As we have previously reported (4
), expression of the LRP5 mutant protein (LRP5G171V
) containing the HBM G171V mutation and an HA epitope tag at its C terminus (Fig. ) did not lead to an increase in LEF-1-dependent transcriptional activity compared to the wild-type (wt) LRP5 (LRP5wt
) (Fig. ). Additionally, the G171V mutation did not result in further potentiation of the activity stimulated by coexpressed Wnt1 in an autocrine paradigm (Fig. ). LEF-1 is a downstream target transcription factor of the canonical Wnt signaling pathway. Its activity, measured by a luciferase reporter gene assay, has been widely used to gauge the canonical Wnt activity (11
). Thus, LRP5G171V
is neither constitutively active nor more competent in transducing Wnt signaling. Surprisingly, the corresponding mutation on LRP6, a substitution of Val at residue G158, rendered it unable to act synergistically with Wnt-1 (Fig. ), thus probably inactivating the receptor.
FIG. 1. LRP5G171V is less susceptible to DKK1-mediated inhibition of the activity of coexpressed Wnt. (A) Effects of the G171V mutation on canonical Wnt signaling activity. HEK cells were transfected with plasmids, as indicated in the figure, together with a (more ...)
Previously, we have shown that LRP5G171V
was less susceptible to DKK1-mediated inhibition than was LRP5wt
in the absence of Kremen (4
). Kremen is a DKK binding single-transmembrane protein known to facilitate DKK1-induced inhibition (22
). In this study, we tested the effect of this mutation in the presence of Kremen. Coexpression of Kremen1 significantly potentiated DKK-mediated inhibition (Fig. ), confirming the previously reported effect of Kremen (22
). Similar to what we observed in the absence of Kremen, in the presence of both Kremen1 and DKK1, Wnt showed higher activity in HEK cells expressing LRP5G171V
than in those expressing LRP5wt
(Fig. ). To ensure that the difference is not a result of multiplasmid transfection, we examined the protein expression of DKK1, Kremen1, and LRP5 (Fig. ). Similar results of increased resistance to DKK-mediated inhibition of autocrine Wnt1 activity were also observed in NIH 3T3 cells and two osteoblast-like cell lines, MC3T3 and 2T3 (data not shown).
The prevailing hypothesis for explaining why LRP5 G171V is less susceptible to DKK1-mediated inhibition is that the mutation might disrupt the interaction between LRP5 and DKK1. It is reasonable to hypothesize that the first YWTD repeat domain that contains G171 is required for DKK1-mediated antagonism. To test this hypothesis, we generated two LRP5 deletion mutants: LRP5R12, with a deletion of the third and fourth YWTD repeat domains, and LRP5R34, with a deletion of the first and second YWTD repeat domains (Fig. ). As previously reported for LRP6 (23
), LRP5R12, but not LRP5R34, could still potentiate Wnt-stimulated LEF-1 activity (Fig. ), suggesting that LRP5R12 retains the Wnt coreceptor function. However, DKK1 could not inhibit Wnt signaling when LRP5R12 was present even if Kremen was coexpressed (Fig. ). This suggests that the last two YWTD repeat domains may be required for DKK1-mediated inhibition. To further delineate the sequence that is required for DKK1-mediated inhibition, we generated an additional LRP5 mutant, LRP5R124, in which the third YWTD repeat domain was deleted (Fig. ). Like LRP5R12, LRP5R124 is also resistant to DKK1-mediated inhibition (Fig. ), indicating that the third YWTD repeat domain is required for DKK1-mediated inhibition.
FIG. 2. The third YWTD repeat domain is required for DKK-mediated antagonism. (A) Schematic representation of wt LRP5 and its mutants. (B) Identification of the YWTD repeat domain required for DKK-mediated inhibition. HEK cells were transfected with the LEF activity (more ...)
