Atrial natriuretic peptide (ANP) receptor consists of a single polypeptide containing an extracellular ANP-binding domain, a single transmembrane sequence, and an intracellular region containing a kinase-homologous domain and a guanylate cyclase (GCase) domain. Studies of the ANP receptor reported to date have been performed mostly by deletion mutagenesis, focusing mainly on the function of the kinase- homologous domain that modulates receptor activity. consequently, our knowledge of the structure and function of the extracellular ANP- binding domain is essentially nil. The overall goal of this proposal is to understand how ANP interacts with the binding domain, what the nature of the activation signal generated by such interaction is, and how this activation signal is transmitted into the intracellular domain. In our studies of the ANP receptor in bovine adrenal membranes, we observed proteolytic processing of the receptor by endogenous membrane-bound proteases. Further studies of this phenomenon led us to identify a unique """"""""hinge""""""""-like structure in the receptor molecule that undergoes a distinct conformational change upon ANP binding and apparently plays a critical role in receptor signaling. We propose to examine the roles of individual structures in the hinge- region in signaling by producing site-directed mutations and evaluating their effects on ANP-binding, GCase-activation, and the ANP-induced conformational change in the hinge-region. To facilitate structural characterization, we have produced the extracellular ANP-binding domain of the receptor in a soluble form by deleting the transmembrane sequence and the intracellular domain. We have also developed a new affinity-labeling procedure, termed stepwise affinity-labeling, that allows highly specific and nearly quantitative chemical labeling of the ANP-binding site. By this method and by the technique of differential chemical modification, we propose to identify and determine several segments of the receptor sequence that constitute the binding-site structure. Utilizing the structural information, we further propose to create site-directed mutations of binding-site residues to evaluate their roles in ANP-binding and GCase-activation. These studies will generate information necessary for ultimate understanding of the mechanisms of the receptor-ligand interaction and receptor activation.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL054329-03
Application #
2735259
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1996-07-01
Project End
2000-06-30
Budget Start
1998-07-01
Budget End
1999-06-30
Support Year
3
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Cleveland Clinic Lerner
Department
Type
DUNS #
017730458
City
Cleveland
State
OH
Country
United States
Zip Code
44195
Misono, Kunio S; Philo, John S; Arakawa, Tsutomu et al. (2011) Structure, signaling mechanism and regulation of the natriuretic peptide receptor guanylate cyclase. FEBS J 278:1818-29
Ogawa, Haruo; Qiu, Yue; Philo, John S et al. (2010) Reversibly bound chloride in the atrial natriuretic peptide receptor hormone-binding domain: possible allosteric regulation and a conserved structural motif for the chloride-binding site. Protein Sci 19:544-57
Ogawa, Haruo; Qiu, Yue; Huang, Liming et al. (2009) Structure of the atrial natriuretic peptide receptor extracellular domain in the unbound and hormone-bound states by single-particle electron microscopy. FEBS J 276:1347-55
Misono, Kunio S; Ogawa, Haruo; Qiu, Yue et al. (2005) Structural studies of the natriuretic peptide receptor: a novel hormone-induced rotation mechanism for transmembrane signal transduction. Peptides 26:957-68
Qiu, Yue; Ogawa, Haruo; Miyagi, Masaru et al. (2004) Constitutive activation and uncoupling of the atrial natriuretic peptide receptor by mutations at the dimer interface. Role of the dimer structure in signalling. J Biol Chem 279:6115-23
Ogawa, Haruo; Qiu, Yue; Ogata, Craig M et al. (2004) Crystal structure of hormone-bound atrial natriuretic peptide receptor extracellular domain: rotation mechanism for transmembrane signal transduction. J Biol Chem 279:28625-31
Ogawa, Haruo; Zhang, Xiaolun; Qiu, Yue et al. (2003) Crystallization and preliminary X-ray analysis of the atrial natriuretic peptide (ANP) receptor extracellular domain complex with ANP: use of ammonium sulfate as the cryosalt. Acta Crystallogr D Biol Crystallogr 59:1831-3
Miyagi, M; Zhang, X; Misono, K S (2000) Glycosylation sites in the atrial natriuretic peptide receptor: oligosaccharide structures are not required for hormone binding. Eur J Biochem 267:5758-68
Miyagi, M; Misono, K S (2000) Disulfide bond structure of the atrial natriuretic peptide receptor extracellular domain: conserved disulfide bonds among guanylate cyclase-coupled receptors. Biochim Biophys Acta 1478:30-8
Misono, K S (2000) Atrial natriuretic factor binding to its receptor is dependent on chloride concentration: A possible feedback-control mechanism in renal salt regulation. Circ Res 86:1135-9

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