Structure of the atrial natriuretic peptide receptor extracellular domain in the unbound and hormone-bound states by single-particle electron microscopy.
Atrial natriuretic peptide (ANP) plays a major role in blood pressure and volume regulation. ANP activities are mediated by a cell surface, single-span transmembrane receptor linked to its intrinsic guanylate cyclase activity. The crystal structures of the dimerized ANP receptor extracellular domain (ECD) with and without ANP have revealed a novel hormone-induced rotation mechanism occurring in the juxtamembrane region that appears to mediate signal transduction [Ogawa H, Qiu Y, Ogata CM & Misono KS (2004) J Biol Chem 279, 28625-28631]. However, the ECD crystal packing contains two major intermolecular contacts that suggest two possible dimer pairs: 'head-to-head' (hh) and 'tail-to-tail' (tt) dimers associated via the membrane-distal and membrane-proximal subdomains, respectively. The existence of these two potential dimer forms challenges the proposed signaling mechanism. In this study, we performed single-particle electron microscopy (EM) to determine the ECD dimer structures occurring in the absence of crystal contacts. EM reconstruction yielded the dimer structures with and without ANP in only the hh dimer forms. We further performed steady-state fluorescence spectroscopy of Trp residues, one of which (Trp74) occurs in the hh dimer interface and none of which occurs in the tt dimer interface. ANP binding caused a time-dependent decrease in Trp emission at 350 nm that was attributable to partially buried Trp74 in the unbound hh dimer interface becoming exposed to solvent water upon ANP binding. Thus, the results of single-particle EM and Trp fluorescence studies have provided direct evidence for hh dimer structures for unbound and ANP-bound receptor. The results also support the proposed rotation mechanism for transmembrane signaling by the ANP receptor.
Ogawa
Haruo
H
Department of Biochemistry, University of Nevada, Reno, NV 89557, USA.
Qiu
Yue
Y
Huang
Liming
L
Tam-Chang
Suk-Wah
SW
Young
Howard S
HS
Misono
Kunio S
KS
D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D019281:Dimerization; D008854:Microscopy, Electron / Q000379:methods; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D051381:Rats; D017461:Receptors, Atrial Natriuretic Factor / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D014364:Tryptophan; D017461:Receptors, Atrial Natriuretic Factor