Since deletion of the entire third YWTD repeat domain may cause gross conformational changes in LRP5, we went on to search for point mutations in this domain that can disrupt DKK1-mediated inhibition. Based on the three-dimensional structure of the third YWTD repeat domain deduced from that of the LDL receptor (12
), we generated 19 LRP5 mutants containing Ala substitution mutations on the surface of the third YWTD repeat domain (Fig. ). The ability of these mutant LRP5 proteins to resist DKK1-mediated inhibition was determined and is shown in Fig. . Nine of the mutants showed altered sensitivity to DKK1-mediated inhibition (by more than 5%), and they all contain mutations that are localized on the same surface (Fig. ). Among these mutations, E721 mutation showed the strongest effect, followed by W781 and then Y719 (Fig. ). Mutation of E721-corresponding residues in the first and second YWTD repeat domains (D111 and D418, respectively) did not significantly alter the sensitivity to DKK-mediated inhibition (data not shown). All the mutants that are resistant to DKK1-mediated inhibition also resist DKK2-mediated inhibition (data not shown). Thus, all these data support the conclusion that the third YWTD repeat domain is required for DKK-mediated inhibition.
FIG. 3. Amino acid residues in the third YWTD repeat domain are required for DKK inhibition. (A) Schematic representation of Ala substitution mutations in the third YWTD repeat domain. The space-filling model of the third YWTD repeat domain was deduced based (more ...)
An obvious explanation for the requirement of the third YWTD repeat domain for DKK1-mediated inhibition is that this domain is responsible for DKK1 binding. We measured direct binding of DKK1-AP fusion protein to LRP5 expressed on the surface of HEK cells, as described previously (22
). As shown in Fig. , DKK1-AP showed a saturating binding curve to HEK cells expressing LRP5. We could measure this binding only when Mesd, an LRP5/6 chaperone that was shown to facilitate the folding and trafficking of LRP5/6 (5
), was coexpressed (data not shown). To our surprise, LRP5E721
still showed significant binding of DKK1 and did better than LRP5G171V
(Fig. ). It thus appears paradoxical that LRP5E721
, which is highly resistant to DKK1-mediated inhibition compared to LRP5G171V
(Fig. ), shows better binding of DKK1 than does LRP5G171V
(Fig. ). To determine whether the third YWTD repeat domain can indeed bind DKK1, we examined the binding of DKK1-AP to HEK cells expressing R34 or R34E (R34E is R34 carrying the E721 mutation). While R34 showed significant binding of DKK1-AP, R34E failed to do so (Fig. ), demonstrating that R34 is capable of binding DKK1 and that E721 is required for the binding. One possible explanation of the aforementioned paradox is that the third YWTD repeat domain is not the only site for DKK binding on LRP5; thus, LRP5E721
still retains the ability to bind DKK1. This possibility was confirmed by the observation that R12 could also bind DKK1 (Fig. ). Although both R12 and R34 can bind DKK1, their affinities for DKK1 appear to be at least fivefold lower than that of the full-length LRP5 (estimated from half-maximal binding). Although the maximal binding to cells expressing R12 or R34 appeared to be comparable to or probably even higher than that of LRP5wt
(the binding to R12 or R34 did not appear to reach saturation at the maximal possible inputs), the expression levels of R12 and R34 estimated by Western analysis (Fig. ) are approximately twice that of LRP5wt
. Thus, it is reasonable to conclude that there is more than one binding site for DKK1 on LRP5.
FIG. 4. Binding of DKK1-AP to LRP5 and its mutants. (A) HEK cells were transfected with the Mesd plasmid and LRP5 plasmids indicated in the figure and incubated on ice with CM prepared from HEK cells expressing mDKK1-AP. The AP activity was determined as described (more ...)
