title: Identification and characterization of two novel PTCH1 splice variants.
abstract: Patched-1 (PTCH1), one of the key molecules involved in the Hedgehog (HH) signaling pathway, acts as the receptor of the HH ligand. PTCH1 also inhibits the positive signal transducer Smoothened (SMO). Several PTCH1 splice variants have been identified and confirmed to play critical roles in HH pathway regulation. In the present study, two novel alternatively spliced variants of PTCH1 transcripts, designated PTCH1-Δ10 and PTCH1-Δ15, were found in humans, mice and zebrafish using RT-PCR, direct sequencing and ribonuclease protection assays. PTCH1-Δ10 lacks exon 10, which encodes part of the sterol-sensing domain (SSD), while PTCH1-Δ15 lacks 166 bp of exon 15, which causes a frame shift that generates a premature stop codon leading to a truncated PTCH1 protein. Different truncated PTCH1 proteins localized in the cytoplasm were capable of internalizing the N-terminal fragment of Sonic hedgehog (SHH-N), which was visualized using immunofluorescence microscopy. Exon skipping dramatically influenced the steady states of the proteins, with the levels of PTCH1-1B and PTCH1-Δ10 being significantly higher than those of PTCH1-Δ15, as detected using western blot. These results imply that the pronounced inhibitory signaling properties of PTCH1-1B and PTCH1-Δ10 may be partially due to high protein expression in addition to intrinsic functional differences. All isoforms examined worked as functional receptors of SHH. However, the isoforms PTCH1-1B and PTCH1-Δ10 inhibited SMO and the pathway transcription factor glioma 1 (GLI1) to a greater extent than did PTCH1-Δ15. In addition, PTCH1-1B and PTCH1-Δ10 (but not PTCH1-Δ15) can be negative regulators of the HH pathway. These results indicate that the SSD domain and the C-terminal region are essential for maximal repressor function of PTCH1. Additionally, SMO inhibition by PTCH1 occurred through a nonstoichiometric, catalytic mechanism, indicating that this inhibition was less dependent on the dose of the PTCH1 protein. Finally, all these isoforms have been revealed to inhibit GLI1 activation by either the classical HH signaling pathway or a new pathway not reliant on both SMO and apoptosis. Thus, our study clearly demonstrated the unique involvement of the two novel PTCH1 splice variants in HH signal transduction.
authors.affiliation: Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou Higher Education Mega Center, 510006, PR China.
authors.forename: Pei
authors.initials: P
authors.lastname: Yu
authors.affiliation: Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou Higher Education Mega Center, 510006, PR China.
authors.forename: Jinqing
authors.initials: J
authors.lastname: Yang
authors.affiliation: Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou Higher Education Mega Center, 510006, PR China. Electronic address: zhang39@mail.sysu.edu.cn.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D001665:Binding Sites; D004705:Endocytosis / Q000502:physiology; D053823:Hedgehog Proteins / Q000378:metabolism*; D000072081:Patched-1 Receptor / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics*
chemical_list: D053823:Hedgehog Proteins; C000606236:PTCH1 protein, human; D000072081:Patched-1 Receptor; D020033:Protein Isoforms; C000606240:Ptch1 protein, mouse
keywords: Exon skipping; GLI1; Patched-1; SHH; Splice variants; Sterol-sensing domain