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. 2010 Jan 1;24(1):57-71.
doi: 10.1101/gad.1870310.

Genetic and biochemical definition of the Hedgehog receptor

Xiaoyan Zheng  1 , Randall K Mann, Navdar Sever, Philip A Beachy
Affiliations

Genetic and biochemical definition of the Hedgehog receptor

Xiaoyan Zheng et al. Genes Dev. .

Abstract

Although the transporter-like protein Patched (Ptc) is genetically implicated in reception of the extracellular Hedgehog (Hh) protein signal, a clear definition of the Hh receptor is complicated by the existence of additional Hh-binding proteins and, in Drosophila, by the lack of physical evidence for direct binding of Hh to Ptc. Here we show that activity of Ihog (Interference hedgehog), or of its close relative Boi (Brother of Ihog), is absolutely required for Hh biological response and for sequestration of the Hh protein to limit long-range signaling. We demonstrate that Ihog interacts directly with Ptc, is required for presentation of Ptc on the cell surface, and that Ihog and Ptc are both required for high-affinity Hh binding. On the basis of their joint roles in ligand binding, signal transduction, and receptor trafficking, we conclude that Ihog and Ptc together constitute the Drosophila Hh receptor.

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Figures

Figure 1.
Figure 1.
ihog/boi mutations disrupt embryonic patterning and Hh signal transduction in the wing imaginal disc. (A) Generation of ihog mutation by gene targeting. A genomic region within the ihog locus was replaced by the white marker gene, as shown. Primers used in RT–PCR (Supplemental Fig. S3) are represented as blue arrows. (B) Generation of boi mutation by gene targeting. Splicing patterns of three predicted boi transcripts (boi-RA, red; boi-RB, blue; boi-RD, black) are indicated. The boi-RA transcript initiates from an internal initiation site, thus defining a sixth exon unique to boi-RA. The exon present in Boi-RD, but excluded from Boi-RB, is a darker green. A genomic region was replaced by the white marker gene as shown. Primers used in RT–PCR (Supplemental Fig. S3) are located within exons and are shown as blue arrows. Note that the gene zeste (light green) is transcribed from the opposite strand. (C–E) Cuticle preparations of embryos lacking maternal and zygotic ihog and boi either alone (C,D) or in combination (E). (F–H) Wingless protein expression in stage 10 embryos lacking maternal and zygotic ihog and boi either alone (F,G) or in combination (H). (I) Reduction of Ptc expression (red) in ihog homozygous boi heterozygous mutant clones (boi/+; ihog), marked by loss of GFP expression (yellow arrows). (J) Loss of Ptc expression (red) in boi; ihog homozygous mutant clones, marked by loss of GFP expression (yellow arrow).
Figure 2.
Figure 2.
ihog/boi mutations prevent sequestration of the Hh signal. Each set of panels shows a wing imaginal disc immunostained for GFP (green), Ptc or dppZ (red), and Ci (blue, marking cells of A compartment origin in A–D). Homozygous ihog mutant clones (yellow asterisk) are marked by the loss of GFP expression, and their ihog+/ihog+ sister clones (red asterisk) are marked by elevated levels of GFP expression. (A,C) Homozygous ihog and heterozygous boi mutant clones (boi/+; ihog) in the A compartment behave as wild-type in that only cells a short distance from the Hh-secreting posterior cells (lacking Ci expression) express Ptc (A) and dppZ (C). No Ptc or dppZ expression was detected anterior to the mutant clones. (B,D) Homozygous boi; ihog mutant clones (boi; ihog) originating in the A compartment (as judged by expression of Ci and by position of the ihog+/ihog+ sister clone) fail to express Ptc (B) or dppZ (D). However, Ptc and dppZ expression is noted in thin strips of cells immediately anterior to the borders of these boi; ihog