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The deleted in brachydactyly B domain of ROR2 is required for receptor activation by recruitment of Src.

Akbarzadeh S, Wheldon LM, Sweet SM, Talma S, Mardakheh FK, Heath JK - PLoS ONE (2008)

Bottom Line: Native ROR2 phosphorylation is induced by the ligand Wnt5a and is blocked by pharmacological inhibition of Src kinase activity.Activation via tyrosine phosphorylation of ROR2 receptor leads to its internalisation into Rab5 positive endosomes.These findings show that BDB mutant receptors are defective in kinase activation as a result of failure to recruit Src.

View Article: PubMed Central - PubMed

Affiliation: CR-UK Growth Factor Group, School of Biosciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom.

ABSTRACT
The transmembrane receptor 'ROR2' resembles members of the receptor tyrosine kinase family of signalling receptors in sequence but its' signal transduction mechanisms remain enigmatic. This problem has particular importance because mutations in ROR2 are associated with two human skeletal dysmorphology syndromes, recessive Robinow Syndrome (RS) and dominant acting Brachydactyly type B (BDB). Here we show, using a constitutive dimerisation approach, that ROR2 exhibits dimerisation-induced tyrosine kinase activity and the ROR2 C-terminal domain, which is deleted in BDB, is required for recruitment and activation of the non-receptor tyrosine kinase Src. Native ROR2 phosphorylation is induced by the ligand Wnt5a and is blocked by pharmacological inhibition of Src kinase activity. Eight sites of Src-mediated ROR2 phosphorylation have been identified by mass spectrometry. Activation via tyrosine phosphorylation of ROR2 receptor leads to its internalisation into Rab5 positive endosomes. These findings show that BDB mutant receptors are defective in kinase activation as a result of failure to recruit Src.

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Fc-ROR2 WT is tyrosine phosphorylated.A) A schematic diagram of the full length ROR2 and Fc-ROR2 chimeras used in this study. ‘Kinase dead’ (KD) mutants were constructed by mutating three Lysine residues within the ATP binding pocket to Argenine. B) Fc fusion chimeras of the intracellular domains from ROR2 WT, ROR2 KD, ROR2 Y755X and, as a control, the Fc-transmembrane domain of FGFR1 fusion were expressed in chondrocytes. The chimeric receptors were immunoprecipitated using Protein-A-sepharose (IP-PA) and analysed by Western blotting following 10% SDS-PAGE. Receptor phosphorylation was detected by a cocktail of phosphotyrosine antibodies (4G10/pY20; WB-pY, upper panel). The membrane was stripped and reprobed with anti-IgG1 antibody to compare receptor expression levels (WB-Fc, lower panel).
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pone-0001873-g001: Fc-ROR2 WT is tyrosine phosphorylated.A) A schematic diagram of the full length ROR2 and Fc-ROR2 chimeras used in this study. ‘Kinase dead’ (KD) mutants were constructed by mutating three Lysine residues within the ATP binding pocket to Argenine. B) Fc fusion chimeras of the intracellular domains from ROR2 WT, ROR2 KD, ROR2 Y755X and, as a control, the Fc-transmembrane domain of FGFR1 fusion were expressed in chondrocytes. The chimeric receptors were immunoprecipitated using Protein-A-sepharose (IP-PA) and analysed by Western blotting following 10% SDS-PAGE. Receptor phosphorylation was detected by a cocktail of phosphotyrosine antibodies (4G10/pY20; WB-pY, upper panel). The membrane was stripped and reprobed with anti-IgG1 antibody to compare receptor expression levels (WB-Fc, lower panel).

Mentions: To evaluate the intrinsic kinase activity potential of ROR2, the cytoplasmic domain of ROR2 was fused to the dimeric Fc portion of human IgG to create Fc-ROR2 ‘wild type’ (WT), Fc-ROR2 ‘Kinase dead’ and an Fc-ROR2 truncation mutant (Y755X) [14]. Fig. 1A shows a schematic detailing these constructs as well as the original full length, myc-tagged ROR2 constructs. Following expression in T/C28a2 human chondrocytes, robust Fc-ROR2 WT phosphorylation was detected in contrast with a markedly reduced phosphorylation of Fc-ROR2 KD. This suggests that, despite divergence from the RTK consensus sequence [15], ROR2 has intrinsic kinase activity which is elicited upon dimerisation (Fig. 1B, upper panel). This confirmed the recent reports that ROR2 is phosphorylated upon homo-dimerisation [16]. It was noted in these experiments that, although markedly reduced compared to WT, residual receptor phosphorylation was observed in cells expressing Fc-ROR2 KD, raising the possibility there may be other tyrosine kinases or co-receptors involved in ROR2 receptor phosphorylation, although the presence of residual endogenous kinase activity in the mutant cannot be rigorously excluded.