The question that remains is how G171V, a point mutation in the first YWTD repeat domain, reduces the apparent binding of DKK1 so drastically (Fig. ). The characteristics of the DKK1 binding curve for LRP5G171V
suggest that the G171V mutation does not appear to alter the affinity for DKK1, despite reducing the maximal binding by sixfold (Fig. ). Given that both LRP5wt
were expressed at similar levels (Fig. ), the G171V mutation appears to result in the presence of less LRP5 protein on the cell surface. Knowing that Mesd plays an important role in the transport of LRP5 proteins to cell surfaces, we investigated whether the G171V mutation interferes with the function of Mesd. Mesd has previously been shown to interact with LRP5/6 (10
). Consistent with this finding, we detected coimmunoprecipitation of LRP5 and Mesd (Fig. ). Additionally, we detected the interaction of R12 with Mesd (Fig. ). Interestingly, the G171V mutation disrupted the interactions of both LRP5 (Fig. , lanes 1 and 3) and R12 (Fig. , lanes 1 and 2) with Mesd, while the E721 mutation did not affect the interaction (Fig. , lanes 2 and 3). If the interaction between LRP5 and Mesd is important for the function of Mesd (folding and transport of LRP5/6), the G171V mutation should also impede the secretion of LRP5 mutants that lack the transmembrane domains. As expected, the G171V mutation inhibited the secretion of R12T (Fig. ) and R1-4 (Fig. ), which are R12 and the full-length LRP5, respectively, lacking the transmembrane and intracellular domains. R1-4 carrying the E721 mutation did not show inhibited secretion (data not shown). In addition, live cells expressing wt LRP5 and LRP5G171V
were biotinylated on their surfaces, and the levels of LRP5 proteins at the cell surfaces were compared by Western analysis using streptavidin-conjugated horseradish peroxidase after the LRP5 proteins were immunoprecipitated. As shown in Fig. , the level of biotinylated LRP5G171V
is clearly lower than that of wt LRP5 even though the levels of the two LRP5 molecules in the immunocomplexes are the same, confirming that the G171V mutation interferes with cell surface transport of LRP5.
FIG. 5. G171V mutation disrupts LRP5 trafficking. (A and B) Interaction of LRP5 with Mesd. HEK cells were transfected with expression plasmids as indicated. One day later, the cells were lysed and immunoprecipitation (IP) was carried out using an anti-Flag antibody. (more ...)
The G171V mutation was predicted to be due to a hypermorphic allele because it is associated with bone phenotypes opposite to those exhibited by LRP5-null or hypomorphic mutations (4
). Our observation of the poor cell surface presentation of LRP5G171V
is an apparent contradiction to the prediction, because one would normally assume that fewer receptors on the cell surface should have resulted in a lower response to Wnt. In fact, when exogenous Wnt was added, which mimics a paracrine or endocrine paradigm, cells expressing LRP5G171V showed a lower response than did cells expressing wt LRP5 (Fig. ). However, this was not true when Wnt was coexpressed with the LRP5 molecules (Fig. ). In other words, the mutation does not appear to affect the activity of autocrine Wnt, suggesting that Wnt proteins may be able to bind to their receptors and activate the signaling events before the receptors are transported to the cell surfaces. These observations allow us to come up with a hypothesis that may explain how LRP5G171V may give rise to higher Wnt activity in osteoblasts during their differentiation; the mutation may affect DKK-mediated antagonism more than Wnt activity if osteoblasts produce autocrine canonical Wnt proteins during differentiation and there is paracrine production of DKK1 in the bone. We found corroborating evidence for our hypothesis when we examined the expression of all 19 mouse Wnt genes in bone marrow stromal osteoblast cultures. One of the Wnt genes, Wnt7b, showed a marked increase in its expression after induction of differentiation (Fig. ). The ability of Wnt7b to stimulate the LEF-1 reporter gene was examined, and it was shown to be able to stimulate the canonical Wnt pathway (Fig. ). Moreover, we found that Dkk1 is highly expressed in osteocytes, and terminally differentiated osteoblasts, but at a low level in osteoblasts (Fig. ). Therefore, the conditions for our hypothesis to be correct exist in the bone.
LRP5G171V shows reduced response to Wnt3a CM compared to wt LRP5. HEK cells were transfected with the LEF activity reporter plasmids, Kremin1 plasmid, and LRP5 or LRP5G171V expression plasmid. Wnt3a CM was added for 6 h.
FIG. 7. Expression of Wnt7b in osteoblasts and DKK1 in osteocytes. (A) Expression of Wnt7b in BMS osteoblasts. Primary BMS osteoblast cultures were established from 3-month-old mice and induced to undergo osteogenic differentiation. Total-RNA samples were isolated (more ...)