The overall objective of this research proposal is to decode the basic biochemical principles behind manifestation of salt sensitive and salt insensitive hypertension. Atrial natriuretic factor (ANF) and type B natriuretic peptide (BMP) are the key agents that control hypertension. ANF and BMP exhibit their physiological activity through ANF-receptor guanylate cyclase (ANF-RGC). ANF-RGC is a single transmembrane protein composed of modular blocks. The transmembrane module separates the protein into two regions, extracellular and intracellular. The extracellular region contains the ANF-binding domain. The intracellular region is composed of the following sequential modular blocks: the ATP-regulated module (ARM), the kinase homology domain (KHD), the dimerization and the catalytic domains. The ANF-RGC signal transduction mechanism is initiated by binding of ANF to the extracellular domain and culminates in the production of a second messenger cyclic GMP by the intracellular catalytic domain. The intermediate steps are unknown, however. The ARM is a critical transduction module, which stringently controls the ANF-dependent activity of the catalytic module. Its three-dimensional structure has been simulated. This structural model forms the working template for the proposed Specific Aims. The first three proposed aims are designed to elucidate the structure-based events by which ATP transduces the ANF signal into the production of cyclic GMP: 1) """"""""To elucidate the structural details of the ATP binding pocket of the ANF- RGC ARM domain"""""""";2) """"""""To determine the role of serine and threonine residues of the ARM domain in the process of ANF/ATP-dependent activation of ANF-RGC"""""""";3) """"""""To determine the amino acid residues of the ARM domain critical for the ANF/ATP-dependent activation of the catalytic domain"""""""". The fourth specific aim will provide an animal model for studying the basic mechanisms of hypertension and for ATP-related therapeutic treatments of hypertension: """"""""To evaluate the physiological contribution of the ATP signaling event through the creation and analysis of a transgenic mouse with the """"""""knock-in"""""""" mutation D646A"""""""".
These aims will be achieved through the use of a combination of molecular, biochemical, biophysical and immunologica! techniques together with computational modeling. The proposed studies are most fundamental in nature, yet they are directly applicable to the molecular understanding the disease processes resulting in hypertension, heart dystrophy, water balance and fluid secretion.
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