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First published online 28 March 2006
doi: 10.1242/jcs.02845

Journal of Cell Science 119, 1666-1676 (2006)
Published by The Company of Biologists 2006
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Interaction of SH2-Bß with RET is involved in signaling of GDNF-induced neurite outgrowth

Yong Zhang*, Wei Zhu*, Yong-Gang Wang, Xiu-Jie Liu, Li Jiao, Xuan Liu, Zhao-Huan Zhang, Chang-Lin Lu and Cheng He{ddagger}

Department of Neurobiology, Second Military Medical University, Shanghai, 200433, P. R. of China


Figure 1
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  		Fig. 1. RET interacts with SH2-Bß in a two-hybrid screen. (A) Schematic representation of the pGilda-RETIC bait. Cad, Cadherin domain; Cys, cysteine-rich domain; TM, transmembrane domain; TK, tyrosine kinase domain; LexA, LexA fusion vector pGilda. (B) The human SH2-Bß region (amino acids 498-670) containing the SH2 domain was isolated from the two-hybrid screen. PH, pleckstrin homology domain; SH2, Src homology 2 domain. (C,D) Filter assay and liquid culture assay using o-nitrophenyl-D-galactoside (ONPG) was performed for ß-galactosidase activity analysis. Full-length wild-type SH2-Bß was co-transformed into yeast reporter strain EGY48 with the bait encoding the intracellular domain of RET, TrkA or EGF receptor. To determine the binding domain of the interaction between SH2-Bß and RET, RETIC bait was transformed with the SH2 domain, the PH domain of SH2-Bß or R555E (SH2-Bß dominant-negative mutant). Positive and negative controls are described in Materials and Methods.

 

Figure 2
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  		Fig. 2. GDNF stimulates interaction of RET with SH2-Bß in PC12-GFR{alpha}1-RET cells. (A) PC12-GFR{alpha}1-RET cells were transfected with pcDNA3, pcDNA3-myc-SH2-Bß, or pcDNA3-myc-R555E respectively. Cells were treated or not with 100 ng/ml GDNF for 10 minutes before lysed on ice and collected. Then, cell lysates were immunoprecipitated (IP) with anti-myc or anti-RET antibodies, followed by immunoblotting (IB) with anti-RET or anti-myc antibodies. Cell lysates were also subjected to immunoblotting with anti-RET or anti-myc antibodies. (B) Cultured PC12 cells or PC12-GFR{alpha}1-RET cells were treated or not with 100 ng/ml GDNF for 10 minutes before they were lysed and collected. Cell lysates were immunoprecipitated with anti-RET or anti-SH2-Bß antibodies, followed by immunoblotting with anti-SH2-Bß or anti-RET antibodies. Expression of RET and SH2-Bß in cells was determined by immunoblotting with anti-RET or anti-SH2-Bß antibodies. IP, immunoprecipitation; IB, immunoblot.

 

Figure 3
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  		Fig. 3. Identification of the binding site in RET crucial for interaction with SH2-Bß. HEK293T cells were co-transfected with wild-type RET or the indicated RET mutants, together with GFR{alpha}1 and SH2-Bß; cells were then stimulated with 100 ng/ml GDNF for 10 minutes. Cell lysates were immunoprecipitated with anti-RET or anti-SH2-Bß antibodies, followed by immunoblotting with anti-SH2-Bß or anti-RET antibodies. Mutation of Tyr981 to Phe severely attenuates the interaction between RET and SH2-Bß. WT, wild type; K, lysine; M, methionine; Y, tyrosine; F, phenylalanine.

 

Figure 4
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  		Fig. 4. Endogenous SH2-Bß interacts with RET in rat-tissue homogenates. Different tissue homogenates from mesencephalon, spinal cord, kidney and muscle were prepared as described in Materials and Methods, and then immunoprecipitated with anti-RET or anti-SH2-Bß antibodies, followed by immunoblotting with anti-SH2-Bß or anti-RET antibodies. Protein expressions in tissues were visualized by immunoblotting with anti-SH2-Bß or anti-RET antibodies.

 

Figure 5
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  		Fig. 5. SH2-Bß and RET are colocolized in rat mesencephalic neurons. (A) The tissue slices from rat mesencephalon were first immunostained with anti-SH2-Bß antibodies (green) and then immunostained with anti-RET antibodies (red). The merged images indicate that SH2-Bß and RET can colocolize in red nucleus neurons (yellow) of rat mesencephalon. Bars, 40 µm (top), 20 µm (bottom). (B) Subcellular localization of SH2-Bß in primary cultured rat mesencephalic neurons. The distribution of SH2-Bß (green) is shown by immunofluorescence, its colocalization with either the dendrite marker MAP-2 or the axon marker tau-1 (red) is shown in the merged images (yellow). Bar, 20 µm.

 

Figure 6
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  		Fig. 6. SH2-Bß is involved in GDNF-induced neuronal differentiation of PC12-GFR{alpha}1-RET cells. (A) PC12-GFR{alpha}1-RET cells were co-transfected with pEGFP-N2 (encoding GFP) in the ratio of 1:10 with pcDNA3 as control, pcDNA3-myc-SH2-Bß or R555E. The cells were visualized using fluorescence microscopy (magnification 20x) based on the expression of the GFP. (B) Quantification of neuronal differentiation of PC12-GFR{alpha}1-RET cells. Values are the mean ± s.d. of three independent experiments performed in triplicate culture wells. Three fields were examined from each well. *, significant difference between SH2-Bß/GFP and pcDNA3/GFP control (P<0.05, factorial ANOVA); ##, significant difference between R555E/GFP and control (P<0.01, factorial ANOVA). (C) Decrease in GDNF-induced differentiation of PC12-GFR{alpha}1-RET cells by SH2-Bß-siRNA. PC12-GFR{alpha}1-RET cells cultured on 24-well plates were transfected with pEGFP-N2 alone or together with missense RNA (20 µM) as control, or with SH2-Bß-siRNA (20 µM). (D) Quantification of neuronal differentiation of PC12-GFR{alpha}1-RET cells. Values are the mean ± s.d. of three independent experiments. **, significant difference between RNAi group and missense RNA group (P<0.01, factorial ANOVA). (E) Effect of overexpression or knock-down of SH2-Bß on GDNF-induced activation of ERK and Akt in PC12-GFR{alpha}1-RET cells. PC12-GFR{alpha}1-RET cells were transfected with plasmids pcDNA3 (control), pcDNA3-myc-SH2-Bß, pcDNA3-myc-R555E, missense RNA or SH2-Bß-siRNA. Cells were stimulated with or without 100 ng/ml GDNF for 10 minutes and then lysed. Equal amounts of total protein of cell lysates were immunoblotted with anti-SH2-Bß, anti-pERK, anti-ERK, anti-pAkt or anti-Akt antibody.

 

Figure 7
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  		Fig. 7. SH2-Bß promotes neurite outgrowth in cultured mesencephalic neurons. Cultured mesencephalic neurons were co-transfected with pEGFP-N2 in the ratio of 1:10 together with pcDNA3 (control), pcDNA3-myc-SH2-Bß or R555E. Images were taken under fluorescence microscopy (magnification 20x) to identify GFP-positve neurons. (B) Quantification of neurite outgrowth in cultured mesencephalic neurons. Metamorph image-analysis software was used to quantify the length of the longest neurite. Data are presented as the mean ± s.d. of three independent experiments performed in triplicate experiments. **, significant difference between SH2-Bß/GFP and pcDNA3/GFP control (P<0.01, factorial ANOVA); ##, significant difference between R555E/GFP and control (P<0.01, factorial ANOVA).

 

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