normalization of glucose levels. Structurally, we show that oligomer formation of Acrp30 critically depends on disulfide bond formation mediated by Cys-39. Mutation of Cys-39 results in trimers that are subject to proteolytic cleavage in the collagenous domain. Surprisingly, Acrp30(C39S) or wild-type Acrp30 treated with dithiothreitol are significantly more bioactive than the higher order oligomeric forms of the protein with respect to reduction of serum glucose levels. Furthermore, treatment of primary hepatocytes with trimeric and higher order forms of Acrp30 confirms that the increased bioactivity seen in vivo is reflected in an augmented potency to reduce glucose output in the presence of gluconeogenic stimuli. Combined, these results shed new light on the regulation of this complex protein and suggest a new model for in vivo activation of the protein, implicating a serum reductase activity.Adipose has been under appreciated as an endocrine tissue for decades because of the prevalent opinion that it served merely as storage for lipids. Recently, however, the importance of adipocytes to whole body energy homeostasis and metabolism has been underscored by several reports focusing on secreted products of adipocytes (1-4). There has been increased interest in adipose tissue as an endocrine organ, and several of these secreted proteins, termed adipokines, are currently undergoing extensive study regarding roles as divergent as feeding behavior to cardiovascular protection. For instance, leptin, the gene disrupted in ob/ob mice, has central roles in the hypothalamus, as well as peripheral effects in liver, muscle, and endothelial cells (5). Other adipose-secreted products, such as tumor necrosis factor ␣ and adipsin (complement factor D), have well established functions in innate immunity (6 -9). The recently identified adipokine resistin has been implicated as a modulator of insulin sensitivity and is also being studied for its effects on metabolism (4, 10).Acrp30 (also known as adiponectin, AdipoQ, and GBP28) is an adipokine exclusively synthesized and secreted by adipocytes (11-14). Acrp30 has recently been shown to influence glucose homeostasis and insulin se...

No abstract

Laminin is a large (900 kDa) mosaic protein composed of many distinct domains with different structures and functions. Globular and rodlike domains are arranged in an extended four-armed, cruciform shape that is well suited for mediating between distant sites on cells and other components of the extracellular matrix. The alpha-helical coiled-coil domain of the long arm is involved in the specific assembly of the three chains (A, B1, B2, and possible variants) of laminin and is the only domain composed of multiple chains. It is terminated by a large globular domain composed of five homologous subdomains formed by the COOH-terminal part of the A chain. Sites for receptor-mediated cell attachment and promotion of neurite outgrowth reside in the terminal region of the long arm. A second cell attachment site, a cell signaling site with mitogenic action, binding sites for the closely associated glycoprotein nidogen/entactin, and regions involved in calcium-dependent aggregation are localized in the short arms. These domains, which to a large extent are composed of Cys-rich repeats with limited homology to EGF, are the most highly conserved regions in laminins of different origin. At present, most structural and functional data have been collected for a laminin expressed by a mouse tumor, which can be readily isolated in native form and dissected into functional fragments by limited proteolysis. Increasing information on laminins from different species and tissues demonstrates considerable variations of structure. Isoforms of laminin assembled from different chains are focally and transiently expressed and may serve distinct functions at early stages of development even before being laid down as major components of basement membranes.

Electron microscopy of ECADCOMP, a recombinant E-cadherin ectodomain pentamerized by the assembly domain of cartilage oligomeric matrix protein, has been used to analyze the role of cis-dimerization and trans-interaction in the homophilic association of this cell adhesion molecule. The Ca 2⍣ dependency of both interactions was investigated. Low Ca 2⍣ concentrations (50 μM) stabilized the rod-like structure of E-cadherin. At medium Ca 2⍣ concentration (500 μM), two adjacent ectodomains in a pentamer formed cis-dimers. At high Ca 2⍣ concentration (>1 mM), two cis-dimers from different pentamers formed a trans-interaction. The X-ray structure of an N-terminal domain pair of E-cadherin revealed two molecules per asymmetric unit in an intertwisted X-shaped arrangement with closest contacts in the Ca 2⍣ -binding region between domains 1 and 2. Contrary to previous data, Trp2 was docked in the hydrophobic cavity of its own molecule, and was therefore not involved in cis-dimerization of two molecules. This was supported further by W2A and A80I (a residue involved in the hydrophobic cavity surrounding Trp2) mutations in ECADCOMP which both led to abrogation of the trans-but not the cisinteraction. Structural and biochemical data suggest a link between Ca 2⍣ binding in the millimolar range and Trp2 docking, both events being essential for the trans-association.

No abstract