The deleted in brachydactyly B domain of ROR2 is required for receptor activation by recruitment of Src.

Akbarzadeh S, Wheldon LM, Sweet SM, Talma S, Mardakheh FK, Heath JK - PLoS ONE (2008)

Fc-ROR2 WT is tyrosine phosphorylated.A) A schematic diagram of the full length ROR2 and Fc-ROR2 chimeras used in this study. ‘Kinase dead’ (KD) mutants were constructed by mutating three Lysine residues within the ATP binding pocket to Argenine. B) Fc fusion chimeras of the intracellular domains from ROR2 WT, ROR2 KD, ROR2 Y755X and, as a control, the Fc-transmembrane domain of FGFR1 fusion were expressed in chondrocytes. The chimeric receptors were immunoprecipitated using Protein-A-sepharose (IP-PA) and analysed by Western blotting following 10% SDS-PAGE. Receptor phosphorylation was detected by a cocktail of phosphotyrosine antibodies (4G10/pY20; WB-pY, upper panel). The membrane was stripped and reprobed with anti-IgG1 antibody to compare receptor expression levels (WB-Fc, lower panel).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2268744&req=5

pone-0001873-g001: Fc-ROR2 WT is tyrosine phosphorylated.A) A schematic diagram of the full length ROR2 and Fc-ROR2 chimeras used in this study. ‘Kinase dead’ (KD) mutants were constructed by mutating three Lysine residues within the ATP binding pocket to Argenine. B) Fc fusion chimeras of the intracellular domains from ROR2 WT, ROR2 KD, ROR2 Y755X and, as a control, the Fc-transmembrane domain of FGFR1 fusion were expressed in chondrocytes. The chimeric receptors were immunoprecipitated using Protein-A-sepharose (IP-PA) and analysed by Western blotting following 10% SDS-PAGE. Receptor phosphorylation was detected by a cocktail of phosphotyrosine antibodies (4G10/pY20; WB-pY, upper panel). The membrane was stripped and reprobed with anti-IgG1 antibody to compare receptor expression levels (WB-Fc, lower panel).
Mentions: To evaluate the intrinsic kinase activity potential of ROR2, the cytoplasmic domain of ROR2 was fused to the dimeric Fc portion of human IgG to create Fc-ROR2 ‘wild type’ (WT), Fc-ROR2 ‘Kinase dead’ and an Fc-ROR2 truncation mutant (Y755X) [14]. Fig. 1A shows a schematic detailing these constructs as well as the original full length, myc-tagged ROR2 constructs. Following expression in T/C28a2 human chondrocytes, robust Fc-ROR2 WT phosphorylation was detected in contrast with a markedly reduced phosphorylation of Fc-ROR2 KD. This suggests that, despite divergence from the RTK consensus sequence [15], ROR2 has intrinsic kinase activity which is elicited upon dimerisation (Fig. 1B, upper panel). This confirmed the recent reports that ROR2 is phosphorylated upon homo-dimerisation [16]. It was noted in these experiments that, although markedly reduced compared to WT, residual receptor phosphorylation was observed in cells expressing Fc-ROR2 KD, raising the possibility there may be other tyrosine kinases or co-receptors involved in ROR2 receptor phosphorylation, although the presence of residual endogenous kinase activity in the mutant cannot be rigorously excluded.

Bottom Line: Native ROR2 phosphorylation is induced by the ligand Wnt5a and is blocked by pharmacological inhibition of Src kinase activity.Activation via tyrosine phosphorylation of ROR2 receptor leads to its internalisation into Rab5 positive endosomes.These findings show that BDB mutant receptors are defective in kinase activation as a result of failure to recruit Src.

View Article: PubMed Central - PubMed

Affiliation: CR-UK Growth Factor Group, School of Biosciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom.

ABSTRACT
The transmembrane receptor 'ROR2' resembles members of the receptor tyrosine kinase family of signalling receptors in sequence but its' signal transduction mechanisms remain enigmatic. This problem has particular importance because mutations in ROR2 are associated with two human skeletal dysmorphology syndromes, recessive Robinow Syndrome (RS) and dominant acting Brachydactyly type B (BDB). Here we show, using a constitutive dimerisation approach, that ROR2 exhibits dimerisation-induced tyrosine kinase activity and the ROR2 C-terminal domain, which is deleted in BDB, is required for recruitment and activation of the non-receptor tyrosine kinase Src. Native ROR2 phosphorylation is induced by the ligand Wnt5a and is blocked by pharmacological inhibition of Src kinase activity. Eight sites of Src-mediated ROR2 phosphorylation have been identified by mass spectrometry. Activation via tyrosine phosphorylation of ROR2 receptor leads to its internalisation into Rab5 positive endosomes. These findings show that BDB mutant receptors are defective in kinase activation as a result of failure to recruit Src.

Show MeSH
Related in: MedlinePlus