Alanine exchanges of polar amino acids in the transmembrane domains of a platelet-activating factor receptor generate both constitutively active and inactive mutants

Isao Ishii, Takashi Izumi, Hiroaki Tsukamoto, Hideaki Umeyama, Michio Ui, Takao Shimizuf

Research output: Contribution to journalArticle

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Abstract

To determine ligand-binding sites of a platelet-activating factor (PAF) receptor, alanine-scanning mutagenesis was carried out. All 23 polar amino acids in the putative 7-transmembrane (TM) domains of a guinea pig PAF receptor were individually replaced with alanine. The ligand-binding properties of mutant receptors were determined after transient expression in COS-7 cells. Mutants in TM II (N58A, D63A), TM III (N100A, T101A, S104A) and TM VII (D289A) displayed higher PAF-binding affinities than seen with the wild-type receptor. In contrast, mutants in TM V (H188A), TM VI (H248A, H249A, Q252A), and TM VII (Q276A, T278A) showed lower affinities. Representative mutants were then stably expressed in Chinese hamster ovary cells to observe PAF-induced cellular signals (arachidonate release, phosphatidylinositol hydrolysis, adenylyl cyclase inhibition). An N100A mutant with the highest affinity was constitutively active and was responsive to lyso-PAF, an inactive derivative of PAF. One nanomolar PAF induced no signals in low affinity mutants, an EC50 value for the wild-type receptor. Three histidines (His-188, His-248, His-249) might form a binding pocket for the phosphate group of PAF, since zinc effectively inhibited ligand binding. Based on these results, a three-dimensional molecular model of PAF and its receptor was generated using bacteriorhodopsin as a reference protein.

Original languageEnglish
Pages (from-to)7846-7854
Number of pages9
JournalJournal of Biological Chemistry
Volume272
Issue number12
DOIs
Publication statusPublished - 1997
Externally publishedYes

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Platelet Activating Factor
Alanine
Amino Acids
Ligands
Bacteriorhodopsins
Mutagenesis
Molecular Models
COS Cells
Phosphatidylinositols
Cricetulus
Histidine
Adenylyl Cyclases
Zinc
Ovary
Hydrolysis
Guinea Pigs
Phosphates
Binding Sites
Cells
platelet activating factor receptor

ASJC Scopus subject areas

  • Biochemistry

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Alanine exchanges of polar amino acids in the transmembrane domains of a platelet-activating factor receptor generate both constitutively active and inactive mutants. / Ishii, Isao; Izumi, Takashi; Tsukamoto, Hiroaki; Umeyama, Hideaki; Ui, Michio; Shimizuf, Takao.

In: Journal of Biological Chemistry, Vol. 272, No. 12, 1997, p. 7846-7854.

Research output: Contribution to journalArticle

Ishii, Isao ; Izumi, Takashi ; Tsukamoto, Hiroaki ; Umeyama, Hideaki ; Ui, Michio ; Shimizuf, Takao. / Alanine exchanges of polar amino acids in the transmembrane domains of a platelet-activating factor receptor generate both constitutively active and inactive mutants. In: Journal of Biological Chemistry. 1997 ; Vol. 272, No. 12. pp. 7846-7854.
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