Crystal Structure of Hormone-bound Atrial Natriuretic Peptide Receptor Extracellular Domain

Abstract

A cardiac hormone, atrial natriuretic peptide (ANP), plays a major role in blood pressure and volume regulation. ANP activities are mediated by a single span transmembrane receptor carrying intrinsic guanylate cyclase activity. ANP binding to its extracellular domain stimulates guanylate cyclase activity by an as yet unknown mechanism. Here we report the crystal structure of dimerized extracellular hormone-binding domain in complex with ANP. The structural comparison with the unliganded receptor reveals that hormone binding causes the two receptor monomers to undergo an intermolecular twist with little intramolecular conformational change. This motion produces a Ferris wheel-like translocation of two juxtamembrane domains in the dimer with essentially no change in the interdomain distance. This movement alters the relative orientation of the two domains by a shift equivalent to counterclockwise rotation of each by 24°. These results suggest that transmembrane signaling by the ANP receptor is initiated via a hormone-induced rotation mechanism. A cardiac hormone, atrial natriuretic peptide (ANP), plays a major role in blood pressure and volume regulation. ANP activities are mediated by a single span transmembrane receptor carrying intrinsic guanylate cyclase activity. ANP binding to its extracellular domain stimulates guanylate cyclase activity by an as yet unknown mechanism. Here we report the crystal structure of dimerized extracellular hormone-binding domain in complex with ANP. The structural comparison with the unliganded receptor reveals that hormone binding causes the two receptor monomers to undergo an intermolecular twist with little intramolecular conformational change. This motion produces a Ferris wheel-like translocation of two juxtamembrane domains in the dimer with essentially no change in the interdomain distance. This movement alters the relative orientation of the two domains by a shift equivalent to counterclockwise rotation of each by 24°. These results suggest that transmembrane signaling by the ANP receptor is initiated via a hormone-induced rotation mechanism. Atrial natriuretic peptide (ANP) 1The abbreviations used are: ANP, atrial natriuretic peptide; ANPR, the extracellular hormone-binding domain of the ANP receptor; GCase, guanylate cyclase; BNP, B-type natriuretic peptide; CNP, C-type natriuretic peptide; EPO, erythropoietin; MES, 4-morpholineethanesulfonic acid. is a hormone produced in the cardiac atrium and secreted into the circulation in response to atrial distension. ANP stimulates salt excretion (1de Bold A.J. Borenstein H.B. Veress A.T. Sonnenberg H. Life Sci. 1981; 28: 89-94Crossref PubMed Scopus (2650) Google Scholar) and dilates arterial vessels (2Currie M.G. Geller D.M. Cole B.R. Boylan J.G. YuSheng W. Holmberg S.W. Needleman P. Science. 1983; 221: 71-73Crossref PubMed Scopus (582) Google Scholar, 3Grammer R.T. Fukumi H. Inagami T. Misono K.S. Biochem. Biophys. Res. Commun. 1983; 116: 696-703Crossref PubMed Scopus (125) Google Scholar). Through these activities, ANP plays a major role in the regulation of blood pressure and salt-fluid volume homeostasis. Transgenic animals devoid of the ANP gene develop salt-sensitive hypertension (4John S.W. Krege J.H. Oliver P.M. Hagaman J.R. Hodgin J.B. Pang S.C. Flynn T.G. Smithies O. Science. 1995; 267: 679-681Crossref PubMed Scopus (567) Google Scholar), and those lacking the ANP receptor gene develop salt-insensitive essential hypertension accompanied by severe cardiac hypertrophy, fibrosis, and dilatation (5Oliver P.M. Fox J.E. Kim R. Rockman H.A. Kim H.S. Reddick R.L. Pandey K.N. Milgram S.L. Smithies O. Maeda N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14730-14735Crossref PubMed Scopus (498) Google Scholar), implicating the ANP and ANP receptor systems in cardiovascular pathophysiology. An analogous hormone, B-type natriuretic peptide (BNP), is also produced and secreted mainly by the heart and has hormonal activities similar to ANP (6Nakao K. Ogawa Y. Suga S. Imura H. J. Hypertens. 