Mutations in CDON, encoding a hedgehog receptor, result in holoprosencephaly and defective interactions with other hedgehog receptors

Abstract

Holoprosencephaly (HPE), a common human congenital anomaly defined by a failure to delineate the midline of the forebrain and/or midface, is associated with diminished Sonic hedgehog (SHH)-pathway activity in development of these structures. SHH signaling is regulated by a network of ligand-binding factors, including the primary receptor PTCH1 and the putative coreceptors, CDON (also called CDO), BOC, and GAS1. Although binding of SHH to these receptors promotes pathway activity, it is not known whether interactions between these receptors are important. We report here identification of missense CDON mutations in human HPE. These mutations diminish CDON's ability to support SHH-dependent gene expression in cell-based signaling assays. The mutations occur outside the SHH-binding domain of CDON, and the encoded variant CDON proteins do not display defects in binding to SHH. In contrast, wild-type CDON associates with PTCH1 and GAS1, but the variants do so inefficiently, in a manner that parallels their activity in cell-based assays. Our findings argue that CDON must associate with both ligand and other hedgehog-receptor components, particularly PTCH1, for signaling to occur and that disruption of the latter interactions is a mechanism of HPE.

Figure 1

CDON HPE Variants Do Not Support SHH-Dependent Gene Expression (A) Schematic diagram of CDON and the location of HPE-associated variants. The missense CDON variants from human HPE cases (hCDON) and the corresponding residues engineered into a rat Cdon (rCDON) cDNA are shown at the right. The p.Thr684Ser and p.Thr681Ser (T684S and T681S), p.Pro689Ala and p.Pro686Ala (P689A and P686A), and p.Val691Met and p.Val688Met (V691M and V688M) variants are in the linker region between Fn repeats 1 and 2. The p.Val780Glu and p.Val777Glu (V780E and V777E) and the p.Thr790Ala and p.Ile787Ala (T790A and I787A) variants are in Fn repeat 2. The p.Ser940Arg and p.Ser937Arg (S940R and S937R) variants are in the linker region between Fn repeat 3 and the transmembrane (TM) region. SHH binds to Fn repeat 3. (B and C) qRT-PCR analysis of Ptch1 (B) and Gli1 (C) expression in Cdon^−/− MEFs transfected with 2 μg of the indicated CDON vectors plus or minus treatment with SHH. Expression was normalized to Gapdh. (D) qRT-PCR analysis of expression of CDON variants in Cdon^−/− MEFs from same lysates analyzed in (B and C). Error bars represent the means of triplicate determinations ±SD. ^∗∗p < 0.01 as compared to wild-type CDON.

Figure 2

Overexpression Reveals Partial Loss of Function in Some CDON Variants (A and B) qRT-PCR analysis of Ptch1 (A) and Gli1 (B) expression in Cdon^−/− MEFs transfected with 5 μg of the indicated CDON vectors plus or minus treatment with SHH. Expression was normalized to Gapdh. Error bars represent the means of triplicate determinations ±SD. ^∗∗p < 0.01, ^∗p < 0.05 as compared to WT CDON. (C) Immunoblot analysis of CDON expression in 10T1/2 cells under the conditions used in (A and B). CDON variants are designated as in Figure 1.

Figure 3

CDON Variants Display Diminished Interaction with Other SHH-Binding Proteins (A) Cos7 cells were transfected with an expression vector encoding flag-tagged PTCH1 (+) or a control vector (−) and expression vectors encoding CDON, the indicated CDON variants, or a control vector (pBp) as indicated. Lysates were immunoprecipitated with antibodies to flag epitope and immunoblotted with antibodies to flag or CDON. Lysates were also blotted as a control. (B) Results from three independent experiments were quantified for the amount of CDON that immunoprecipitated with PTCH1. (C) 293T cells were transfected with an expression vector encoding BOC (+) or a control vector (−) and expression vectors encoding CDON, the indicated CDON variants, or a control vector (pBp) as indicated. Lysates were immunoprecipitated with antibodies to the CDON intracellular region and immunoblotted with antibodies to CDON or BOC. Lysates were also blotted as a control. (D) Results from three independent experiments were quantified for the amount of BOC that immunoprecipitated with CDON. (E) 10T1/2 or C2C12 cell lysates were immunoprecipitated with antibodies to CDON and immunoblotted with antibodies to CDON and GAS1. Lysates were immunoblotted as a control. (F) 293T cells were transfected with an expression vector encoding GAS1 (+) or a control vector (−) and expression vectors encoding CDON, the indicated CDON variants, or a control vector (pBp) as indicated. Lysates were immunoprecipitated with antibodies to the CDON intracellular region and immunoblotted with antibodies to CDON or GAS1. Lysates were also blotted as a control. (G) Results from three independent experiments were quantified for the amount of GAS1 that immunoprecipitated with CDON. CDON variants are designated as in Figure 1. Error bars represent the means of triplicate determinations ±SD. ^∗∗p < 0.01, ^∗p < 0.05 as compared to wild-type CDON.

Figure 4

CDON, BOC, and GAS1 Have Roughly Additive Activity in Promoting SHH-Dependent Gene Expression (A and B) qRT-PCR analysis of Ptch1 and Gli1 expression in Cdon^−/− MEFs transfected with 2 μg of the indicated CDON, BOC, and GAS1 expression vectors plus or minus treatment with SHH. Expression was normalized to Gapdh. (C) qRT-PCR analysis of expression of CDON variants in Cdon^−/− MEFs from same RNA samples analyzed in (A and B). (D) Immunoblot analysis of CDON, BOC, and GAS1 expression in Cdon^−/− MEFs transfected with 5 μg of CDON, BOC, and GAS1 expression vectors. CDON variants are designated as in Figure 1. Error bars represent the means of triplicate determinations ±SD. ^∗∗p < 0.01 as compared to wild-type CDON.

Figure 5

CDON Fn Repeats Bind GAS1, BOC, and CDON Itself (A) Cos7 cells were transfected with plasmids encoding CDON-Fc fusion proteins containing the entire ectodomain (CDON-Fc), the 5 Ig repeats (CDONIg1-5)-Fc) or the 3 Fn repeats (CDON(Fn1-3)-Fc) and GAS1 as indicated; lysates were pulled down with protein A-agarose and immunoblotted with antibodies to Fc or GAS1. (B) As in (A), except CDON-Fc proteins contained the indicated CDON Fn repeats. (C) 293T cells were transfected with plasmids encoding CDON-Fc fusion proteins containing the indicated CDON Fn repeats and BOC as indicated, lysates were pulled down with protein A-agarose and immunoblotted with antibodies to Fc or BOC. (D) 293T cells were transfected with plasmids encoding CDON-Fc fusion proteins containing the indicated CDON Fn repeats and full-length CDON as indicated; lysates were pulled down with protein A-agarose and immunoblotted with antibodies to Fc or CDON. (E and F) qRT-PCR analysis of Ptch1 (E) and Gli1 (F) expression in Cdo^−/− MEFs transfected with 2 μg of vectors encoding WT CDON or CDON deletion variants lacking the indicated Fn domains plus or minus treatment with SHH. Expression was normalized to Gapdh. (G) qRT-PCR analysis of expression of CDON and CDON deletion mutants in the same lysates analyzed in (E and F). Error bars represent the means of triplicate determinations ±SD. ^∗∗p < 0.01, ^∗p < 0.05 as compared to wild-type CDON.