The crystal packing of the extracellular hormone binding domain of the atrial natriuretic peptide (ANP) receptor contains two possible dimer pairs, the head-tohead (hh) and tail-to-tail (tt) dimer pairs associated through the membrane-distal and membrane-proximal subdomains, respectively. The tt-dimer structure has been proposed previously (van den Akker, F., Zhang, X., Miyagi, M., Huo, X., Misono, K. S., and Yee, V. C. (2000) Nature 406, 101-104). However, no direct evidence is available to identify the physiological dimer form. Here we report site-directed mutagenesis studies of residues at the two alternative dimer interfaces in the full-length receptor expressed on COS cells. The Trp 74 to Arg mutation (W74R) or D71R at the hh-dimer interface caused partial constitutive guanylate cyclase activation, whereas mutation F96D or H99D caused receptor uncoupling. In contrast, mutation Y196D or L225D at the ttinterface had no such effect. His 99 modification at the hh-dimer interface by ethoxyformic anhydride abolished ANP binding. These results suggest that the hhdimer represents the physiological structure. Recently, we determined the crystal structure of ANPR complexed with ANP and proposed a hormone-induced rotation mechanism mediating transmembrane signaling (H. Ogawa, Y. Qiu, C. M. Ogata, and K. S. Misono, submitted for publication). The observed effects of mutations are consistent with the ANP-induced structural change identified from the crystal structures with and without ANP and support the proposed rotation mechanism for ANP receptor signaling.
Atrial natriuretic peptide (ANP)1 is a hormone secreted from the heart in response to atrial distension. ANP plays a major role in the regulation of blood pressure and electrolytes-fluid volume homeostasis through its natriuretic (1) and vasorelaxant activities (2, 3). ANP also has an antitrophic activity, through which it regulates maintenance and remodeling of the cardiovascular system (4 -7). Transgenic animals lacking the ANP gene develop salt-sensitive hypertension (8), and those lacking the ANP receptor gene develop salt-insensitive essential hypertension accompanied by severe cardiac hypertrophy (9), implicating the ANP and ANP receptor system in cardiovascular pathophysiology. The hormonal actions of ANP are mediated by a cell membrane receptor carrying intrinsic guanylate cyclase (GCase) activity. The receptor occurs as a dimer of a single span transmembrane polypeptide containing an extracellular hormone binding domain and an intracellular domain consisting of an ATP-dependent regulatory domain and a GCase catalytic domain (10). ANP binding stimulates GCase activity and elevates intracellular cGMP levels. cGMP, in turn, mediates hormonal actions via cGMP-regulated ion channels, protein kinases, and phosphodiesterases (11). The ANP receptor belongs to the family of GCase-coupled receptors which includes retinal GCases (12), olfactory GCases (13), and guanylin and enterotoxin receptors (14). These receptors have a similar overall molecular topology and may ...