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Comparative Study
. 2005 Oct 5;24(19):3493-503.
doi: 10.1038/sj.emboj.7600817. Epub 2005 Sep 15.

The role of the cysteine-rich domain of Frizzled in Wingless-Armadillo signaling

Affiliations
Comparative Study

The role of the cysteine-rich domain of Frizzled in Wingless-Armadillo signaling

Michael Povelones et al. EMBO J. .

Abstract

The Frizzled (Fz) receptors contain seven transmembrane helices and an amino-terminal cysteine-rich domain (CRD) that is sufficient and necessary for binding of the ligands, the Wnts. Recent genetic experiments have suggested, however, that the CRD is dispensable for signaling. We engineered fz CRD mutant transgenes and tested them for Wg signaling activity. None of the mutants was functional in cell culture or could fully replace fz in vivo. We also show that replacing the CRD with a structurally distinct Wnt-binding domain, the Wnt inhibitory factor, reconstitutes a functional Wg receptor. We therefore hypothesized that the function of the CRD is to bring Wg in close proximity with the membrane portion of the receptor. We tested this model by substituting Wg itself for the CRD, a manipulation that results in a constitutively active receptor. We propose that Fz activates signaling in two steps: Fz uses its CRD to capture Wg, and once bound Wg interacts with the membrane portion of the receptor to initiate signaling.

