The identification of LDL receptor-related proteins, LRP5 and LRP6 (32
) and their Drosphila
orthologue Arrow (44
), as Wnt coreceptors has led to important biochemical insights into the role of this receptor in canonical signaling. Mao et al. (28
) demonstrated that the intracellular domain of wild-type LRP5 receptor recruits Axin, a component of the β-catenin degradation complex (36
), to the membrane in response to Wnt triggering. However, the lack of any recognizable catalytic motif in the LRP5 and LRP6 intracellular domain (12
) and the fact that this domain interacts with Axin in both yeast and mammalian cells (28
) suggested that posttranslational modifications triggered by Wnt were not required for LRP5-Axin interactions. Mutational analysis independently revealed that an LRP5 deletion mutant lacking the external domain but preserving the transmembrane and cytoplasmic domains was constitutively active, implying that the external domain negatively regulates LRP5 function (28
). Our present studies have extended these observations to LRP6 and have led to the elucidation of a novel mechanism for ligand activation of a transmembrane receptor.
In striking contrast to the general mechanism of receptor activation by oligomerization (27
), we demonstrated that wild-type LRP6 forms an inactive homomeric complex, whereas constitutively active LRP6 mutants were monomeric. These results suggested that the extracellular domain of the wild-type receptor might inhibit signaling by oligomerization. We mapped LRP6 oligomerization to the YWTD-EGF repeat region of the external domain through analysis of a series of LRP6 mutants. An inverse correlation between receptor oligomerization and signaling was confirmed by use of a chimeric GyrB-LRP6 receptor also lacking the LRP6 external domain, which was replaced by GyrB. This mutant exhibited constitutive signaling activity, which was inhibited by coumermycin-induced forced dimerization. Axin interaction with the cytoplasmic domain of this chimera was also inhibited by coumermycin-induced receptor dimerization. All these results support a model in which LRP6 dimerization mediated by the extracellular YWTD-EGF repeat region inhibits interaction of the LRP6 intracellular domain with Axin as well as LRP6 signaling functions. This model is consistent with the possibility that LRP5 and LRP6 heterodimers may also occur. If so, such heteromeric receptor complexes may possess different affinities for Wnt ligands or even different intracellular signaling specificity compared to those of LRP5 or LRP6 homomeric oligomers.
Two studies published after submission of this paper reported the requirement of a chaperone protein, Mesd, or of the Drosophila
ortholog Boca for proper folding and cell surface location of functional LRP6 or arrow receptors (13
). The majority of exogenously expressed LRP6 in COS-1 cells was shown to accumulate as aggregates in the endoplasmic reticulum through aberrant intermolecular disulfide linkages, presumably due to the lack of sufficient expression of Mesd proteins in these cells (20
). In human embryonic kidney 293 cells, exogenously expressed LRP6 has been reported to function at the cell surface and to mediate Wnt signaling and Dkk-1 binding (3
). Consistent with these reports, we were able to detect the expression of wild-type and various LRP6 mutant receptors at the cell surface by FACS analysis in 293 cells. When expressed in 293 cells under nonreducing conditions, the majority of the LRP6 receptor was not observed as high-molecular-weight aggregates or LRP6 oligomers. These findings establish both that oligomer formation of the wild-type LRP6 receptor does not involve intermolecular disulfide linkages and that chaperone proteins cannot be limiting in 293 cells. By using a cell membrane-impermeable cross-linker, homodimers of wild-type LRP6, LRP6ΔE1-2, and LRP6ΔE3-4 were detected at the cell surface, while the constitutively active LRP6ΔE1-4 mutant failed to be cross-linked under the same conditions. These results strongly support the concept that the functional LRP6 receptor exists as an oligomer at the cell surface and that oligomerization is mediated by the YWTD-EGF repeats.
