Site-directed mutagenesis of cysteinyl and serine residues of human thromboxane A2 receptor in insect cells

Nan Chiang, Wai Ming Kan, Hsin Hsiung Tai

研究成果: Article同行評審

56 引文 斯高帕斯(Scopus)

摘要

A thromboxane A2 receptor cDNA was isolated from a human placenta library by polymerase chain reaction (PCR) and was expressed in insect (Sf21) cells using baculovirus system. The recombinant receptor exhibited [3H]- SQ29548 and [125I]-BOP binding activities with K(d) values of 1.01 ± 0.09 nM and 1.63 ± 0.23 nM, respectively. The receptor binding activity was inhibited by dithiothreitol in a time- and concentration-dependent manner, indicating the involvement of disulfide linkage in ligand binding. The role of the four conserved cysteinyl residues in ligand binding was further examined by site-directed mutagenesis. Each of the four cysteinyl residues was respectively mutated to a serine residue. C102S, C105S, and C183S mutants exhibited no ligand binding activity although successful expression was achieved as revealed by immunoblot analysis, whereas C257S mutant retained most of the binding activity. Homology analysis of all prostanoid receptors indicates that Cys-105 (first extracellular loop) and Cys-183 (second extracellular loop) are conserved and are presumed to form a disulfide bond for receptor stability as suggested by the inhibition of ligand binding by dithiothreitol reduction. Loss of binding activity by C102S mutant revealed that the sulfhydryl group of Cys-102 must play an essential role in ligand binding. Molecular modeling proposed that the Ser-201 is involved in interacting with TXA2 by forming hydrogen bonding. Point mutations of both Ser-201 and a conserved Ser-255 did not affect the ligand binding specificity and affinity for [3H]-SQ29548, but have significantly altered K(d) values for [125I]-BOP. These results indicate that various cysteinyl and serine residues of thromboxane A2 receptor may play different roles in ligand binding.

原文English
頁(從 - 到)9-17
頁數9
期刊Archives of Biochemistry and Biophysics
334
發行號1
DOIs
出版狀態Published - 1996 十月 1

All Science Journal Classification (ASJC) codes

  • 生物物理學
  • 生物化學
  • 分子生物學

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