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Figures

Figure 1
Figure 1
Design fz CRD variants. (A) To test the role of the CRD in Arm signaling, we engineered the following fz constructs into a vector for constitutive expression in Drosophila via the tubulin-α1 promoter (pTub): Wild-type fz (581 amino acids). fz57 and fz81 insert three amino acids (GSG) between residues in the CRD and are predicted to interfere with Wg binding (Hsieh et al, 1999b). fzΔCRD1 specifically deletes the CRD in-frame (amino acids 53–164). This leaves the remainder of the receptor intact, including the native signal sequence and the entire extracellular ‘hinge' region lying between the CRD and the first transmembrane domain. fzΔCRD2 and fz2ΔCRD replace the entire N-terminal portions (up to the open triangles shown in panel B), including the native signal sequences of fz and fz2 with the wg signal sequence and three Flu epitopes (Chen et al, 2004). fzΔCRD3 specifically removes the eight amino acids of the CRD present in fzΔCRD2 but absent in fzΔCRD1, fzWIF and fzSMO specifically exchange the CRD with the WIF domain of hWIF (Hsieh et al, 1999a) or the CRD of Drosophila smoothened (Alcedo et al, 1996; van den Heuvel and Ingham, 1996). wgfzΔCRD and wntDfzΔCRD fusions join the full-length Wg or WntD protein in-frame with the region of Fz C-terminal to the CRD in fzΔCRD1 (amino acids 165–581). Wnt-Fz fusions use the signal sequence native to their respective Wnt protein. (B) Alignment of the Drosophila Fz and Fz2 amino-acid sequence within the CRD. The 10 cysteine residues defining this domain are indicated by shaded vertical bars. Identical amino acids are indicated in red (*). Amino acids with conserved properties are indicated in purple (:) and green (.). Two mutations previously characterized to disrupt the binding between Xenopus Wnt8 and Fz2 CRD (Hsieh et al, 1999b) were engineered into a homologous position in the Fz CRD (gray triangles). FzΔCRD1 removes the entire region shown, leaving the flanking N- and C-terminal sequences unmodified. The breakpoints of FzΔCRD2 and Fz2ΔCRD are indicated with open triangles. FzΔCRD2 includes eight amino acids not present in FzΔCRD1. These eight amino acids are deleted in FzΔCRD3 with the remainder of the construct identical to FzΔCRD2.
Figure 2
Figure 2
Fz protein abundance and surface localization in S2 cells. (A) Whole-cell protein extracts of S2 cells transfected with fz transgenes probed with a polyclonal antibody directed against the ‘hinge' region of Fz (α-Fz, top panel), an epitope, which Fz2 lacks. When the same samples are probed with an HA antibody, we observed that Fz2ΔCRD is expressed comparably to FzΔCRD3, which are both expressed at higher levels than FzΔCRD2. The molecular mass in kDa of a known protein standard is indicated. (B) Cell surface localization of Fz protein was assayed in S2 cells transfected with fz transgenes and stained using an antibody directed against the ‘hinge' region of Fz. Fz2 was not tested as it lacks this epitope. The same settings were used for all samples aside from mock-transfected cells where the gain was intentionally set higher to emphasize lack of signal. Staining is specific, as untransfected cells (*) are not detectable. The scale bar in upper left panel is 10 μm.
Figure 3
Figure 3
Arm signaling of fz CRD variants in S2 cells. (A) To measure Arm signaling, TCF/LEF-dependent luciferase reporter assays were performed on cells cotransfected with pTub (Mock) or a pTub-fz variant and empty pTub (−) or pTub-wg (+). (B) TCF/LEF-dependent luciferase reporter assays were performed on cells cotransfected with pTub (Mock) or a pTub-fz variant and challenged with plain medium (−) or medium containing purified Wg (+).
Figure 4
Figure 4
wgfzΔCRD requires arr to activate Arm signaling in S2 cells. TCF/LEF reporter assays on two sets of cells transfected with empty plasmid (Mock), wntDfzΔCRD or wgfzΔCRD transgenes. One set was cotransfected with control double-stranded RNA (GFP dsRNA) and the second set was cotransfected with arr dsRNA (Arrow dsRNA). There is a 3.7-fold reduction in signaling when cells are cotransfected with arr dsRNA compared to the control dsRNA.
Figure 5
Figure 5
fz CRD mutations show Arm signaling defects in embryos. (A) Cuticle of a wild-type Drosophila embryo. (B) fz,fz2 germline clone zygotically mutant for fz and fz2. The following panels are the same genotype as in (B), zygotically expressing via the tubulin-1α promoter (pTub) one copy of (C) wild-type fz, (D) fz57, (E) fz81 and (F) fzΔCRD1. For each of the transgenes, one insert line was tested.
Figure 6
Figure 6
fz CRD mutations show Arm signaling defects in wings. Shown here are the wing margins of flies possessing different clones as indicated by the vertical label to the right of each panel. Clones that cross the margin elaborate yellow bristles due to the loss of the y+ marker and are indicated with open arrows. Darker bristles that are elaborated by heterozygous cells are indicated with closed arrows. Arrowheads indicate the presence of a bristle formed inappropriately away from the margin. (A) Control clones that do not carry any mutations produce numerous yellow bristles, similar to clones of either fz2 (B) or fz (C). (D) fz,fz2 clones do not generate yellow margin bristles and have large patches along the margin lacking bristles. The bristles generated by surrounding heterozygous tissue show tighter spacing and occasional ectopic bristles that form away from the margin (arrowheads). (E) The margin gaps and ectopic bristle phenotypes of fz,fz2 clones are fully rescued by the ubiquitous expression of one copy of wild-type fz. These wings generate many yellow margin bristles. (F) fz,fz2 clones rescued by fz57 have subtle defects in the spacing of the margin bristles. (G) fz,fz2 clones rescued by fz81 have fewer yellow margin bristles and more severe bristle spacing problems. (H) fz,fz2 clones rescued by fzΔCRD1 have ectopic margin bristles, which are almost invariably dark, indicating they are derived from nearby heterozygous cells. (I) fz,fz2 clones rescued by fzWIF produce numerous yellow bristles. There are places along the margin where a bristle appears to be missing. However, unlike the defects in bristle spacing seen with fz57 and fz81 rescued clones, in fzWIF rescued clones a structure resembling a bristle socket forms in the place of the apparently missing bristle (*). For each of the transgenes, two separate insert lines were tested.
Figure 7
Figure 7
fzWIF activates Arm signaling in vivo. (A) Picture of a wild-type Drosophila wing. The boxed region distal to the posterior crossvein indicates the location of the inset in all panels. Note all wing hairs uniformly point towards the distal portion of the wing. (B) Overexpression of fzWIF generates both Arm-dependent ectopic bristles near the wing margin (arrows) and Arm-independent PCP defects throughout the wing. (C) Overexpression of fz alone only leads to a PCP disruption throughout the wing. (D) Overexpression of fz2 only produces ectopic bristles near the wing margin (arrows).
Figure 8
Figure 8
wgfzΔCRD activates Arm signaling in vivo. (A) Wild-type cuticle pattern displayed by an embryo with one copy of the hairy-gal4 (h) driver but lacking a UAS transgene. (B) Expression of wg in alternating segments results in the formation of ectopic naked cuticle (arrows). (C) Expression of wgfzΔCRD results in the formation of ectopic naked cuticle (arrows). Either expression of wntD (D) or wntDfzΔCRD (E) does not result in naked cuticle formation.

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