The atrial natriuretic peptides are critically important for endocrine and cardiovascular homeostasis. The broad, long-term objectives of this proposal are to understand the mechanisms of signal transduction and the normal physiological roles of atrial natriuretic peptide receptors, and of related receptors having intrinsic guanylyl cyclase activity.
The specific aims are 1. To test whether ANP stimulates the guanylyl cyclase activity of its receptor by inducing its dimerization, and whether the protein tyrosine kinase-like domain of the receptor has protein kinase activity; 2. To test whether the regulation of receptor guanylyl cyclase activity by ATP involves direct binding of the nucleotide to the receptor; 3. To identify related receptor/guanylyl cyclases in the rat and determine their tissue localization and developmental expression; 4. To sequence cDNA clones for putative receptor/guanylyl cyclases from Drosophila and determine their chromosomal location and the developmental expression of the corresponding genes, and to determine whether the gene products have guanylyl cyclase activity. The health-relatedness of the project is in leading to a better understanding of the mechanisms of action of peptides that are important in regulating blood pressure, and that are potential therapeutic agents. The experimental plan is 1. to test whether treatment with ANP increases the apparent size of the receptor as measured by sedimentation analysis of chemical crosslinking; to perform protein kinase assays on receptor in immune complexes; 2. To affinity-label and sequence the site responsible for ATP activation of receptor guanylyl cyclase activity; 3. To identify novel receptor/guanylyl cyclases using the polymerase chain reaction with degenerate oligonucleotides, based on conserved sequences in this receptor family, and 4. To sequence full-length cDNA clones for Drosophila guanylyl cyclases, determine the chromosomal localization and developmental expression of the corresponding genes by hybridization to chromosome squashes and by in situ hybridization and to test whether the clones have enzyme activity when transiently expressed in cultured cells.
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