RESUMO
The Wnt coreceptor LRP6 is required for canonical Wnt signaling. To understand the molecular regulation of LRP6 function, we generated a series of monoclonal antibodies against the extra cellular domain (ECD) of LRP6 and selected a high-affinity mAb (mAb135) that recognizes cell surface expression of endogenous LRP6. mAb135 enhanced Wnt dependent TCF reporter activation and antagonized DKK1 dependent inhibition of Wnt3A signaling, suggesting a role in modulation of LRP6 function. Detailed analysis of LRP6 domain mutants identified Ser 243 in the first propeller domain of LRP6 as a critical residue for mAb135 binding, implicating this domain in regulating the sensitivity of LRP6 to DKK1. In agreement with this notion, mAb135 directly disrupted the interaction of DKK1 with recombinant ECD LRP6 and a truncated form of the LRP6 ECD containing only repeats 1 and 2. Finally, we found that mAb135 completely protected LRP6 from DKK1 dependent internalization. Together, these results identify the first propeller domain as a novel regulatory domain for DKK1 binding to LRP6 and show that mAb against the first propeller domain of LRP6 can be used to modulate this interaction.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas Relacionadas a Receptor de LDL/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Sítios de Ligação/genética , Sítios de Ligação/imunologia , Western Blotting , Linhagem Celular , Endocitose/efeitos dos fármacos , Citometria de Fluxo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas Relacionadas a Receptor de LDL/genética , Proteínas Relacionadas a Receptor de LDL/imunologia , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Mutação , Ligação Proteica/efeitos dos fármacos , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Proteína Wnt3 , Proteína Wnt3ARESUMO
The R-Spondin (RSpo) family of secreted proteins is implicated in the activation of the Wnt signaling pathway. Despite the high structural homology between the four members, expression patterns and phenotypes in knockout mice have demonstrated striking differences. Here we dissected and compared the molecular and cellular function of all RSpo family members. Although all four RSpo proteins activate the canonical Wnt pathway, RSpo2 and 3 are more potent than RSpo1, whereas RSpo4 is relatively inactive. All RSpo members require Wnt ligands and LRP6 for activity and amplify signaling of Wnt3A, Wnt1, and Wnt7A, suggesting that RSpo proteins are general regulators of canonical Wnt signaling. Like RSpo1, RSpo2-4 antagonize DKK1 activity by interfering with DKK1 mediated LRP6 and Kremen association. Analysis of RSpo deletion mutants indicates that the cysteine-rich furin domains are sufficient and essential for the amplification of Wnt signaling and inhibition of DKK1, suggesting that Wnt amplification by RSpo proteins may be a direct consequence of DKK1 inhibition. Together, these findings indicate that RSpo proteins modulate the Wnt pathway by a common mechanism and suggest that coexpression with specific Wnt ligands and DKK1 may determine their biological specificity in vivo.