Parathyroid Hormone Receptor Directly Interacts with Dishevelled to Regulate β-Catenin Signaling and Osteoclastogenesis

Guillermo Romero,W Bruce Sneddon,Yanmei Yang,David Wheeler,Harry C Blair,Peter A Friedman

doi:10.1074/jbc.m110.102970

Guillermo Romero, W Bruce Sneddon + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m110.102970

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Abstract

Bone growth and remodeling depend upon the opposing rates of bone formation and resorption. These functions are regulated by intrinsic seven transmembrane-spanning receptors, the parathyroid hormone receptor (PTH1R) and frizzled (FZD), through their respective ligands, parathyroid hormone (PTH) and Wnt. FZD activation of canonical beta-catenin signaling requires the adapter protein Dishevelled (Dvl). We identified a Dvl-binding motif in the PTH1R. Here, we report that the PTH1R activates the beta-catenin pathway by directly recruiting Dvl, independent of Wnt or LRP5/6. PTH1R coimmunoprecipitated with Dvl. Deleting the carboxyl-terminal PTH1R PDZ-recognition domain did not abrogate PTH1R-Dvl interactions; nor did truncating the receptor at position 480. However, further deletion eliminating the putative Dvl recognition domain abolished PTH1R interactions with Dvl. PTH activated beta-catenin in a time- and concentration-dependent manner and translocated beta-catenin to the nucleus. beta-Catenin activation was inhibited by Dvl2 dominant negatives and by short hairpin RNA sequences targeted against Dvl2. PTH-induced osteoclastogenesis was also inhibited by Dvl2 dominant negative mutants. These findings demonstrate that G protein-coupled receptors other than FZD directly activate beta-catenin signaling, thereby mimicking many of the functions of the canonical Wnt-FZD pathway. The distinct modes whereby FZD and PTH1R activate beta-catenin control convergent or divergent effects on osteoblast differentiation, and osteoclastogenesis may arise from PTH1R-induced second messenger phosphorylation.

Highlights

Direct Interactions between PTH1R and Dvl—Recruitment of Dvl to the plasma membrane is mediated by direct interactions between FZD and the PDZ domain of Dvl proteins
Analysis of the PTH1R sequence revealed the presence of a similar motif (KSWSRW; see Fig. 1A)
The findings described here identify Dvl as a molecular router, integrating signals derived from FZD and PTH1R to

Summary

The abbreviations used are

NF␬B, nuclear factor-␬B; JNK, Jun kinase; PTH, parathyroid hormone; CHO, Chinese hamster ovary; TIRF, total internal reflection fluorescence; TRITC, tetramethylrhodamine isothiocyanate; EGFP, enhanced green fluorescent protein; GPCR, G protein-coupled receptor; CREB, cAMP-response element-binding protein; shRNA, short hairpin RNA; HA, hemagglutinin. Other findings establish that PTH increases ␤-catenin levels in UMR, MC3T3E1, and SAOS cells [16, 17, 20] and that ablation of the Wnt antagonist, secreted frizzled-related protein, blunts the anabolic action of PTH [21]. Cao and co-workers [18] showed that PTH1R signals through LRP6. Further evidence for the interaction of PTH and ␤-catenin pathways in regulating bone turnover comes from studies showing that overexpression of sFRP1 attenuates PTH-dependent bone anabolism [22]. Together, these and other studies imply that the actions of PTH may be partially mediated through ␤-catenin signaling. We describe multiple lines of cross-talk between the two pathways and show that PTH activates ␤-catenin in an LRP- and Wnt-independent manner

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION