Self-association of the CTD. (A) Autoradiogram after PAGE analysis of ³⁵SCTD produced by in vitro translation either before (CTD) or after incubation with glutathione beads (CTD + GST proteins) that were bound by bacteria-generated GST or GST fusions proteins, as indicated. ERR is the cytoplasmic domain of the estrogen receptor-related protein (CG7404) (Ostberg et al. 2003), and was used as an unrelated control. Pull-downs were with 10-μg beads except for lane 5 (5 μg). (B) α-Myc and α-GST immunoprecipitates of S2 cell extracts containing Myc-tagged CTD, Myc-tagged ERR, and GST-CTD fusion proteins, fractionated by PAGE and Western blots and probed with α-Myc and α-GST antibodies as indicated on the right. (Lane 5) Coimunoprecipitation was only apparent with Myc-CTD and GSTCTD. (C) Gel exclusion chromatography of purified MBP (40.4 kDa) and MBP-CTD (60.5 kDa) fusion proteins yielded single peaks of 280-nm absorbance at volumes expected for a MBP monomer and a MBPCTD trimer: 14.3 mL and 11.4 mL, respectively. Molecular weight standards eluted as follows: ferritin (440 kDa, 9.7 mL); aldolase (158 kDa, 11.7 mL); albumin (67 kDa, 13.3 mL); ovalbumin (43 kDa, 14.1 mL); chymotrypsinogen A (25 kDa, 15.2 mL); and RNAse A (13.7 kDa, 16.3 mL). (D) Nedd4 coimmunoprecipitated with the Ptc CTD (residues 1104–1286) but not with the CTD of Smo (residues 559–1036).

Inhibition of Hh signaling by ectopic Patched expression. Wings from wild-type (A) and transgenic (B–F) flies expressing wild-type or mutant Ptc proteins. Wing veins 3 and 4 are indicated. (B–D) Ptc expression in the posterior compartment reduces the 3–4 intervein region, a phenotype that is more extreme when protein with mutant CTD is expressed. Reducing the level of wild-type Ptc (F) increases Hh pathway activation in the anterior compartment when Ptc^Δ1/2C is expressed uniformly (E,F). Wings of ptc^B98/+ flies were indistinguishable from wild type (not shown). All wings are shown anterior up.

The CTD regulates turnover of Patched protein. (A) Western blot analysis of wild-type wing discs and wing discs expressing the indicated proteins under the control of en-Gal4. Thirty discs in each lane. (B) Quantification of wild-type Ptc, Ptc^Δ1/2C, and Ptc^3P by Western analysis in transfected S2 cells after incubation in the presence of cycloheximide for the indicated times. (C) Quantification of wild-type mouse Ptc and mouse Ptc^ΔCTD in transfected COS-7 cells after incubation in the presence of cycloheximide for the indicated times. (D) Pulse-chase analysis of Ptc and Ptc^Δ1/2C in transfected S2 cells labeled with ³⁵S-methionine. (E) Western blot analysis of S2 cells expressing CD2 or CD2-CTD and incubated in the presence or absence of cycloheximide for 3 h with or without NH₄Cl. Blot was probed with α-CD2 antibody.

Patched protein redistribution in response to Hh requires the CTD. (A–D) GFP fluorescence in salivary glands expressing Ptc-GFP (A,B) or Ptc^Δ1/2C-GFP (C,D) under control of SGS-Gal4 in the presence (B,D) or absence (A,C) of Hh. (E–H) Epi-fluorescence monitored in wing discs expressing GFP-Ptc (E), GFP-Ptc^Δ1/2C (F), both GFP-Ptc and Ptc^Δ1/2C (G), both Ptc and GFP-Ptc^Δ1/2C (H), Ptc^3P (I), or Ptc^PPAA (J) in the posterior compartment under control of en-Gal4.

Ptc is a Hh receptor. Distribution of Ptc (green; A,C, D,F,G,I,J,L) and Hh-N (red; B,C,E,F,H,I,K,L) in S2 cells expressing GFP-Ptc (A–C), GFP-Ptc^Δ1/2C (D–F), Ptc^3P (G–I), and Ptc^PPAA (J–L), and exposed to Hh-N conditioned medium. (C,F,I,L) Merged images. Hh was detected with α-Hh antibody. Ptc was detected with α-Ptc antibody in panels G, I, J, and L. Images are confocal micrographs.

Ptc is a multimer. (A) FRET analysis of S2 cells expressing the indicated proteins (CFP, YFP, a CFP-YFP fusion; a CFP-Ptc or YFP-Ptc fusion; a CFP-Ptc^ΔC or YFP-Ptc^ΔC fusion; or a YFP-Disp fusion. (Black bars) Donor FRET from the bleached region; (white bars) donor FRET from unbleached region. (B) Coimmunoprecipitation analysis of S2 cells expressing GFP-Ptc^WT, GFP-Ptc^Δ1/2C, GST-Ptc^Δ1/2C, or GST-SmoC (residues 559–1036), immunoprecipitated with α-GST antibody and probed with either α-GST (left) or α-GFP (right) antibodies.

Model of Ptc homo- and heteromultimers. Ptc monomers can multimerize to form stable trimers that repress the Hh signaling pathway, unless bound by Hh. Ptc monomers with a mutant CTD can form stable trimers that consist entirely of the mutant protein or include wild-type Ptc. Trimers containing CTD mutations can bind Hh, are stable, but are incapable of inactivating the Hh signaling pathway.