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 language | English |
|---|---|
| Pages (from-to) | 7846-7854 |
| Number of pages | 9 |
| Journal | Journal of Biological Chemistry |
| Volume | 272 |
| Issue number | 12 |
| DOIs | |
| Publication status | Published - 1997 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Biochemistry
Cite this
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 journal › Article
}
TY - JOUR
T1 - Alanine exchanges of polar amino acids in the transmembrane domains of a platelet-activating factor receptor generate both constitutively active and inactive mutants
AU - Ishii, Isao
AU - Izumi, Takashi
AU - Tsukamoto, Hiroaki
AU - Umeyama, Hideaki
AU - Ui, Michio
AU - Shimizuf, Takao
PY - 1997
Y1 - 1997
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0030956418&partnerID=8YFLogxK
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U2 - 10.1074/jbc.272.12.7846
DO - 10.1074/jbc.272.12.7846
M3 - Article
VL - 272
SP - 7846
EP - 7854
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 12
ER -