mutant clones, abnormally far from the posterior cell source of the Hh signal, and indicating that sequestration of the Hh signal in the boi; ihog cells failed. White arrows indicate abnormally far-ranging Hh action across mutant clones lacking both Ihog and Boi to induce Ptc or dppZ. Although Ci levels in boi; ihog mutant clones at the compartment boundary are lower than in surrounding cells, these Ci levels are slightly higher than in cells far to the anterior. (E,F) Homozygous boi; ihog; pka-C1 mutant clones originating in the A compartment autonomously express Ptc (E) and dppZ (F), but still fail to sequester the Hh signal. Note expression of Ptc or dppZ in thin strips of cells (blue arrows) immediately anterior to the border (yellow lines) of these boi; ihog; pka-C1 mutant clones, indicating that elevated Ptc expression alone is not sufficient to sequester the Hh signal, and that Ihog family proteins are absolutely required to limit the range of Hh activity. White lines indicate the border of Hh-secreting posterior cells.
Figure 3.
Figure 3.
Ihog/Boi is required for Hh response, not export. Wing discs are immunostained for GFP (green) and Ptc (red). ihog homozygous mutant clones also express active GAL4 (from ActP-Gal4, activated by the MARCM method and indicated by expression of mCD8GFP) and have been induced in boi heterozygous (boi/+; ihog in A,B) or boi hemizygous (boi; ihog in C,D) larvae; cells in these clones simultaneously express a UAS-Hh transgene. Note that in the A compartment Hh is expressed only in GFP-positive cells. (A,B) Ptc expression was induced both inside and outside of the Hh-expressing ihog mutant clone carried by boi heterozygous animals (boi/+; ihog; ActP-Gal4 > UAS-Hh). (C,D) Ptc expression was induced only in cells surrounding the Hh-expressing ihog mutant clones carried by boi hemizygous animals (boi; ihog; ActP–Gal4 > UAS–Hh). boi; ihog mutant cells are thus not capable of responding to the Hh signal, but do produce and release an active Hh signal that stimulates pathway activation in surrounding cells.
Figure 4.
Figure 4.
Cell surface presentation of Ptc is dependent on Ihog. (A) A schematic diagram of the Ihog protein. (B) Drosophila S2R+ cells coexpressing Ptc with wild-type or variant Ihog proteins were lysed, Ihog proteins were immunoprecipitated with anti-Ihog antibody and Protein G beads, and proteins were detected by immunoblotting for Ihog (top panel) or Ptc (bottom panel). Ihog Fn2 but not Fn1 is critical for Ptc binding. Note that two surface residues on the Fn2 domain are mutated in IhogFn2* (see Supplemental Fig. S6). (C) Drosophila S2R+ cells expressing wild-type or variant Ihog proteins were incubated with HhN-conditioned medium, cells were lysed, Ihog proteins were immunoprecipitated with anti-Ihog antibody and Protein G beads, and proteins were detected by immunoblotting for Ihog (top panel) or Hh (bottom panel). Ihog Fn1 but not Fn2 is critical for HhN binding. (D–I) Confocal microscope images showing localization of Ihog (red) and Ptc (green) proteins. Drosophila S2R+ cells were transfected for expression of Ptc and/or Ihog, as indicated. Ihog protein was detected mainly on the cell surface (D), whereas Ptc was localized mainly in intracellular vesicles with a trace of cell surface expression (E). Surface localization of Ptc increased with coexpression of Ihog (F), IhogΔCTD (G), or IhogΔFn1 (H), but not with IhogΔFn2 (I). (J) S2R+ cells transfected for expression of Ptc and Ihog variants were labeled by surface biotinylation. Immunoblots in the top panels show 5% of the biotinylated cell lysate, and the bottom panel shows the biotin-labeled proteins recovered by Streptavidin-agarose beads. Coexpression with Ihog and IhogΔCTD increased levels of Ptc expressed on the surface.
Figure 5.
Figure 5.
