The first propeller domain of LRP6 regulates sensitivity to DKK1

Abstract

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.

Figure 1.

mAb135 recognizes endogenous LRP6 with high-affinity. (A) 293T cells were transfected with indicated HA-tagged LRP6 constructs. Transfected cells were stained with anti-HA or mAb135 antibodies and analyzed by flow cytometry. (B) mAb135 was immobilized on an anti-mouse sensor chip surface and its affinity for hLRP6 ECD-Fc determined by BIAcore analysis. (C) HEK 293 cells were transfected with control or LRP6 specific siRNA duplexes (10 nM) and analyzed for endogenous LRP6 levels by Western blotting with anti-LRP6 polyclonal antibodies. Recognition of endogenous LRP6 by mAb135 in siRNA-treated cells was analyzed by FACS analysis.

Figure 2.

mAb135 enhances Wnt signaling. (A) HEK 293 A6 stable 16TCF reporter cells were treated with media control or Wnt3A (200 ng/ml) in the presence of increasing concentrations of mAb135 IgG, mAb135 Fab, or isotype control IgG. (B) HEK 293 A6 cells were treated with increasing amounts of Wnt3A in the presence of a fixed amount of mAb135 IgG, isotype control IgG (10 μg/ml), or mAb135 Fab (3.3 μg/ml) and analyzed for reporter activation. (C) HEK 293 cells were treated with increasing amounts of Wnt3A in the absence or presence of mAb135 (10 μg/ml) and analyzed for cytosolic beta-catenin by Western blotting.

Figure 3.

mAb135 antagonizes DKK1. HEK293 A6 cells were incubated with increasing amounts of mAb135 IgG (A), mAb135 Fab (B), or isotype control antibody before treament with Wnt3A (200 ng/ml) in the presence or absence of DKK1 (400 ng/ml). HEK293 A6 cells were treated with Wnt3A (200 ng/ml) and increasing amounts of DKK1, after addition of mAb135 IgG or isotype control IgG (C) (10 μg/ml), or mAb135 Fab (D) (3.3 μg/ml), and analyzed for reporter activity. (E) HEK293 cells were treated with media control, Wnt3A (50 ng/ml), or Wnt3A plus DKK1 (400 ng/ml) in the presence or absence of mAb135 (10 μg/ml) and analyzed for cytosolic beta-catenin by Western blotting. (F) HEK293 cells were treated with media control, Wnt3A (50 ng/ml), or Wnt3A plus DKK1 (400 ng/ml) in the presence or absence of increasing amounts of mAb135 and analyzed for phosphorylated LRP6 by Western blotting with phospho-Ser1490 specific antibodies.

Figure 4.

mAb135 maps to an epitope in the first β-propeller domain of LRP6. (A) Diagram of LRP6 propeller domain mutants. (B) HEK293T cells transfected with the indicated LRP6 domain mutants were analyzed for binding of anti-HA mAb or mAb135 by FACS analysis. (C) Diagram of LRP6 propeller domain 1 human/mouse chimeric constructs. (D) HEK293 T-cells were transfected with the indicated human/mouse chimeric LRP6 propeller domain 1 constructs and analyzed for HA and mAb135 binding by FACS analysis. (E) Outline of amino acid differences between human and mouse LRP6 in the C-terminal region of the first propeller domain. (F) HEK293T cells were transfected with indicated LRP6 propeller domain 1 human -> mouse substitution mutants and analyzed for anti-HA and mAb135 binding as described above. (G) HEK293T cells were transfected with indicated LRP6 propeller domain 1 mouse -> human substitution mutants and analyzed for anti-HA and mAb135 binding.

Figure 5.

mAb135 inhibits interaction of recombinant DKK1 and LRP6 ECD proteins. Interaction of recombinant DKK1-his protein with full-length recombinant His-tagged LRP6 ECD (LRP6 ECD) (A) or recombinant LRP6 ECD domain mutants containing WYTD beta-propeller domains 1–2 or 3–4 (LRP6 Dom 1–2 and LRP6 Dom 3–4) (B) were evaluated by analyzing the mobility of DKK1 with or without LRP6 on an analytical Superose 6 size exclusion column, in the presence or absence of a fivefold molar excess of mAb135. Fractions were analyzed for the presence of DKK1 by Western blotting and the elution profile of DKK1 compared with the profile of molecular weight standards. In several experiments, we observed that free DKK1 bound the column and can be eluted with urea, therefore the recovery of free DKK1 was varied as presented in B.

Figure 6.

mAb135 inhibits DKK1-dependent internalization of LRP6. HEK-293 cells were cotransfected with LRP6-HA and Kremen1 constructs, pretreated with mAb135 (5 μg/ml) for 1 h followed by treatment with recombinant DKK1 (200 ng/ml) for 10 min. Distribution of immunostained HA-tagged LRP6 was evaluated by confocal microscopy.