Elsevier

Gene

Volume 391, Issues 1–2, 15 April 2007, Pages 103-112
Gene

Structure-based mutation analysis shows the importance of LRP5 β-propeller 1 in modulating Dkk1-mediated inhibition of Wnt signaling

https://doi.org/10.1016/j.gene.2006.12.014Get rights and content

Abstract

A single point mutation (G to T) in the low-density lipoprotein receptor related protein 5 (LRP5) gene results in a glycine to valine amino acid change (G171V) and is responsible for an autosomal dominant high bone mass trait (HBM) in two independent kindreds. LRP5 acts as a co-receptor to Wnts with Frizzled family members and transduces Wnt-canonical signals which can be antagonized by LRP5 ligand, Dickkopf 1 (Dkk1). In the presence of Wnt1, LRP5 or the HBM variant (LRP5–G171V) induces β-catenin nuclear translocation and activates T cell factor (TCF)-luciferase reporter activity. HBM variant suppresses Dkk1 function and this results in reduced inhibition of TCF activity as compared to that with LRP5. Structural analysis of LRP5 revealed that the HBM mutation lies in the 4th blade of the first β-propeller domain. To elucidate the functional significance and consequence of the LRP5–G171V mutation in vitro, we took a structure-based approach to design 15 specific LRP5 point mutations. These included (a) substitutions at the G171 in blade 4, (b) mutations in blades 2–6 of β-propeller 1, and (c) mutations in β-propellers 2, 3 and 4. Here we show that substitutions of glycine at 171 to K, F, I and Q also resulted in HBM-like activity in the presence of Wnt1 and Dkk1. This indicates the importance of the G171 site rather than the effect of specific amino acid modification to LRP5 receptor function. Interestingly, G171 equivalent residue mutations in other blades of β-propeller 1 (A65V, S127V, L200V, A214V and M282V) resulted in LRP5–G171V-like block of Dkk1 function. However G171V type mutations in other β-propellers of LRP5 did not result in resistance to Dkk1 function. These results indicate the importance of LRP5 β-propeller 1 for Dkk1 function and Wnt signaling. These data and additional comparative structural analysis of the LRP5 family member LDLR suggest a potential functional role of the first β-propeller domain through intramolecular interaction with other domains of LRP5 wherein Dkk1 can bind. Such studies may also lead to a better understanding of the mechanisms underlying the reduced function of Dkk1-like inhibitory ligands of LRP5 with HBM-like mutations and its relationship to increased bone density phenotypes.

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)

    1

    Wyeth Research, Cambridge, MA 02140.

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    Epitome Biosystems, Waltham, MA 02451, USA.

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    Novartis Institute for BioMedical Research Inc., Cambridge, MA 02139, USA.

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