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Proc. Natl. Acad. Sci. USA, August 28, 2018 115 (35) 8787-8792 | https://doi.org/10.1073/pnas.1805901115
Biophysical and functional characterization of Norrin signaling through Frizzled4
Injin Banga,1, Hee Ryung Kimb,1, Andrew H. Beavenc,2, Jinuk Kima, Seung-Bum Koa, Gyu Rie Leed,3, Hasup Leed,4, Wonpil Ime,f, Chaok Seokd, Ka Young Chungb,5, and Hee-Jung Choia,5
aDepartment of Biological Sciences, Seoul National University, 08826 Seoul, Republic of Korea; bSchool of Pharmacy, Sungkyunkwan University, 16419 Suwon, Republic of Korea; cDepartment of Chemistry, The University of Kansas, Lawrence, KS 66047; dDepartment of Chemistry, Seoul National University, 08826 Seoul, Republic of Korea; eDepartment of Biological Sciences, Lehigh University, Bethlehem, PA 18015; and fDepartment of Bioengineering, Lehigh University, Bethlehem, PA 18015
1I.B. and H.R.K. contributed equally to this work.
aDepartment of Biological Sciences, Seoul National University, 08826 Seoul, Republic of Korea; bSchool of Pharmacy, Sungkyunkwan University, 16419 Suwon, Republic of Korea; cDepartment of Chemistry, The University of Kansas, Lawrence, KS 66047; dDepartment of Chemistry, Seoul National University, 08826 Seoul, Republic of Korea; eDepartment of Biological Sciences, Lehigh University, Bethlehem, PA 18015; and fDepartment of Bioengineering, Lehigh University, Bethlehem, PA 18015
1I.B. and H.R.K. contributed equally to this work.
2Present address: Department of Biomedical Engineering, University of Minnesota,Minneapolis, MN 55455.
3Present address: Department of Biochemistry, University of Washington, Seattle, WA 98195.
4Present address: Organic Material Lab, Samsung Advanced Institute of Technology, Gyeonggi-do, 443-803 Suwon, Republic of Korea.
5To whom correspondence may be addressed.
Abstract
Wnt signaling is initiated by Wnt ligand binding to the extracellular ligand binding domain, called the cysteine-rich domain (CRD), of a Frizzled (Fzd) receptor. Norrin, an atypical Fzd ligand, specifically interacts with Fzd4 to activate β-catenin-dependent canonical Wnt signaling. Much of the molecular basis that confers Norrin selectivity in binding to Fzd4 was revealed through the structural study of the Fzd4CRD-Norrin complex. However, how the ligand interaction, seemingly localized at the CRD, is transmitted across full-length Fzd4 to the cytoplasm remains largely unknown. Here, we show that a flexible linker domain, which connects the CRD to the transmembrane domain, plays an important role in Norrin signaling. The linker domain directly contributes to the high-affinity interaction between Fzd4 and Norrin as shown by ∼10-fold higher binding affinity of Fzd4CRD to Norrin in the presence of the linker. Swapping the Fzd4 linker with the Fzd5 linker resulted in the loss of Norrin signaling, suggesting the importance of the linker in ligand-specific cellular response. In addition, structural dynamics of Fzd4 associated with Norrin binding investigated by hydrogen/deuterium exchange MS revealed Norrin-induced conformational changes on the linker domain and the intracellular loop 3 (ICL3) region of Fzd4. Cell-based functional assays showed that linker deletion, L430A and L433A mutations at ICL3, and C-terminal tail truncation displayed reduced β-catenin-dependent signaling activity, indicating the functional significance of these sites. Together, our results provide functional and biochemical dissection of Fzd4 in Norrin signaling.
Frizzled 4, linker domain, Norrin, Dishevelled, signaling
논문정보
- Research article
- 2018년 08월 (BRIC 등록일 2018-09-27)
- 국내(교신)+국외 연구진
- 생명과학 > 구조생물 및 생물물리학
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