1992; 10: 907-912Crossref PubMed Scopus (198) Google Scholar). The activities of ANP and BNP are mediated by the ANP receptor or the A-type natriuretic peptide receptor carrying intrinsic guanylate cyclase (GCase) catalytic activity. Binding of the hormone to the receptor stimulates GCase catalytic activity, thereby elevating intracellular cGMP levels. cGMP, in turn, mediates the hormonal actions through cGMP-regulated ion channels, protein kinases, and phosphodiesterases. The ANP receptor occurs as a dimer of a single span transmembrane polypeptide, each containing an extracellular hormone-binding domain and an intracellular domain consisting of a protein kinase-like, ATP-dependent regulatory domain and a GCase catalytic domain (7Chinkers M. Garbers D.L. Chang M.S. Lowe D.G. Chin H.M. Goeddel D.V. Schulz S. Nature. 1989; 338: 78-83Crossref PubMed Scopus (887) Google Scholar). The molecular mechanism by which ANP binding to the extracellular domain stimulates the catalytic activity of the intracellular GCase domain is not understood. A closely related receptor, the B-type natriuretic peptide receptor, mediates actions of C-type natriuretic peptide (CNP), which occurs mostly in the brain. CNP and the B-type receptor are thought to play a role in the central nervous system-mediated control of blood pressure and salt-fluid balance (6Nakao K. Ogawa Y. Suga S. Imura H. J. Hypertens. 1992; 10: 907-912Crossref PubMed Scopus (198) Google Scholar, 8Koller K.J. Lowe D.G. Bennett G.L. Minamino N. Kangawa K. Matsuo H. Goeddel D.V. Science. 1991; 252: 120-123Crossref PubMed Scopus (660) Google Scholar, 9Nakao K. Ogawa Y. Suga S. Imura H. J. Hypertens. 1992; 10: 1111-1114Crossref PubMed Scopus (209) Google Scholar). The B-type receptor has ∼60% sequence identity with the A-type receptor and has a similar overall molecular topology. It is likely then that the B-type receptor has a signaling mechanism similar to that of the A-type receptor. Yet another related receptor, the natriuretic peptide clearance-receptor, lacks the GCase domain and is not GCase-coupled (10Fuller F. Porter J.G. Arfsten A.E. Miller J. Schilling J.W. Scarborough R.M. Lewicki J.A. Schenk D.B. J. Biol. Chem. 1988; 263: 9395-9401Abstract Full Text PDF PubMed Google Scholar). The clearance-receptor binds ANP, BNP, and CNP as well as some of their biologically inactive fragments with equally high affinity and removes the excesses of these peptides from the circulation (11Maack T. Suzuki M. Almeida F.A. Nussenzveig D. Scarborough R.M. McEnroe G.A. Lewicki J.A. Science. 1987; 238: 675-678Crossref PubMed Scopus (852) Google Scholar, 12Cohen D. Koh G.Y. Nikonova L.N. Porter J.G. Maack T. J. Biol. Chem. 1996; 271: 9863-9869Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The clearance-receptor has not been linked to any of the known hormonal actions of natriuretic peptides. The GCase-coupled A-type and B-type natriuretic peptide receptors belong to the family of membrane-bound receptor GCases that include guanylin and enterotoxin receptors (13Schulz S. Green C.K. Yuen P.S. Garbers D.L. Cell. 1990; 63: 941-948Abstract Full Text PDF PubMed Scopus (521) Google Scholar), retinal GCases (14Lowe D.G. Dizhoor A.M. Liu K. Gu Q. Spencer M. Laura R. Lu L. Hurley J.B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5535-5539Crossref PubMed Scopus (238) Google Scholar), and olfactory cell GCases (15Fulle H.J. Vassar R. Foster D.C. Yang R.B. Axel R. Garbers D.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3571-3575Crossref PubMed Scopus (233) Google Scholar). These receptor GCases, in turn, belong to the superfamily of single span transmembrane receptors for which the mechanism of transmembrane signaling has not been well defined. To elucidate the signaling mechanism of the ANP receptor, we have expressed and purified the extracellular hormone-binding domain of the receptor (ANPR) in a soluble form (16Misono K.S. Sivasubramanian N. Berkner K. Zhang X. Biochemistry. 1999; 38: 516-523Crossref PubMed Scopus (40) Google Scholar) and have characterized its biochemical properties, including the disulfide bond structure (17Miyagi M. Misono K.S. Biochim. Biophys. Acta. 2000; 1478: 30-38Crossref PubMed Scopus (34) Google Scholar), glycosyl structure (18Miyagi M. Zhang X. Misono K.S. Eur. J. Biochem. 