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

Nan Chiang, Wai-Ming Kan, Hsin Hsiung Tai

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Abstract

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.

Original languageEnglish
Pages (from-to)9-17
Number of pages9
JournalArchives of Biochemistry and Biophysics
Volume334
Issue number1
DOIs
Publication statusPublished - 1996 Oct 1

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Prostaglandin H2 Receptors Thromboxane A2
Mutagenesis
Site-Directed Mutagenesis
Serine
Insects
Ligands
Dithiothreitol
Disulfides
Sf9 Cells
Molecular modeling
Baculoviridae
Polymerase chain reaction
Hydrogen Bonding
Point Mutation
Placenta
Prostaglandins
Hydrogen bonds
Complementary DNA
Polymerase Chain Reaction

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology

Cite this

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title = "Site-directed mutagenesis of cysteinyl and serine residues of human thromboxane A2 receptor in insect cells",
abstract = "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.",
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Site-directed mutagenesis of cysteinyl and serine residues of human thromboxane A2 receptor in insect cells. / Chiang, Nan; Kan, Wai-Ming; Tai, Hsin Hsiung.

In: Archives of Biochemistry and Biophysics, Vol. 334, No. 1, 01.10.1996, p. 9-17.

Research output: Contribution to journalArticle

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