Analysis of the Sterol Sensing Domain Containing proteins during development

跨膜蛋白补丁（Ptc）结合刺猬蛋白（Hh）并调节Smoothened （Smo）的活性。Ptc含有一个固醇感应结构域（SSD），存在于参与胆固醇稳态或运输的蛋白质中。我们早前报道发现在果蝇的SSD中有两个错义突变表现出明显的显性负作用。我们在体内测试和分析了基底细胞痣综合征人类PTCH1基因错义突变的功能，产生了携带相应果蝇补丁突变的转基因果蝇。当我们错误表达G500V、D504Y、G500R这三个突变时，它们的表现与野生型补丁相似。然而，它们都不能挽救ptc突变胚胎。这一发现表明，并非所有的SSD突变都以显性阴性作用，PTCH1中的GxxxD基序对该蛋白的功能至关重要。为了进一步阐明SSD的功能，我们检索了果蝇和小鼠的基因组，在果蝇中发现了一个新的SSD基因（Ptr: CG11212），在小鼠中发现了四个新的SSD基因（Ptchd1、Ptchd2、Ptchd3、Ptchd4）。对所有基因进行克隆和测序。用RT-PCR和原位杂交分析其表达。在E13胚胎时，Ptchd1在小脑原基中表达，在E11胚胎后，Ptchd2在髓脑和脊髓中表达，而Ptchd3在成年睾丸精细胞中表达受限。果蝇的Ptr表达是动态的。转录本在胚层被检测到，除了在囊胚期的中胚层区域。后来这个表达变成了分段式的。抗Ptr抗体分析表明，Ptr蛋白定位于卵巢的囊胚母细胞和卵母细胞，而Ptr蛋白则在5期胚胎的中心体中检测到。这些表达模式预示着Ptr家族基因的一个功能参与生殖细胞的形成。

Transmembrane protein Patched (Ptc) binds to Hedgehog (Hh) proteins and regulates the activity of Smoothened (Smo). Ptc contains a sterol-sensing domain (SSD) found in proteins involved in cholesterol homeostasis or trafficking. We earlier reported finding that two missense mutations in the SSD of Drosophila patched exhibit a clear dominant negative effect. Here we tested and analyzed in vivo the function of Basal Cell Nevus Syndrome missense mutations of human PTCH1 gene generating transgenic flies carrying corresponding Drosophila patched mutations. When we missexpressed all three mutations, G500V D504Y G500R, they behaved similar to wild-type patched. However none of them could rescue ptc mutant embryos. This finding suggested that not all SSD mutations act with a dominant negative and that the GxxxD motif in PTCH1 is essential for the protein function.To further elucidate SSD functions, we searched Drosophila and mouse genomes and identified a new SSD containing a gene in Drosophila (Ptr: CG11212) and four new SSD containing genes in mouse (Ptchd1 Ptchd2 Ptchd3 Ptchd4). All genes were cloned and sequenced. Their expressions were then analyzed by RT-PCR and in situ hybridization. Ptchd1 expression was observed in cerebellar primordium at E13 embryos, Ptchd2 expression in myelencephalon and spinal cord after E11, while Ptchd3 expression was restricted in spermatocyte of adult testis.Drosophila Ptr expression is very dynamic. Transcripts are detected at the germ layers except in the future mesoderm region at blastoderm stage. Later the expression becomes segmental. Anti-Ptr antibody analyses showed that Ptr protein was localized in cystoblasts and oocytes in ovary, while Ptr is detected later in centrosomes at stage 5 embryos. These expression patterns predict that one of the functions of Ptr family genes is involved in germ cell formation.