The molecular interaction involved in the ligand binding of the rat angiotensin II receptor (AT1A) was studied by site-directed mutagenesis and receptor model building. The three-dimensional structure of AT1A was constructed on the basis of a multiple amino acid sequence alignment of seven transmembrane domain receptors and angiotensin II receptors and after the beta 2 adrenergic receptor model built on the template of the bacteriorhodopsin structure. These data indicated that there are conserved residues that are actively involved in the receptor-ligand interaction. Eleven conserved residues in AT1, His166, Arg167, Glu173, His183, Glu185, Lys199, Trp253, His256, Phe259, Thr260, and Asp263, were targeted individually for site-directed mutation to Ala. Using COS-7 cells transiently expressing these mutated receptors, we found that the binding of angiotensin II was not affected in three of the mutations in the second extracellular loop, whereas the ligand binding affinity was greatly reduced in mutants Lys199-->Ala, Trp253-->Ala, Phe259-->Ala, Asp263-->Ala, and Arg167-->Ala. These amino acid residues appeared to provide binding sites for Ang II. The molecular modeling provided useful structural information for the peptide hormone receptor AT1A. Binding of EXP985, a nonpeptide angiotensin II antagonist, was found to be involved with Arg167 but not Lys199.

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OBJECTIVE -To clarify the role of the T-lymphocyte-associated-4 (CTLA-4) polymorphism in the susceptibility to child-onset type 1 diabetes with regard to its clinical characteristics and complications with autoimmune thyroid disease (AITD) in the Japanese population.RESEARCH DESIGN AND METHODS -The CTLA-4 49 A/G polymorphism was detected by the PCR-restriction fragment-length polymorphism (RFLP) method in 97 type 1 diabetic subjects and 20 patients with Graves' disease, a cohort which included 4 patients who also had type 1 diabetes.RESULTS -The genotypes and allele frequencies of this polymorphism did not differ between the type 1 diabetic subjects and the control subjects. The G allele frequency was 63.9% in the type 1 diabetic subjects. The G allele frequency in the subgroup of patients with a high titer of autoantibodies to the GAD antibody (Ab) was 72.9% (P ϭ 0.0499 vs. control subjects); in the subgroup of patients without HLA DRB1*0405, it was 72.6% (P ϭ 0.0271 vs. control subjects); and in the subgroup of patients with a residual ␤-cell function, it was 78.6% (P ϭ 0.0391 vs. control subjects). The G allele frequency in the patients with Graves' disease was also significantly higher at 78.1% (P ϭ 0.0405 vs. control subjects). Furthermore, the frequency in our diabetic subjects complicated with Graves' disease was even higher (87.5%).CONCLUSIONS -We have demonstrated that a distinct association exists between the G allele of CTLA-4 and high values of GAD Ab, residual ␤-cell function, and the absence of HLA-DRB1*0405.

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Pseudohypoaldosteronism (PHA) is characterized by urinary salt-wasting in infancy resulting from a congenital resistance to aldosterone involving the genes for the mineralocorticoid receptor (MR) and the amiloride-sensitive sodium channel (ENaC). We identified, in a Japanese patient with sporadic PHA, three homozygous substitutions in the MR gene: G215-->C215, A754-->G754 (Ile180-->Val180), C938-->T938 (Ala241-->Val241), which had previously been reported to occur in healthy populations. Luciferase activities induced by MR with either G215-->C215, Ile180-->Val180, or Ala241-->Val241 substitution were significantly lower than those for wild-type MR with aldosterone at concentrations ranging from 10(-11) to 10(-9) M, 10(-8) M, or 10(-11) to 10(-6) M, respectively. A homozygous A-->G substitution of the donor splice site of alphaENaC intron 4 was found in the patient. The corresponding cDNA exhibited a normal structure, suggesting that this substitution does not alter the splice. The results suggest that each of three MR polymorphisms identified in our patient is functionally and structurally heterogeneous. We hypothesize that two or more "functional" polymorphisms, any of which exhibits only slight effects on MR or ENaC function and is alone incapable causing PHA, may in the right allelic combination induce the negative salt-conservation characteristic of PHA.