Analysis of the Sterol Sensing Domain Containing proteins during development

2006 Fiscal Year Final Research Report Summary

• Project/Area Number: 17570182
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Developmental biology
• Research Institution: Hyogo College of Medicine
• Principal Investigator: NAKANO Yoshiro Hyogo College of Medicine, Faculty of Medicine, Assistant Professor (30360267)
• Co-Investigator(Kenkyū-buntansha): YAMAMOTO Daisuke Tohoku University, Graduate School of Life Science, Professor (50318812) ; UMEDA Masato Kyoto University, Institute for Chemical Research, Professor (10185069)
• Project Period (FY): 2005 – 2006
• Keywords: sterol-sensing / patched / hedgehog / patched-related

Research Abstract

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.