Recently, GATA4 and NKX2.5 were reported as the disease genes of atrial septal defect (ASD) but the relationship between the locations of their mutations and phenotypes is not clear. We analyzed GATA4 and NKX2.5 mutations in 16 familial ASD cases, including four probands with atrioventricular conduction disturbance (AV block) and two with pulmonary stenosis (PS), by PCR and direct sequencing, and examined their phenotypes clinically. Five mutations, including two GATA4 and three NKX2.5 mutations, were identified in 31.3% of the probands with ASD, and three of them were novel. The two GATA4 mutations in the probands without AV block were S52F and E359Xfs (c.1075delG) that was reported previously, and three NKX2.5 mutations in the probands with AV block were A88Xfs (c.262delG), R190C, and T178M. Additionally, we observed some remarkable phenotypes, i.e., dextrocardia with E359Xfs (c.1075delG) and cribriform type ASD with R190C, both of which are expected to be clues for further investigations. Furthermore, progressive, most severe AV block was closely related with a missense mutation in a homeodomain or with a nonsense/frame-shift mutation of NKX2.5 for which classification has not been clearly proposed. This pinpoints essential sites of NKX2.5 in the development of the conduction system.

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The molecular recognition theory suggests that binding sites of interacting proteins, for example, peptide hormone and its receptor binding site, were originally encoded by and evolved from complementary strands of genomic DNA. To test this theory, we screened a rat kidney complementary DNA library twice: first with the angiotensin II (AII) followed by the vasopressin (AVP) antisense oligonucleotide probe, expecting to isolate cDNA clones of the respective receptors. Surprisingly, the identical cDNA clone was isolated twice independently. Structural analysis revealed a single receptor polypeptide with seven predicted transmembrane regions, distinct AII and AVP putative binding domains, a Gs protein-activation motif, and an internalization recognition sequence. Functional analysis revealed specific binding to both AII and AVP as well as AII- and AVP-induced coupling to the adenylate cyclase second messenger system. Site-directed mutagenesis of the predicted AII binding domain obliterates AII binding but preserves AVP binding. This corroborates the dual nature of the receptor and provides direct molecular genetic evidence for the molecular recognition theory.

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Elucidation of the dual ET-1/AngII receptor provides further molecular genetic evidence in support of the molecular recognition theory and identifies for the first time a molecular link between the ET-1 and AngII hormonal systems that could underlie observed similar physiological responses elicited by ET-1 and AngII in different organ systems. The prominent expression of the ET-1/AngII receptor mRNA in brain and heart tissues suggests an important role in cardiovascular function in normal and pathophysiological states.