Wnts include a large family of secreted hydrophobic proteins that regulate essential developmental processes, including embryonic development, cell growth, migration, and differentiation (1
). When Wnt signaling is perturbed, cancer or degenerative diseases result (2
). Thus, understanding the ways in which Wnt signaling is regulated is of great importance.
The large number of different Wnt family members evokes such questions as why so many Wnt proteins are necessary and whether they all signal through the same mechanism. Some clues come from studies in cell culture and intact organisms, which have shown that various intracellular signaling pathways become activated either directly or indirectly in response to different Wnt proteins (7
). Ongoing questions in the field, thus, are how do distinct Wnt family members elicit differential cellular outcomes and which Wnts, if not all, activate each distinct pathway?
Whereas most Wnt signaling has been attributed to the activation of the Frizzled and low density lipoprotein receptor-related protein 5 and 6 (Lrp5/6) coreceptor proteins leading to β-catenin stabilization (a pathway called Wnt/β-catenin or canonical Wnt signaling), emerging evidence suggests that some Wnts can bind to alternative receptors to activate noncanonical signal transduction cascades (10
). For example, Wnt5a has been shown to interact with one such alternative Wnt receptor, Ror2, leading to the inhibition of Wnt/β-catenin signaling (12
). Although it has been shown that this inhibition can occur at the level of β-catenin stabilization or T cell factor/lymphoid enhancer factor gene transcription, much remains to be understood regarding the cytosolic pathway that is activated in response to Wnt5a binding. Thus, research into the mechanism by which Ror2 functions will further our understanding of this novel regulatory pathway.
The Ror2 receptor belongs to the receptor tyrosine kinase (RTK)2
). This large protein family plays an important role in regulating diverse cellular processes ranging from the cell cycle, cell migration, as well as cell proliferation and differentiation (16
). The Ror2 protein and its homolog Ror1 play essential roles during development (17
). Mutations of the Ror2 receptor, resulting in protein misfolding or premature truncation, have been associated with human diseases such as dominant Brachydactyly B and recessive Robinow syndrome (18
). These autosomal syndromes are associated with a variety of phenotypes that vary in their degree of severity depending on which specific mutation has occurred (A
). Defects associated with these diseases include brachydactyly (a shortening of the digits), skeletal dysplasia, congenital heart disease, and craniofacial abnormalities (19
). Ror2 null mice exhibit skeletal abnormalities similar to the human syndromes. In addition, these mice display heart and lung defects and die perinatally, suggesting that Ror2 may play a broad role in the embryonic development of various tissues (20
FIGURE 1. Deletion of mRor2 extracellular and intracellular domains results in decreased Wnt5a-mediated inhibition of Wnt/β-catenin signaling. A, diagram of the major domains of the mRor2 receptor. B, extracellular domain deletion mutants of mRor2 (ΔCRD (more ...)
Despite the increased interest in the Ror2 receptor, relatively few efforts have been made to determine which specific residues are required for Wnt5a-mediated Ror2 inhibitory signaling (23
), hereafter referred to as Wnt5a/Ror2 signaling. To that end, we performed mutation/deletion analysis of the receptor and have determined which specific intracellular domains and residues are necessary for Ror2-mediated Wnt5a signaling.
Although there is strong in vitro evidence suggesting that Ror2 can inhibit Wnt/β-catenin signaling, this has not been tested in vivo. Thus, to determine whether Ror2 can inhibit Wnt/β-catenin signaling in the context of a whole organism, we analyzed Ror2 protein expression, using a monoclonal antibody we generated, and the effect of Ror2 loss on Wnt/β-catenin signaling in Wnt reporter mice. We find that the absence of Ror2 leads to enhanced Wnt/β-catenin signaling, specifically in cells that have lost Ror2 expression. Our data, taken together, suggest that Ror2 can inhibit canonical Wnt signaling in vivo and point to Ror2 as a potential therapeutic target for human disease.