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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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ROR2 receptor exhibits increased tyrosine phosphorylation and Src association upon Wnt5a stimulation.A) Chondrocytes were either transfected with pcDNA3.1 (V, control) or pcDNA3.1 containing the myc-tagged, wild type, ROR2 construct (ROR2-myc WT). Following 1 hr of serum starvation in KHB, cells were either stimulated with 0.1% BSA carrier (C) or Wnt5a (1.6 µg/ml) for 5, 30 or 60 min. Control vector-transfected cells (V) were not stimulated. The receptor was immunoprecipitated using anti-myc antibody (IP-myc) and analysed by SDS-PAGE and subsequent Western blotting. The membrane was probed with an anti-phosphotyrosine cocktail (WB-pY), followed by stripping and re-probing with anti-myc antibody (WB-myc). The fold-increase of ROR2 receptor phosphorylation was determined by normalizing Wnt5a-induced phosphorylation to carrier-induced phosphorylation (mean±s.e.m., n = 3, lower panel). B) Chondrocytes expressing ROR2-myc WT were stimulated with either 0.1% BSA (C) or Wnt5a (1.6 µg/ml) for 30 min. The receptor was immunoprecipitated using anti-myc antibody (IP-myc) and probed with a phospho-Src antibody (WB-pSrc-Y416) to detect activated Src. The membrane was stripped and reprobed with anti-c-Src (WB-cSrc) and anti-myc (WB-myc).
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pone-0001873-g002: ROR2 receptor exhibits increased tyrosine phosphorylation and Src association upon Wnt5a stimulation.A) Chondrocytes were either transfected with pcDNA3.1 (V, control) or pcDNA3.1 containing the myc-tagged, wild type, ROR2 construct (ROR2-myc WT). Following 1 hr of serum starvation in KHB, cells were either stimulated with 0.1% BSA carrier (C) or Wnt5a (1.6 µg/ml) for 5, 30 or 60 min. Control vector-transfected cells (V) were not stimulated. The receptor was immunoprecipitated using anti-myc antibody (IP-myc) and analysed by SDS-PAGE and subsequent Western blotting. The membrane was probed with an anti-phosphotyrosine cocktail (WB-pY), followed by stripping and re-probing with anti-myc antibody (WB-myc). The fold-increase of ROR2 receptor phosphorylation was determined by normalizing Wnt5a-induced phosphorylation to carrier-induced phosphorylation (mean±s.e.m., n = 3, lower panel). B) Chondrocytes expressing ROR2-myc WT were stimulated with either 0.1% BSA (C) or Wnt5a (1.6 µg/ml) for 30 min. The receptor was immunoprecipitated using anti-myc antibody (IP-myc) and probed with a phospho-Src antibody (WB-pSrc-Y416) to detect activated Src. The membrane was stripped and reprobed with anti-c-Src (WB-cSrc) and anti-myc (WB-myc).

Mentions: As discussed above, members of the Wnt family of ligands have been implicated in ROR2 signalling. Having established that ROR2 could exhibit intrinsic kinase activity (albeit of an unusual kind) we were interested in discovering if Wnt ligands could elicit activation of the kinase activity of native ROR2 receptors. Chondrocytes transfected with full length myc-tagged ROR2 (ROR2-myc WT) were stimulated with either BSA carrier or Wnt5a for 5, 30 or 60 min. The receptor was immunoprecipitated and receptor phosphorylation was detected by phosphotyrosine immunoblotting (Fig. 2A, upper panel). A marked increase in receptor tyrosine phosphorylation after 30 min of Wnt5a stimulation was detected compared to carrier stimulated controls. These findings show that Wnt5a ligand elicits tyrosine phosphorylation of native ROR2, confirming that Wnt5a is, directly or indirectly, a bona fide activator of ROR2 signalling, acting via stimulation of intrinsic tyrosine kinase activity.


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)

ROR2 receptor exhibits increased tyrosine phosphorylation and Src association upon Wnt5a stimulation.A) Chondrocytes were either transfected with pcDNA3.1 (V, control) or pcDNA3.1 containing the myc-tagged, wild type, ROR2 construct (ROR2-myc WT). Following 1 hr of serum starvation in KHB, cells were either stimulated with 0.1% BSA carrier (C) or Wnt5a (1.6 µg/ml) for 5, 30 or 60 min. Control vector-transfected cells (V) were not stimulated. The receptor was immunoprecipitated using anti-myc antibody (IP-myc) and analysed by SDS-PAGE and subsequent Western blotting. The membrane was probed with an anti-phosphotyrosine cocktail (WB-pY), followed by stripping and re-probing with anti-myc antibody (WB-myc). The fold-increase of ROR2 receptor phosphorylation was determined by normalizing Wnt5a-induced phosphorylation to carrier-induced phosphorylation (mean±s.e.m., n = 3, lower panel). B) Chondrocytes expressing ROR2-myc WT were stimulated with either 0.1% BSA (C) or Wnt5a (1.6 µg/ml) for 30 min. The receptor was immunoprecipitated using anti-myc antibody (IP-myc) and probed with a phospho-Src antibody (WB-pSrc-Y416) to detect activated Src. The membrane was stripped and reprobed with anti-c-Src (WB-cSrc) and anti-myc (WB-myc).
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Related In: Results  -  Collection

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pone-0001873-g002: ROR2 receptor exhibits increased tyrosine phosphorylation and Src association upon Wnt5a stimulation.A) Chondrocytes were either transfected with pcDNA3.1 (V, control) or pcDNA3.1 containing the myc-tagged, wild type, ROR2 construct (ROR2-myc WT). Following 1 hr of serum starvation in KHB, cells were either stimulated with 0.1% BSA carrier (C) or Wnt5a (1.6 µg/ml) for 5, 30 or 60 min. Control vector-transfected cells (V) were not stimulated. The receptor was immunoprecipitated using anti-myc antibody (IP-myc) and analysed by SDS-PAGE and subsequent Western blotting. The membrane was probed with an anti-phosphotyrosine cocktail (WB-pY), followed by stripping and re-probing with anti-myc antibody (WB-myc). The fold-increase of ROR2 receptor phosphorylation was determined by normalizing Wnt5a-induced phosphorylation to carrier-induced phosphorylation (mean±s.e.m., n = 3, lower panel). B) Chondrocytes expressing ROR2-myc WT were stimulated with either 0.1% BSA (C) or Wnt5a (1.6 µg/ml) for 30 min. The receptor was immunoprecipitated using anti-myc antibody (IP-myc) and probed with a phospho-Src antibody (WB-pSrc-Y416) to detect activated Src. The membrane was stripped and reprobed with anti-c-Src (WB-cSrc) and anti-myc (WB-myc).
Mentions: As discussed above, members of the Wnt family of ligands have been implicated in ROR2 signalling. Having established that ROR2 could exhibit intrinsic kinase activity (albeit of an unusual kind) we were interested in discovering if Wnt ligands could elicit activation of the kinase activity of native ROR2 receptors. Chondrocytes transfected with full length myc-tagged ROR2 (ROR2-myc WT) were stimulated with either BSA carrier or Wnt5a for 5, 30 or 60 min. The receptor was immunoprecipitated and receptor phosphorylation was detected by phosphotyrosine immunoblotting (Fig. 2A, upper panel). A marked increase in receptor tyrosine phosphorylation after 30 min of Wnt5a stimulation was detected compared to carrier stimulated controls. These findings show that Wnt5a ligand elicits tyrosine phosphorylation of native ROR2, confirming that Wnt5a is, directly or indirectly, a bona fide activator of ROR2 signalling, acting via stimulation of intrinsic tyrosine kinase activity.

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