Sequence requirements for function of Ihog proteins in Hh transduction. Each set of panels shows a wing disc immunostained for GFP (green) and Ptc (red). The yellow outlines indicate marked clones in the A compartment near the A/P boundary, where Ptc normally is highly expressed. (A) Loss of Ptc expression in boi hemizygous larvae containing MARCM clones (indicated by expression of mCD8GFP) that lack ihog function (boi; ihog). (B–F) Hemizygous boi larvae with MARCM clones (indicated by expression of mCD8GFP) lacking ihog function (boi; ihog) and specifically expressing wild-type Ihog (B), wild-type Boi (C), or various altered Ihog proteins as indicated (D–F). Note that Ptc expression is rescued by IhogΔCTD, but not by variants with altered or missing Fn1 or Fn2 domains. Note that two surface residues on Fn2 domain are mutated in IhogFn2* (see Supplemental Fig. S6).
Figure 6.
Figure 6.
Presentation of Ptc on the cell surface is insufficient for high-affinity Hh binding. (A) S2R+ cells transfected for expression of Ptc, Ptc1180, or Ptc1130 together with control dsRNA showed mild increase in binding of HhN-Ren (Renilla luciferase-tagged form of HhN) as compared with cells expressing GFP. Cells expressing Ptc, Ptc1180, or Ptc1130 together with dsRNA against ihog showed significantly reduced binding to HhN-Ren, whereas cells expressing Ptc, Ptc1180, or Ptc1130 together with Ihog and control dsRNA showed dramatically enhanced binding to HhN-Ren. Note that the fold increase in HhN-Ren binding was calculated by normalizing to cells transfected for control expression of GFP together with dsRNA against ihog. Similar to full-length Ptc protein, constitutively surface-localized Ptc1180 and Ptc1130 (Supplemental Fig. S8; Lu et al. 2006) also require Ihog for high-affinity HhN binding. (B) Cell-free binding of HhN to membrane vesicles containing Ihog, Ptc, or the two combined. HhN-Ren binding to vesicles was enhanced dramatically with vesicles from Hi5 cells expressing both Ihog and Ptc. The inset shows levels of Ihog and Ptc present in vesicles from cells expressing either protein alone or in combination. (C–F) Each set of panels shows a wing disc immunostained for GFP (green), Hh (red), and Ptc (blue). MARCM clones (indicated by expression of mCD8GFP) lacking ihog and induced in boi heterozygous (boi/+; ihog in C,D) or boi hemizygous (boi; ihog in E,F) larvae simultaneously express a UAS-Ptc1130 transgene driven by ActP-Gal4. (C,D) HhNp protein accumulated dramatically in boi/+; ihog clones expressing Ptc1130. Note that high-level HhNp accumulation occurred only in the cells that immediately abut Hh-expressing P cells (blue arrows), but not in other boundary cells outside the clone that lack expression of Ptc1130 (yellow arrows). We also noted such accumulations at the boundaries of Ptc1130-expressing clones within the posterior compartment. (E,F) No HhN accumulated in boi; ihog clones expressing Ptc1130. Note that HhN traveled through the Ptc1130-expressing boi; ihog mutant clones and accumulated in wild-type cells immediately anterior to the mutant clone (yellow asterisks).
Figure 7.
Figure 7.
A complex of Ihog, Ptc, and HhN. Immobilized HhN preferentially binds detergent-solubilized Ptc in the presence of Ihog. GluGlu-tagged HhN protein was purified using Sepharose beads containing the anti-GluGlu monoclonal antibody, and these beads were incubated with detergent-solubilized extracts from S2R+ cells expressing Ptc variants (lanes 1–3), or Ptc variants coexpressed with HA-tagged Ihog (lanes 4–6). Precipitation of Ptc by Sepharose HhN-GluGlu beads depended critically on the presence of Ihog, and was much reduced by deletion of the large extracellular Ptc loops (Δloop1,2 or Δloop7,8). (Lane 7) In comparison, precipitation of Ihog with anti-HA beads precipitated significantly less Ptc.
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