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Fig. 6. SULF1 and SULF2 regulate GDNF binding to heparin and the GDNF signaling activity. (A) SULF1 reduces GDNF binding to heparin. Heparin conjugated to agarose beads was digested by SULF1 or inactive QSULF1(C-A) control. Various amounts of GDNF were incubated with enzyme-digested heparin beads (20 µl) to allow binding. The amount of GDNF bound to the beads was assayed by western blot. The intensity of individual bands was quantified by Multi-analysis software (Bio-Rad). Numbers listed beneath each lane are normalized quantification of the individual bands from three independent experiments. (B) SULF1 has no effect on GDNF binding to GFR1. Heparin predigested either by SULF1 or inactive QSULF1(C-A) was added to a mixture of GDNF (10 ng) and GFR1-Fc (1 µg) to allow GDNF-heparin-GFR1-Fc ternary complex formation. The complex was pulled down by protein A-agarose beads. The amount of GDNF bound to GFR1 was assayed by western blot and normalized to the amount of GFR1. (C-E) SULF2 enhances the GDNF signaling activity. NG108-15 cells that were stably transfected with the control vector or the SULF2 expression vector were stimulated by GDNF for 5 minutes, or by GDNF (5 ng/ml) for various lengths of time. The activation of GDNF signaling pathway was analyzed by assaying the phosphorylation of RET (at tyrosine, p-RET) and of the downstream AKT (p-AKT) by western blot. Total RET or Erks were used as loading control. Data shown are controlled for loading and then normalized to the basal level of control cells. (F) SULF2 had no effect on NGF signaling in PC12 cells. Serum-starved PC12 cells that stably expressed SULF2 or inactive QSULF1(C-A) were treated with NGF. The activation of NGF signaling was analyzed by assaying the phosphorylation of downstream Erks (p-Erk). Data presented are the mean and standard deviation of a minimum of three independent experiments. ^**, P<0.01, ^* P<0.05 (two-tailed Student's t-test). (G) Sulf double-mutant esophagi have reduced MAPK phosphorylation in the intrinsic neurons. E14.5 esophageal sections were immunostained with an antibody against phosphorylated MAPK. Arrowheads point to phosphorylated MAPK immunoreactivity in neuronal cell bodies within the muscle layers. Asterisks mark the endothelial cells with phosphorylated MAPK immunoreactivity.

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