Study of identification and characterization of crustacean hyperglycemic hormone receptor: ligand activation, binding, and specificityof receptor guanylyl cyclases

• Main Authors: Shao-Yen Huang, 黃劭彥
• Other Authors: Chi-Ying Lee
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/02528457081743785768

碩士 === 國立彰化師範大學 === 生物學系 === 100 === In the purpose, analysis of 2 mGC genes, PcGC-M2 and PcGC-M2B, were cloned from the muscle of the crayfish in our previous studies and evaluated for their binding affinities of polymorphic CHH peptides to PcGC-M2 and PcGC-M2B are compared. The 2 mGC proteins are identical in containing the same extracellular binding and transmembrane domains, while PcGC-M2 also contains a kinase homology domain and a cyclase catalytic domain and PcGC-M2B only a truncated kinase homology domain. Immunohistochemical localization of PcGC-M2 in membrane of abdominal muscle in Procambarus clarkii. The results of semi-quantitative PCR showed the transcript levels that expressed as a ratio of PcGC-M2 to PcGC-M2B, significantly increased after eyestalk ablation in CHH target tissues, muscle and hepatopancreas. When expressed in human embryonic kidney-293T cells, PcGC-M2 and PcGC-M2B are cell-surface glycoproteins and capable of forming a PcGC-M2/PcGC-M2B heterodimer. In the GC activity, CHH is able to stimulate the GC activity of PcGC-M2 in a dose-dependent manner. The activity of PcGC-M2 was decreased when co-expressed with PcGC-M2B, which by itself lacks GC activity. In CHH binding assay, CHH is able to bind to PcGC-M2, PcGC-M2B, or PcGC-M2/PcGC-M2B. Through the results of the displacement assay indicated D-Phe3 CHH has higher affinity than L-Phe3 CHH or CHH-L to PcGC-M2. According to the results, the PcGC-M2 and PcGC-M2B, in either homodimeric or heterodimeric form, are CHH receptors. The effects of regulating mechanism of CHH on physiological events, that depended on different affinities of CHH family to receptor and that sensitivity of CHH target tissues to CHH regulation varies as a function of the ratio of levels of PcGC-M2 to PcGC-M2B.