We report the expression cloning and characterization of GDNFR-alpha, a novel glycosylphosphatidylinositol-linked cell surface receptor for glial cell line-derived neurotrophic factor (GDNF). GDNFR-alpha binds GDNF specifically and mediates activation of the Ret protein-tyrosine kinase (PTK). Treatment of Neuro-2a cells expressing GDNFR-alpha with GDNF rapidly stimulates Ret autophosphorylation. Ret is also activated by treatment with a combination of GDNF and soluble GDNFR-alpha in cells lacking GDNFR-alpha, and this effect is blocked by a soluble Ret-Fc fusion protein. Ret activation by GDNF was also observed in cultured embryonic rat spinal cord motor neurons, a cell type that responds to GDNF in vivo. A model for the stepwise formation of a GDNF signal-transducing complex including GDNF, GDNFR-alpha, and the Ret PTK is proposed.

The receptor for glial cell line-derived neurotrophic factor (GDNF) consists of GFR␣-1 and Ret. Neurturin is a GDNF-related neurotrophin whose receptor is presently unknown. Here we report that neurturin can bind to either GFR␣-1 or GFR␣-2, a novel receptor related to GFR␣-1. Both GFR␣-1 and GFR␣-2 mediate neurturininduced Ret phosphorylation. GDNF can also bind to either GFR␣-1 or GFR␣-2, and activate Ret in the presence of either binding receptor. Although both ligands interact with both receptors, cells expressing GFR␣-1 bind GDNF more efficiently than neurturin, while cells expressing GFR␣-2 bind neurturin preferentially. Crosslinking and Ret activation data also suggest that while there is cross-talk, GFR␣-1 is the primary receptor for GDNF and GFR␣-2 exhibits a preference for neurturin. We have also cloned a cDNA that apparently codes for a third member of the GFR␣ receptor family. This putative receptor, designated GFR␣-3, is closely related in amino acid sequence and is nearly identical in the spacing of its cysteine residues to both GFR␣-1 and GFR␣-2. Analysis of the tissue distribution of GFR␣-1, GFR␣-2, GFR␣-3, and Ret by Northern blot reveals overlapping but distinct patterns of expression. Consistent with a role in GDNF function, the GFR␣s and Ret are expressed in many of the same tissues, suggesting that GFR␣s mediate the action of GDNF family ligands in vivo.

Previous studies have suggested that the daidzein metabolite equol rather than daidzein itself contributes to the beneficial effect of soya foods in the prevention of CVD. The aim of the present study is to examine the proportion of equol excretion in Chinese adults and compare plasma lipids and carotid artery intima -media thickness (IMT) between equol excretors and non-excretors, and to evaluate the effect of soya isoflavone intakes on serum lipids and IMT in either equol excretors or non-excretors. Subjects (n 572; women n 362, men n 210) were recruited for the present study. An overnight urine sample was provided by each subject on their usual diet to quantify urinary concentrations of daidzein and equol. Far-wall IMT was determined by B-mode ultrasound in the right carotid at two sites, carotid bulb (CB-IMT) and common carotid artery (CCA-IMT), and fasting serum lipids were measured. Habitual dietary intakes were estimated with a FFQ, and soya isoflavone intake derived from the FFQ was assessed. Of the 572 subjects, the proportion of equol excretors on their usual diet was 25·0 % (n 143). Compared with non-excretors, equol excretors showed significantly lower serum TAG (238·2 (95 % CI 270·4, 25·9) %, P¼0·012) and CCA-IMT (2 4·9 (95 % CI 2 9·7, 2 0·3) %, P¼ 0·033). Equol excretors with higher daily isoflavone intakes (2 5·4 mg/d) had significantly lower IMT (2 16·2 %, P¼ 0·035) and tended to have higher HDL-cholesterol (P¼0·055) than did those with lower daily isoflavone intakes (1·5 mg/d), while no association was observed between soya isoflavone intakes and serum lipids or IMT in non-excretors. In conclusion, the benefits of soya isoflavones in preventing CVD may be apparent among equol excretors only.

Drug-induced cardiac arrhythmia, specifically Torsades de pointes, is associated with QT/QTc interval prolongation, thus prolongation of the QT interval is considered as a biomarker for Torsades de pointes risk (N Engl J Med 350:1013-1022, 2004). Specific inhibition of human ether-a-go-go-related gene (hERG) potassium channels has been recognized as the main mechanism for QT prolongation (Cardiovasc Res 58:32-45, 2003). This mechanism has been demonstrated for a variety of smallmolecule agents, which access the inner pore of the hERG channel preferentially from inside the cell. Peptide inhibitors of hERG, such as BeKm-1, interact with the extracellular amino acid residues close to the external pore region of the channel. In this study, the isolated rabbit heart was used to assess whether BeKm-1 could induce QTc prolongation like dofetilide and N- [4-[[1-[2-(6-methyl-2-pyridinyl) ethyl]-4-piperidinyl]carbonyl]phenyl]methanesulfonamide (E-4031). Five hearts were perfused with 10 and 100 nM BeKm-1 sequentially. ECG parameters and left ventricular contractility were measured with spontaneously beating hearts. Both concentrations of BeKm-1 prolonged QTc intervals significantly and concentration-dependently (4.7 and 16.3% at 10 and 100 nM, respectively). When evaluated for their inhibitory effect in a hERG functional assay, BeKm-1, dofetilide, and E-4031 caused QTc prolongation at concentrations that caused significant hERG channel inhibition. Lastly, two polyclonal anti-hERG antibodies were also assessed in the hERG channel assay and found to be devoid of any inhibitory effect. These results indicated that the isolated rabbit heart assay can be used to measure QTc changes caused by specific hERG inhibition by peptides that specifically block the external pore region of the channel.

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