NATRIURETIC PEPTIDE RECEPTOR--STRUCTURE AND SIGNALING

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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.