2000; 267: 5758-5768Crossref PubMed Scopus (36) Google Scholar), and requirement for chloride ion for its binding with ANP (19Misono K.S. Circ. Res. 2000; 86: 1135-1139Crossref PubMed Scopus (41) Google Scholar). We have also crystallized the ANPR without the hormone (apoANPR) and determined its x-ray structure (20van den Akker F. Zhang X. Miyagi M. Huo X. Misono K.S. Yee V.C. Nature. 2000; 406: 101-104Crossref PubMed Scopus (142) Google Scholar). The apoANPR was originally described as occurring in a tail-to-tail dimer form associated through its membrane-proximal domain (20van den Akker F. Zhang X. Miyagi M. Huo X. Misono K.S. Yee V.C. Nature. 2000; 406: 101-104Crossref PubMed Scopus (142) Google Scholar). However, it was later recognized that the crystal packing of apoANPR also contained an alternative dimer pair, a head-to-head dimer, associated through the membrane-distal domain. Both the tail-to-tail and head-to-head dimer interfaces involve a large buried surface area (1,680 and 1,100 Å2, respectively) and multiple residue contacts. Thus, from the crystallographic data alone, it has not been possible to distinguish which dimer form represents the physiological ANP receptor dimer structure. We have recently reported site-directed mutagenesis studies of the residue involved in the two possible dimer interfaces in the full-length ANP receptor expressed on COS cells. We have found that certain mutations at the head-to-head dimer interface cause the receptor to become either uncoupled or constitutively GCase active, whereas mutations at the tail-to-tail dimer interface cause no such effect (21Qiu Y. Ogawa H. Miyagi M. Misono K.S. J. Biol. Chem. 2004; 279: 6115-6123Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). These results strongly suggest that the extracellular domain of the native ANP receptor on the cell surface assumes the head-to-head dimer structure and that the tail-to-tail apoANPR dimer previously described represents an artificial crystallographic dimer pair occurring only in the crystal packing. The head-to-head dimer structure for the apoANPR is similar to that proposed for the extracellular domain of the natriuretic peptide clearance-receptor (22He X. Chow D. Martick M.M. Garcia K.C. Science. 2001; 293: 1657-1662Crossref PubMed Scopus (141) Google Scholar). In the present study, we have determined the crystal structure of the ANPR complexed with the hormone ANP. The comparison of the complex structure, also occurring in the head-to-head configuration, with the unbound structure reveals a structural change caused by ANP binding and suggests a structural basis for transmembrane signaling by the ANP receptor. Crystallization of ANPR·ANP Complex and Data Collection—Expression and purification of the ANPR (16Misono K.S. Sivasubramanian N. Berkner K. Zhang X. Biochemistry. 1999; 38: 516-523Crossref PubMed Scopus (40) Google Scholar) and crystallization of the ANPR·ANP complex were carried out as described elsewhere (23Ogawa H. Zhang X. Qiu Y. Ogata C.M. Misono K.S. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 1831-1833Crossref PubMed Scopus (7) Google Scholar). Briefly, the ANPR was expressed in Chinese hamster ovary cells and purified by ANP affinity chromatography. The ANPR was obtained N-glycosylated (18Miyagi M. Zhang X. Misono K.S. Eur. J. Biochem. 2000; 267: 5758-5768Crossref PubMed Scopus (36) Google Scholar). The ANPR was treated with sialidase to reduce heterogeneity in the glycosyl structure and was again purified by ANP affinity chromatography. The complex of the ANPR (10 mg/ml) and an ANP peptide consisting of residues 7–27 (sequence: Cys-Phe-Gly-Gly-Arg-Ile-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg) was crystallized by hanging drop vapor diffusion at room temperature with 1.6-2.0 m ammonium sulfate in 0.1 m MES buffer, pH 6.5, containing 10 mm NaCl. These conditions differ from those used for crystallizing the apoANPR. Crystals were dialyzed against high concentrations of ammonium sulfate solution and were frozen in liquid propane. The crystals had the space group of P61 with unit cell parameters a = b = 100.1 Å, and c = 259.8 Å. Two ANPR molecules are present in an asymmetric unit with a VM (Mathews coefficient) of 3.9 Å3/dalton. Data were collected at 100 K at Advanced Photon Source beamline 19-ID and National Synchrotron Light Source beamlines X4A and X25. Data were processed and scaled using the HKL package (24Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 306-326Google Scholar). Structure Determination and Refinement—The structure was solved by molecular replacement with the program CNS (25Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve W. Jiang J.