Constitutive activation and uncoupling of the atrial natriuretic peptide receptor by mutations at the dimer interface. Role of the dimer structure in signalling

• Published in: The Journal of biological chemistry Vol. 279; no. 7; pp. 6115 - 6123
• Main Authors: Qiu, Yue, Ogawa, Haruo, Miyagi, Masaru, Misono, Kunio S
• Format: Journal Article
• Language: English
• Published: United States 13.02.2004
• Subjects: Animals; Arginine - chemistry; atrial natriuretic peptide receptors; COS Cells; Crystallography, X-Ray; Cyclic GMP - metabolism; Diethyl Pyrocarbonate - chemistry; Dimerization; DNA, Complementary - metabolism; Histidine - chemistry; Hydrogen Bonding; Kinetics; Mass Spectrometry; Models, Molecular; Mutagenesis, Site-Directed; Mutation; Peptides - chemistry; Protein Conformation; Receptors, Atrial Natriuretic Factor - chemistry; Receptors, Atrial Natriuretic Factor - genetics; Signal Transduction; Time Factors; Transfection; Trypsin - pharmacology; Tryptophan - chemistry
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M310225200

The crystal packing of the extracellular hormone binding domain of the atrial natriuretic peptide (ANP) receptor contains two possible dimer pairs, the head-to-head (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 Trp74 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 tt-interface had no such effect. His99 modification at the hh-dimer interface by ethoxyformic anhydride abolished ANP binding. These results suggest that the hh-dimer 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.