|Budget Amount *help
¥3,200,000 (Direct Cost : ¥3,200,000)
Fiscal Year 1999 : ¥1,200,000 (Direct Cost : ¥1,200,000)
Fiscal Year 1998 : ¥2,000,000 (Direct Cost : ¥2,000,000)
1. Screening of a constitutively active angiotensin II type 1 mutant receptor
We have found the first constitutively active mutant receptor of Angiotensin II type 1 (AT1) receptor in vitro, in which Asn111 was mutated to Gly (Asn111Gly). Balmforth AJ et a1. also reported another constitutively active mutant, Asn295Ser (J. Biol. Chem. 272 : 4245-4251, 1997), and they proposed the side-chain interaction between Asn111 and Asn295. However, recent reports (Biochemistry 37 : 15791-15798, 1998, Mol Pharmacol 54 : 427-34, 1998) showed that Asn295Ser was not constitutively active mutant. The aim of this study was to find the new amino acid residue interacting to Asn111 by mutation resulting in the new constitutively active mutant. The ten amino acid residues, Phe204, Phe205, Phe206 in the fifth transmembrane domain and Phe249, Phe251, Ser252, Trp253, Gln257, Phe259, Phe261 in the sixth transmembrane domain were mutated to Ala by site-directed mutagenesis and these mutants were analyzed by binding property and inositol phosphate metabolism. Phe204Ah Phe249Ala. Trp253Ala' and Gln257Ala showed low affinity for peptide ligands. Non of ten mutants showed high basal activity in the absence of angiotensin II analogs. At least, these ten mutants were not constitutively active.
2. Screening of mutation at Asn111 in patients with cardiovascular diseases
The sequence ofAsn111 codon (AAC) was analyzed in 104 patients in our hospital with essential hypertension, myocardial infarction, angina pectoris, arterial disease, congestive heart failure. No mutation was found among all patients.