-S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16967) Google Scholar), using one ANPR molecule in the apoANPR dimer structure (Protein Data Base accession number 1DP4) as the search model (Table I). Molecular replacement calculations were also performed using the individual membrane-proximal and membrane-distal domains as the model, which gave essentially the same structure. Refinement was done with the program REFMAC (26Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13870) Google Scholar) without imposing non-crystallographic symmetry restraints. Rebuilding and correction of the model was guided by σA weighted 2 Fo - Fc map with the program TURBO-FRODO (BioGraphics). The structure of bound ANP was traced in a single unique conformation occurring in 2-fold symmetry orientations (Supplemental Fig. 1). The ANP structure was refined as alternate conformations with equal occupancy. Residues 427–435 of monomer A and residues 253–256 and 426–435 of monomer B had insufficient electron density to be assigned. The stereochemistry of the final structure was analyzed with PROCHECK (27Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). Assignment of the secondary structures was done using program DSSP (28Kabsch W. Sander C. Biopolymers. 1983; 22: 2577-2637Crossref PubMed Scopus (12332) Google Scholar).Table ICrystallographic data and refinement statisticsData collectionResolution (Å) (last shell)2.95 (3.04–2.95)Measured reflections225,118Unique reflections30,886Completeness (%)93.7 (89.8)I/σ(I)30.9 (3.2)RmergeaRmerge = Σhkl|I – 〈I〉|/ΣhklI, where I is the intensity of unique reflection hkl and 〈I〉 is the average over symmetry-related observation of unique reflection hkl (%)5.4 (55.4)Refinement statistics (I/σ(I) > 1)Resolution range (Å)87.71–2.95Number of reflections25,503RcrystbRcryst = Σ|Fobs – Fcalc|/Σ|Fobs|, where Fobs and Fcalc are the observed and calculated structure factors, respectively23.9 (26.9)RfreecRfree is R with 7.5% of reflections sequestered before refinement26.9 (30.1)Average B-factors (Å2)ANP receptor52.4ANP44.0Oligosaccharides70.1Chlorides53.1Root mean square deviation from idealityBond length (Å)0.009Bond angles (°)1.5Bonded B-factors (Å2)(Main chain, side chain)1.6, 2.6Ramachandran plot (%)(Favored, allowed, generous, disallowed)84.2, 15.4, 0.4, 0a Rmerge = Σhkl|I – 〈I〉|/ΣhklI, where I is the intensity of unique reflection hkl and 〈I〉 is the average over symmetry-related observation of unique reflection hklb Rcryst = Σ|Fobs – Fcalc|/Σ|Fobs|, where Fobs and Fcalc are the observed and calculated structure factors, respectivelyc Rfree is R with 7.5% of reflections sequestered before refinement Open table in a new tab Overall Structure of the ANPR·ANP Complex—The extracellular hormone-binding domain of the ANPR consisting of residues 1–435 was expressed and purified as described (16Misono K.S. Sivasubramanian N. Berkner K. Zhang X. Biochemistry. 1999; 38: 516-523Crossref PubMed Scopus (40) Google Scholar). ANPR was crystallized with an ANP peptide with the sequence and are ANP residues 7–27 (23Ogawa H. Zhang X. Qiu Y. Ogata C.M. Misono K.S. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 1831-1833Crossref PubMed Scopus (7) Google Scholar). This peptide is K.S. R.T. Fukumi H. Inagami T. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar, Res. 1990; 10: PubMed Scopus Google Scholar). The structure of receptor complex was determined to (Table I). The asymmetric unit of the crystal two ANPR molecules bound with one molecule of ANP, a the ANP molecule has no binding of ANP to the receptor is ANP occurs in two alternate conformations of equal related by a 2-fold of symmetry (Supplemental Fig. 1). The 2-fold symmetry in the electron density for ANP suggests that these two alternate bound conformations are equally in the crystal packing. The ANPR monomer has membrane-distal and membrane-proximal each consisting of a central by The membrane-distal domain a bound chloride ion (20van den Akker F. Zhang X. Miyagi M. Huo X. Misono K.S. Yee V.C. Nature. 