Distinct Consequences of Sterol Sensor Mutations in Drosophila and Mouse patched Homologs

Abstract

The membrane protein Patched (Ptc) is a critical regulator of Hedgehog signaling. Ptc is among a family of proteins that contain a sterol sensor motif. The function of this domain is poorly understood, but some proteins that contain sterol sensors are involved in cholesterol homeostasis. In the SREBP cleavage-activating protein (SCAP), sterols inhibit the protein's activity through this domain. Mutations in two highly conserved residues in the SCAP sterol sensor have been identified that confer resistance to sterol regulation. We introduced the analogous mutations in the sterol sensor motif of fly Ptc and mouse Ptc1 and examined their effect on protein activity. In contrast to SCAP, the sterol sensor mutations had different affects on Drosophila Ptc; Ptc Y442C retained function, while Ptc D584N conferred dominant negative activity. In the wing imaginal disc, Ptc D584N overexpression induced Hedgehog targets by stabilizing Cubitus interruptus and inducing decapentaplegic. However, Ptc D584N did not induce collier, a gene that requires high levels of Hedgehog signaling. In mouse Ptc1, the Y438C and D585N mutations did not stimulate signaling in Shh-responsive cell lines but did complement murine ptc1(-/-) cells. The results suggest that mutations in sterol sensor motifs alter function differently between sterol sensor family members.

Functional analysis in Drosophila indicates that the NBCCS/PTCH1 mutation G509V results in activation of smoothened through a dominant‐negative mechanism

“…However, the results presented here agree with other studies that indicate the SSD is critical for Ptc function. Substitutions within TM3 (G477R) and TM6 (D584N) have both been observed to result in dominant-negative activity of Drosophila Ptc (Strutt et al, 2001;Johnson et al, 2002). Our data (Figs.…”

“…Investigation of the D584N substitution in both Drosophila and murine cell culture also resulted in the similar conclusions of dominant-negative activity in flies and close to wild-type activity in the cell culture assays (Strutt et al, 2001;Johnson et al, 2002). This finding may indicate a fundamental difference in Patched signalling between Drosophila and vertebrates, or it may reflect the differences in the assays used.…”

“…An Asp 584 to Asn substitution in TM6 (D584N) has been shown to act as a dominant-negative allele in Drosophila with the ability to up-regulate Smo protein levels when ectopically expressed (Martin et al, 2001). In contrast to the clear dominant-negative effect observed in Drosophila, the corresponding mutation in mouse Ptc1 did not significantly alter Ptc function in an in vitro assay (Johnson et al, 2002). This result suggests the possibility that SSD mutations may affect fly and mouse Ptc homologues differently.…”

“…The SSD is a region of five consecutive membrane-spanning domains that is phylogenetically conserved across a range of proteins with roles in sterol and vesicle trafficking. SSD mutations in TM3 and TM6 of Drosophila Ptc have been shown to result in antimorphic or dominant-negative alleles, suggesting that the TM4 mutations may also be dominantnegative (Strutt et al, 2001;Johnson et al, 2002). An Asp 584 to Asn substitution in TM6 (D584N) has been shown to act as a dominant-negative allele in Drosophila with the ability to up-regulate Smo protein levels when ectopically expressed (Martin et al, 2001).…”

A large complex containing Patched and Smoothened initiates Hedgehog signaling inDrosophila

“…Ptc D584N has an aspartic acid changed to asparagine at position 584 in the sterol sensing domain (SSD), is also a dominant negative and activates the pathway, although not as strongly as Ptc 1130X (Johnson et al, 2002). The amount of Ptc immunoprecipitated (~1%) by Sxl and Ci appeared to decrease compared with the wild type ( Fig.…”