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Biological Effects of Recombinant PAC1-EC1(N) on the Viability of Cell Lines with Different PAC1 Isoforms |
LI Juan, YU Rong-jie, WANG Jing-jing, HUANG Lin, LIU Xiao-fei |
Bioengineering Institute of Jinan University, Guangzhou 510632, China |
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Abstract PAC1 is a specific receptor for pituitary adenylate cyclase-activating polypeptide (PACAP).The N terminal first extracellular region of the PAC1 (PAC1-EC1) is involved in the regulation for the activation of PAC1. To study the effects of the EC1 domain of the PAC1 normal (N) isoform on the cell lines expressing different PAC1 isoforms, the recombinant protein PAC1-EC1 (N) with 6-His-tag at the C-terminus was expressed in an engineered Escherichia coli strain and purified by Ni-NTA affinity chromatography. Mass spectrum, SDS-PAGE and Western blot were used to characterize the recombinant PAC1-EC1(N). Western blot were used to identify the expression pattern of PAC1 isoforms in three cell lines including rabbit corneal stromal cells, human neuroblastoma cells SH-SY5Y and human prostate cancer cells 22RV1. MTT assays were used to detect different biological effects of the recombinant PAC1-EC1 (N) on the cell lines with different PAC1 isoforms. The results showed that PAC1-EC1(N) had significantly different biological effects on the cell lines with different PAC1 isoforms. PAC1-EC1(N) stimulated the viability of rabbit corneal stromal cells expressing only one PAC1 isoform with a smaller molecular weight, but inhibited the viability of human neuroblastoma cells SH-SY5Y expressing one type of PAC1 isoform with a larger molecular weight. Meanwhile, PAC1-EC1(N) caused complex biological effects on human prostate cancer 22RV1 cells with three types of PAC1 isoforms. These results showed that PAC1-EC1(N) exerts different effects on the viabilities of the cells with different expression patterns of PAC1 isoforms, indicating that the PAC1 owns a complex self-regulation mechanism of activation.
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Received: 03 March 2011
Published: 28 June 2011
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