Ligand activation of tyrosine kinase receptors involves ligand-mediated receptor oligomerization, which induces activation of the kinase domains (45
). By generation of an LRP6-FGFR chimera it was possible to establish that oligomerization mediated by the LRP6 external domain caused sufficiently close juxtaposition of the intracellular tyrosine kinase domains to cause their chronic activation. We demonstrated that both coexpressed and exogenously added Wnt induced a conformational switch which separated the intracellular domains of the LRP6-FGFR chimera from the close proximity required to maintain kinase activation without dissociation of the oligomeric receptor complex. Thus, Wnt signaling through LRP6 invokes a new paradigm in which Wnts act to relieve allosteric inhibition imposed on the intracellular domain of an inactive receptor oligomer. This is in direct contrast to the general mechanism of ligand-induced receptor clustering responsible for the activation of hormone and growth factor receptors, lymphokine receptors, T-cell and B-cell receptors, and many other families of cell surface receptors (27
). According to this model, the Axin-binding site in the intracellular domain of the LRP receptor would be sequestered within the LRP6 oligomer, presumably through mutual steric hindrance. Wnt ligands would alter the conformation of this receptor oligomer to relieve allosteric inhibition, allowing access to substrates such as Axin and producing an active LRP6 receptor.
Previous studies have shown that forced dimerization of CD45, a member of the receptor-like protein tyrosine phosphatase (RPTP) family involved in T-cell receptor (TCR) signaling, inhibits its activity and results in the loss of TCR signaling (15
). Similarly, dimerization inhibits the activity of RPTPα (22
). It has been proposed on the basis of crystal structure that dimerization negatively regulates phosphatase activity through interaction of an inhibitory wedge on one monomer with the catalytic cleft in the other to block substrate access (6
). While inhibitory ligands for these receptors have been postulated to negatively regulate RPTPs by causing their dimerization, such ligands have not been identified (45
). In fact, a recent study suggests that CD45 may not require an inhibitory ligand to form a dimer. Instead, CD45 phosphatase activity may be regulated by the differential dimerization of alternatively spliced isoforms (48
Genetic and biochemical studies have provided strong evidence that the seven transmembrane-spanning frizzled receptor and the single transmembrane-spanning LRP5 or LRP6 receptor cooperate in Wnt canonical signaling (41
). Previous studies have indicated that the frizzled receptor itself forms a dimer through interactions involving its cysteine-rich domain, and these interactions are independent of Wnt (2
). Specific mutations within the frizzled cytoplasmic tail have been shown to inhibit canonical signaling (42
), implying that it possesses intracellular signaling functions. Tamai et al. (41
) reported that Wnt binds to frizzled and LRP6 independently and recruits these two receptors into a complex. However, the interaction between Wingless
was not detectable in Drosophila
). Although we were able to detect specific Wnt3a/LRP6 complexes by overexpression and coimmunoprecipitation as has been previously reported (23
), this approach does not determine the affinity of the interaction or whether this interaction involves other components of the receptor complex. Our findings as well as those of others (28
) indicate that the NH2-terminally truncated LRP mutants lacking the extracellular domain are capable of canonical signaling independent of either a Wnt ligand or frizzled receptor. Thus, the mechanism by which frizzled cooperates with LRP to transduce the Wnt signal remains to be elucidated. Nonetheless, it is likely that at physiological Wnt ligand and receptor levels the engagement of both frizzled and LRP coreceptors is required to achieve efficient intracellular coupling to the canonical pathway. Based on our results, it is conceivable that an LRP mutant lacking the intracellular domain might act to increase Wnt canonical signaling by forming a receptor heterodimer with one free cytoplasmic tail. However, there are reports that the LRP extracellular domain inhibits canonical signaling (29
). If so, such inhibition may reflect the complexity of in vivo signaling involving LRP interactions not only with Wnt ligands but also with frizzled coreceptors.
Missense mutations in the YWTD-EGF repeat region of LRP5 have been found both in the human autosomal-recessive disorder osteoporosis-pseudoglioma syndrome (18
) and the autosomal-dominant high-bone-mass trait (9
). Our present findings suggest possible functional consequences of these mutations in disease. Such mutations could alter the association or dissociation of LRP oligomers, which might influence the conformational switch mediated by Wnt ligands or the ability of the inhibitory ligand, Dkk-1, to downregulate the receptor through interactions with LRP (3
) and a recently identified coreceptor, kremen (30
). In fact, one report indicates that the YWTD-EGF repeat mutation at codon 171 in the high-bone-mass trait interferes with Dkk-1 function (9
). Constitutive activation of canonical signaling due to mutations in certain Wnt pathway components is etiologically involved in a number of human tumors (36
). Our present findings suggest that genetic lesions in the LRP receptor, which impaired its ability to form oligomers, might activate canonical signaling, making LRP a potential candidate for oncogenic activation in tumors.