Molecular cloning and expression of a membrane form guanylyl cyclase in the crayfish, Procambarus clarkii
| Autor: | Hui-Fen Liu, 劉慧芬 |
|---|---|
| Rok vydání: | 2004 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 92 In this study, two membrane form guanylyl cyclases (mGC) were cloned from the abdominal muscle of the crayfish Procambarus clarkii－PcGC-M2 (5,158 bp) and PcGC-M2’ (4,989 bp). PcGC-M2, a typical mGC, contains an open reading frame encoding 1,403 amino acid residues. Sequence analysis indicated that PcGC-M2 is composed of an extracellular domain (ECD), a transmembrane domain (TM), a kinase homology domain (KHD), a guanylyl cyclase catalytic domain (GCD) and a C-terminal tail. PcGC-M2’ lacks a stretch of 169 bp (corresponding to nucleotides 2610－2778) in the KHD of PcGC-M2, hence resulting in a 766-residue protein composed only of ECD, TM, and a partial KHD. Except PcGC-M2’, PcGC-M2 is most closely related (34 % identity) to Drosophila (DrGC-1) and Anopheles gambiae (AgaGC) membrane form GCs. These invertebrate mGCs also share a unique distribution pattern of conserved cystine residues in the ECD. The KHD of PcGC-M2 contains 26 residues that are conserved in protein kinases and a putative ATP binding site－Gly-X-X-X-Gly. Furthermore, RT-PCR analysis demonstrated that PcGC-M2 and PcGC-M2’ were present in various crustacean hyperglycemic hormone (CHH) target tissues, including muscle, hepatopancreas, heart, reproductive organs, gill and antennal gland, implying that PcGC-M2 and PcGC-M2’ participate in the signaling cascade activated by CHH. It is suggested that both PcGC-M2 and PcGC-M2’ encode proteins that contain CHH binding sites. Furthermore, PcGC-M2 and PcGC-M2’ may modulate CHH-activated cGMP signaling by forming homodimers and heterodimers. Finally, PcGC-M2 and PcGC-M2’ were expressed in eukaryotic cells (insect Schneider 2 cells). Immunoblotting analysis showed that proteins with expected sizes (153 and 80 kDa, respectively) were expressed at low levels. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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