Biochemical characterization of Wnt-Frizzled interactions using a soluble, biologically active vertebrate Wnt protein

被引：287

作者：

Hsieh, JC

Rattner, A

Smallwood, PM

Nathans, J

机构：

[1] Johns Hopkins Univ, Sch Med, Dept Mol Biol & Genet, Baltimore, MD 21205 USA

[2] Johns Hopkins Univ, Sch Med, Dept Neurosci & Ophthalmol, Baltimore, MD 21205 USA

[3] Johns Hopkins Univ, Sch Med, Howard Hughes Med Inst, Baltimore, MD 21205 USA

来源：

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA | 1999年 / 96卷 / 07期

关键词：

D O I：

10.1073/pnas.96.7.3546

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

Biochemical studies of Wnt signaling have been hampered by difficulties in obtaining large quantities of soluble, biologically active Wnt proteins. In this paper, we report the production in Drosophila S2 cells of biologically active Xenopus Wnt8 (XWnt8). Epitope- or alkaline phosphatase-tagged XWnt8 proteins are secreted by concentrated S2 cells in a form that is suitable for quantitative biochemical experiments with yields of 5 and 0.5 mg per liter, respectively. Conditions also are described for the production in 293 cells of an IgG fusion of the cysteine-rich domain (CRD) of mouse Frizzled 8 with a yield of 20 mg/liter. We demonstrate the use of these proteins for studying the interactions between soluble XWnt8 and various Frizzled proteins, membrane anchored or secreted CRDs, and a set of insertion mutants in the CRD of Drosophila Frizzled 2. In a solid phase binding assay, the affinity of the XWnt8-alkaline phosphatase Fusion for the purified mouse Frizzled 8-CRD-IgG fusion is approximate to 9 nM.

引用

页码：3546 / 3551

页数：6

共 57 条

[41] FGF BINDING AND FGF RECEPTOR ACTIVATION BY SYNTHETIC HEPARAN-DERIVED DISACCHARIDES AND TRISACCHARIDES
ORNITZ, DM
HERR, AB
NILSSON, M
WESTMAN, J
SVAHN, CM
WAKSMAN, G
[J]. SCIENCE, 1995, 268 (5209) : 432 - 436
[42] SECRETED INT-1 PROTEIN IS ASSOCIATED WITH THE CELL-SURFACE
PAPKOFF, J
SCHRYVER, B
[J]. MOLECULAR AND CELLULAR BIOLOGY, 1990, 10 (06) : 2723 - 2730
[43] A family of secreted proteins contains homology to the cysteine-rich ligand-binding domain of frizzled receptors
Rattner, A
Hsieh, JC
Smallwood, PM
Gilbert, DJ
Copeland, NG
Jenkins, NA
Nathans, J
[J]. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1997, 94 (07) : 2859 - 2863
[44] Glycosaminoglycans can modulate extracellular localization of the wingless protein and promote signal transduction
Reichsman, F
Smith, L
Cumberledge, S
[J]. JOURNAL OF CELL BIOLOGY, 1996, 135 (03) : 819 - 827
[45] Wnt signaling and an APC-related gene specify endoderm in early C-elegans embryos
Rocheleau, CE
Downs, WD
Lin, RL
Wittmann, C
Bei, YX
Cha, YH
Ali, M
Priess, JR
Mello, CC
[J]. CELL, 1997, 90 (04) : 707 - 716
[46] Stabilization of beta-catenin by genetic defects in melanoma cell lines
Rubinfeld, B
Robbins, P
ElGamil, M
Albert, I
Porfiri, E
Polakis, P
[J]. SCIENCE, 1997, 275 (5307) : 1790 - 1792
[47] Salic AN, 1997, DEVELOPMENT, V124, P4739
[48] The Caenorhabditis elegans gene lin-17, which is required for certain asymmetric cell divisions, encodes a putative protein similar to seven-transmembrane protein similar to the Drosophila Frizzle protein
Sawa, H
Lobel, L
Horvitz, HR
[J]. GENES & DEVELOPMENT, 1996, 10 (17) : 2189 - 2197
[49] Identification and characterization of a conserved family of protein serine/threonine phosphatases homologous to Drosophila retinal degeneration C (rdgC)
Sherman, PM
Sun, H
Macke, JP
Williams, J
Smallwood, PM
Nathans, J
[J]. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1997, 94 (21) : 11639 - 11644
[50] HEPARIN-INDUCED OLIGOMERIZATION OF FGF MOLECULES IS RESPONSIBLE FOR FGF RECEPTOR DIMERIZATION, ACTIVATION, AND CELL-PROLIFERATION
SPIVAKKROIZMAN, T
LEMMON, MA
DIKIC, I
LADBURY, JE
PINCHASI, D
HUANG, J
JAYE, M
CRUMLEY, G
SCHLESSINGER, J
LAX, I
[J]. CELL, 1994, 79 (06) : 1015 - 1024

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