Structure-based mutation analysis shows the importance of LRP5 β-propeller 1 in modulating Dkk1-mediated inhibition of Wnt signaling
Introduction
Low-density lipoprotein receptor related protein 5 (LRP5) has emerged as a key regulator of bone metabolism through the Wnt signaling pathway. The High Bone Mass phenotype (HBM) has been associated with the LRP5–G171V mutation in two independent pedigrees (Boyden et al., 2002, Little et al., 2002). Six additional missense mutations (D111Y, G171R, A214T, A214V, A242T and T253I) in LRP5 were identified in patients who also showed an increased bone density (Van Wesenbeeck et al., 2003). The importance of LRP5 in the development and maintenance of postnatal bone is highlighted in another disease called osteoporosis pseudoglioma syndrome (OPPG). OPPG is characterized by premature osteoporosis that leads to bone deformities and fractures as well as juvenile-onset blindness and is caused by frameshift or nonsense mutations in LRP5 (Gong et al., 2001). The role of LRP5 in the regulation of bone density is demonstrated by LRP5 knockout mice and transgenic mice over expressing the human HBM gene where both recapitulate loss or gain of function human phenotypes respectively (Kato et al., 2002, Babij et al., 2003). Subsequently, various mutations in human LRP5 have been identified as contributing factors to both increased and decreased bone mineral density suggesting that other allelic forms of LRP5 may contribute to a spectrum of bone density phenotypes (Johnson, 2004, Whyte et al., 2004).
LRP5, a member of the LDL receptor family, functionally acts as a co-receptor with Frizzled for secreted Wnts to activate the canonical Wnt signaling pathway. Its in vitro activity can be monitored by increased signal of T cell factor (TCF) responsive reporters in vitro. LRP5 is also a receptor for Dickkopf (Dkk1), a secreted inhibitor of LRP5-mediated Wnt signaling (Mao et al., 2001). We and others have shown that the LRP5–G171V (HBM) mutation partially relieves the inhibitory effect of Dkk1 on Wnt signaling (Boyden et al., 2002, Bhat et al., 2003, Zhang et al., 2004, Ai et al., 2005b) that results in activation of the LRP5–Wnt–TCF signaling. In order to understand the potential molecular mechanism of Wnt signaling by the LRP5, we generated a structural model of the LRP5 extracellular region based on the structure of the LDL Receptor (LDLR) (Jeon et al., 2001). Using this model, we created a panel of mutants in specific domains of the extracellular region of LRP5 each with the potential to result in an HBM-like function. In this report, we present the effect of these mutations on LRP5 and Dkk1 functions using canonical Wnt β-catenin–TCF signal analysis in bone cells.
Section snippets
LRP5 structural model
A set of YWTD-propeller sequence segments from mouse and human LRP5 sequences was assembled and aligned using CLUSTALW (Thompson et al., 1994), together with the sequences corresponding to the modeled YWTD-propeller structures (Springer, 1998). A tertiary-structure homology model for the first propeller of human LRP5 using the 1lpx model as a template (Kopp and Schwede, 2004) was built with InsightII (Accelrys Inc., San Diego, CA) and examined using its graphics display programs and Rasmol (
The LRP5 structural model shows residue G171 resides in β-propeller 1, blade 4
LRP5 is a type I transmembrane receptor of 1615 amino acids and consists of a long extracellular region (1376 aa), a single membrane spanning segment (22 aa) and a cytoplasmic region (207 aa) (Hey et al., 1998). A schematic of human LRP5 was constructed based on LDL receptor modeling, and homology alignments of LDLR, mouse and human LRP5/6 extracellular domains (Fig. 1A). The extracellular domain contains four β-propellers (∼ 265 aa each) which alternate with four EGF-receptor-like cysteine rich
Structure-based prediction of LRP5 mutants correlates with functional outcome
This study represents the first systematic attempt to evaluate the structure–function relationship of human LRP5 utilizing molecular modeling. It also provides insight into the impact of various LRP5 mutations on Wnt signaling and Dkk1 function. The 3-D model for LRP5 generated by utilizing β-propeller models from the LDLR (Jeon et al., 2001) allowed us to recognize the structural context of the G171V mutation (Fig. 1B and C), which resulted in the HBM phenotype in humans and transgenic mice.
Acknowledgements
We are grateful to Drs. Charles Richard and Chris Miller for enthusiastic and stimulating discussions during these studies and to Dr. Shun-Ichi Harada for valuable comments on the manuscript.
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Cited by (0)
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Wyeth Research, Cambridge, MA 02140.
- 2
Epitome Biosystems, Waltham, MA 02451, USA.
- 3
Novartis Institute for BioMedical Research Inc., Cambridge, MA 02139, USA.