Numerous studies have demonstrated that estrogens induce rapid and transient activation of the Src͞Erk phosphorylation cascade. Activation of this cascade triggers vital cellular functions including cell proliferation and differentiation. However, the details of the molecular mechanism of this process remain to be elucidated. We have identified a previously uncharacterized nuclear receptor-interacting protein designated as modulator of nongenomic activity of estrogen receptor (MNAR). Here we show that MNAR modulates estrogenreceptor (ER) interaction with members of the Src family of tyrosine kinases, which leads to a stimulation of Src enzymatic activity and activation of Erk1 and Erk2 kinases. We also show that MNAR, through activation of the Src͞Erk phosphorylation cascade, affects ER transcriptional activity and ultimately ER-mediated gene expression. These data reveal that MNAR mediates the crosstalk between two important classes of signal transducing molecules and suggest that ER ''genomic'' and ''nongenomic'' activities are interrelated.

, and the complex formation between eNOS and heat shock protein 90, resulting in a marked reduction of NO production. Adenovirus-mediated overexpression of a constitutively active version of AMPK reversed these changes. In db/db diabetic mice, both APPL1 expression and adiponectin-induced vasodilation were significantly decreased compared with their lean littermates. Taken together, these results suggest that APPL1 acts as a common downstream effector of AdipoR1 and -R2, mediating adiponectin-evoked endothelial NO production and endothelium-dependent vasodilation. Diabetes 56: [1387][1388][1389][1390][1391][1392][1393][1394] 2007 E ndothelial dysfunction, characterized by decreased production and/or bioactivity of nitric oxide (NO) and impaired endothelium-dependent vasodilation, is a key mediator that links obesity, diabetes, and cardiovascular diseases (1). Dysfunction of the endothelium in conduit arteries is a well-established antecedent of hypertension and atherosclerosis, whereas dysfunction of peripheral vascular endothelium at the arteriolar and capillary level contributes to the pathogenesis of insulin resistance and the metabolic syndrome (2). On the other hand, insulin resistance aggravates endothelial dysfunction. Therapeutic interventions in animal models and humans have demonstrated that improving endothelial function ameliorates insulin resistance, while increasing insulin sensitivity alleviates endothelial dysfunction (3).Adiponectin, an insulin-sensitizing adipokine secreted predominantly from adipocytes, possesses potent protective effects against endothelial dysfunction (4). Unlike most adipokines, plasma levels of adiponectin are decreased in obese individuals and patients with insulin resistance, type 2 diabetes, and cardiovascular diseases. An independent association between serum levels of adiponectin and endothelium-dependent vasodilation has been repeatedly documented (5-7). Hypoadiponectinemia has been closely linked to impairment in endotheliumdependent vasodilation in both normal subjects and patients with hypertension and type 2 diabetes. Consistent with these clinical findings, adiponectin-deficient mice exhibit reduced endothelium-dependent vasodilation on an atherogenic diet (6), increased neointimal hyperplasia after acute vascular injury (8,9), and elevated blood pressure compared with their wild-type littermates (10). On the other hand, both adenovirus-mediated overexpression of full-length adiponectin and transgenic overexpression of globular adiponectin result in a marked alleviation of atherosclerotic lesion in apolipoprotein E-deficient mice (11) and also cause a significant amelioration of endothelial dysfunction and hypertension (10) in obese mice.The endothelium-protective functions of adiponectin are mediated, at least in part, by its ability to increase the production of NO, a vasodilator synthesized by endothelial NO synthase (eNOS) from the precursor L-arginine (4,7,12). NO protects the vascular system by enhancing vasodilation and inhibiting platelet aggregation,...

The double-stranded RNA (dsRNA)-activated protein kinase (PKR) plays a major role in the innate immune response in humans. PKR binds dsRNA non-sequence specifically and requires a minimum of 15 bp dsRNA for one protein to bind and 30 bp dsRNA to induce protein dimerization and activation by autophosphorylation. PKR phosphorylates eIF2α, a translation initiation factor, resulting in the inhibition of protein synthesis. We investigated the mechanism of PKR activation by an RNA hairpin with a number of base pairs intermediate between these 15 to 30 bp limits: HIV-I TAR RNA, a 23 bp hairpin with three bulges that is known to dimerize. To test whether RNA dimerization affects PKR dimerization and activation, TAR monomers and dimers were isolated from native gels and assayed for RNA and protein dimerization. To modulate the extent of dimerization, we included TAR mutants with different secondary features. Native gel mixing experiments and analytical ultracentrifugation indicate that TAR monomers bind one PKR monomer and that TAR dimers bind two or three PKRs, demonstrating that RNA dimerization drives the binding of multiple PKR molecules. Consistent with functional dimerization of PKR, TAR dimers activated PKR while TAR monomers did not, and RNA dimers with fewer asymmetrical secondary structure defects, as determined by enzymatic structure mapping, were more potent activators. Thus, the secondary structure defects in the TAR RNA stem function as antideterminants to PKR binding and activation. Our studies support that dimerization of a 15-30 bp hairpin RNA, which effectively doubles its length, is a key step in driving activation of PKR and provide a model for how RNA folding can be related to human disease. Keywords protein kinase; RNA folding; innate immunity; analytical ultracentrifugation; RNA-protein interaction

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