2000; 406: 101-104Crossref PubMed Scopus (142) Google Scholar) for ANP binding (19Misono K.S. Circ. Res. 2000; 86: 1135-1139Crossref PubMed Scopus (41) Google Scholar). The two ANPR also related by non-crystallographic 2-fold form a head-to-head dimer through their membrane-distal domains Structure the structural basis for ANP receptor the structure of the ANPR dimer complex was with that of the apoANPR dimer The structural change a shift in the relative of the two ANPR monomers as in Fig. 2 in is no intramolecular conformational change in each individual ANPR monomer mean square deviation of ANP the two ANPR molecules undergo a twist motion on a In the ANP binding to a on that causes the dimer interface to and the membrane-proximal domains to the bound ANP from the motion is accompanied by a rotation of each again on In the juxtamembrane twist motion of the two monomers results in of the two membrane-proximal domains by in The the two domains is essentially the residues its and the juxtamembrane signaling X. T. Misono K.S. Biochemistry. 1999; 38: PubMed Scopus Google Scholar), a structure that is and plays a role in transmembrane The movement of these residues that the two juxtamembrane domains undergo a This translocation alters the relative orientation of the two juxtamembrane likely transmembrane conformational change by binding in the ANPR and in the natriuretic peptide clearance-receptor extracellular of the structure of the ANPR that of unbound ANPR in ANP binding causes little intramolecular conformational change mean square deviation of of the clearance-receptor extracellular domain structure in the form the unbound form (22He X. Chow D. Martick M.M. Garcia K.C. Science. 2001; 293: 1657-1662Crossref PubMed Scopus (141) Google Scholar). In the clearance-receptor, binding causes each to at the structure from to the membrane-proximal domain to the bound with the hormone-induced structural change in the ANP receptor from that found in the natriuretic peptide clearance-receptor CNP binding (22He X. Chow D. Martick M.M. Garcia K.C. Science. 2001; 293: 1657-1662Crossref PubMed Scopus (141) Google Scholar). In the clearance-receptor, the structural change occurs each receptor CNP binding causes each monomer structure to at the intramolecular the membrane-distal and membrane-proximal domains This causes the membrane-proximal domain to the bound the membrane-distal domain and its dimerized structure essentially (Supplemental Fig. This motion the two membrane-proximal domains not change their relative the ANP receptor, the clearance-receptor lacks the GCase domain (10Fuller F. Porter J.G. Arfsten A.E. Miller J. Schilling J.W. Scarborough R.M. Lewicki J.A. Schenk D.B. J. Biol. Chem. 1988; 263: 9395-9401Abstract Full Text PDF PubMed Google Scholar) and is not known to any of the known hormonal activities of natriuretic peptides. The clearance-receptor binds ANP, BNP, and CNP as well as some of their biologically inactive fragments with equally high affinity in to the of these peptides from the circulation (11Maack T. Suzuki M. Almeida F.A. Nussenzveig D. Scarborough R.M. McEnroe G.A. Lewicki J.A. Science. 1987; 238: 675-678Crossref PubMed Scopus (852) Google Scholar, 12Cohen D. Koh G.Y. Nikonova L.N. Porter J.G. Maack T. J. Biol. Chem. 1996; 271: 9863-9869Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The in the motion in the ANP receptor from that in the clearance-receptor the that the ANP receptor mediates hormonal whereas the clearance-receptor the conformational at the intramolecular in the clearance-receptor (22He X. Chow D. Martick M.M. Garcia K.C. Science. 2001; 293: 1657-1662Crossref PubMed Scopus (141) Google Scholar) be for its the dimer interface in the apoANPR structure in intermolecular are through of one monomer and of the two and and and and and ANP the dimer interface in the and and The on the and to the receptor We have found that of to or in the full-length ANP receptor as expressed on COS cells produced a receptor that is constitutively GCase (21Qiu Y. Ogawa H. