Fig. 4: Mammalian GFRα1 (mGFRα1) pentameric interface is required for GDNF-dependent adhesion in cell-clustering and neuronal dendritic spine assays.

a Cell adhesion assay using HEK293T cells transiently transfected with control pcDNA plasmid or mGFRα1^FL in the presence and absence of GDNF. White scale bar is 100 μm. b Effect of mutational analysis of mGFRα1 pentameric interface in HEK293T cell adhesion assay. The level of cell adhesion promoted by transfected proteins with and without GDNF treatment evaluated as the percentage of GFP+ cells present in aggregates more than 5 cells/field ± s.e.m. n ≥3 biologically independent experiments. c Schematic illustration to demonstrate impact of cis GFRα1 pentamer interface mutations on the ability of GFRα1 to act as a cell adhesion molecule. GPI-anchored GFRα1 coloured green and soluble GDNF yellow. For GFRα1 wild-type, adhesion is driven through both GDNF-dependent interactions in trans and GFRα1 homophilic contribution in cis (top). For GFRα1 mutants that target the pentamer interface, GDNF mediates adhesion through bridging GFRα1 molecules in trans (bottom). d The presence of soluble RET^ECM interferes with GDNF-induced adhesion of GFRα1-expressing HEK293T cells. HEK293T cells expressing mGFRα1 and GFP were preincubated with human RET^ECM for 2 h in the absence of GDNF. GDNF was then added for an additional 2 h at room temperature. The bar graph indicate the percentage of GFP⁺ cells in aggregates greater than 5 cells ± s.e.m. n = 3 biologically independent experiments. e In the presence of GDNF, mGFRα1 mutants reduce spine density of dissociated hippocampal neurons. Hippocampal neurons transfected with GFP-expressing plasmid in combination with indicated constructs at 15 DIV, maintained in the absence or presence of GDNF as indicated in the figure for 72 h. Arrows indicated dendritic spines along the dendritic shaft. Scale bar: 50 μm. f Quantification of total dendritic spines along 100 µm of dendritic length of hippocampal neurons. Mean ± s.e.m. from three independent experiments (n = 14–32 neurons/condition). Dashed line indicates dendritic spine density on neurons transfected with empty vector cultured in the absence of GDNF. b *p = 0.0154, **p = 0.0016, ***p = 0.0002, ****p < 0.0001; (d) *p = 0.0037, **p = 0.007, ***p = 0.0002; (f) *p = 0.03, **p = 0.0236, ***p = 0.0041, ****p = 0.0012, ^#p < 0.0001. One-way ANOVA, followed by Tukey´s multiple comparison test.