Hypertension is a global disease that is one of the major causes of death in the United States and the rest of the industrial world. Understanding the mechanisms of regulation of blood pressure is therefore one of the most important challenges in health science research. Atrial natriuretic peptide (ANP) is an endogenous and potent hypotensive factor that elicits its physiological effects by binding to a guanylyl cyclase-linked atrial natriuretic peptide receptor (GC-A) that synthesizes the intracellular messenger cyclic GMP in response to ANP binding. GC-A is a member of a unique class of cell surface receptors that contain an extracellular ligand binding domain, a single membrane spanning region, and a guanylyl cyclase catalytic domain. It also contains a protein kinase-like domain whose function is not yet established. The ligand-induced activation of GC-A is regulated by both ATP and the phosphorylation state of the enzyme. My recent work on highly purified recombinant GC-A expressed in Sf9 cells, suggests that the receptor contains an ATP-binding domain that is required for transduction of the ANP binding signal to the guanylyl cyclase catalytic domain. These results, which have never been previously reported for a purified guanylyl cyclase receptor, suggest that GC-A contains all of the necessary components for signal transduction.
The specific aims of the proposal are as follows: (1) To define the effects of the GC-A phosphorylation state on ATP/ANP regulation. The mol phosphate/mol of enzyme and the site(s) of ANP-induced dephosphorylation will be determined. Mutations of the identified phosphorylation site(s) will provide evidence of the importance of each site in signaling by GC-A. It also will be determined whether or not dephosphorylation or mutation at a specific phosphorylation-site alters the potency of ANP/ATP to activate GC-A. (2) To define the molecular basis of ATP-dependent signal transduction. Purified GCA (the phosphorylation state will be altered dependent on the results from specific aim (1)) will be used to study ATP binding and nucleotide specificity. It also will be determined whether or not GC-A contains intrinsic ATPase or protein kinase activities. Various GC-A mutants will be used to confirm whether any such activity originates from GC-A. We have been able to inactivate and covalently radiolabel GC-A with an analogue of ATP and will determine the site(s) of binding. These studies will provide information on the molecular mechanisms of regulation of GC-A and therefore will help elucidate the mechanisms by which ANP modulate hypotension and other activities in the body.
| Wong, S K; Ma, C P; Foster, D C et al. (1995) The guanylyl cyclase-A receptor transduces an atrial natriuretic peptide/ATP activation signal in the absence of other proteins. J Biol Chem 270:30818-22 |
