Constitutive activation and uncoupling of the atrial natriuretic peptide receptor by mutations at the dimer interface. Role of the dimer structure in signalling.
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.
Qiu
Yue
Y
Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
Ogawa
Haruo
H
Miyagi
Masaru
M
Misono
Kunio S
KS
D000818:Animals; D001120:Arginine / Q000737:chemistry; D019556:COS Cells; D018360:Crystallography, X-Ray; D006152:Cyclic GMP / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004047:Diethyl Pyrocarbonate / Q000737:chemistry; D019281:Dimerization; D006639:Histidine / Q000737:chemistry; D006860:Hydrogen Bonding; D007700:Kinetics; D013058:Mass Spectrometry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D017461:Receptors, Atrial Natriuretic Factor / Q000737:chemistry / Q000235:genetics*; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D014357:Trypsin / Q000494:pharmacology; D014364:Tryptophan / Q000737:chemistry
D018076:DNA, Complementary; D010455:Peptides; D006639:Histidine; D014364:Tryptophan; D001120:Arginine; D014357:Trypsin; D017461:Receptors, Atrial Natriuretic Factor; D006152:Cyclic GMP; D004047:Diethyl Pyrocarbonate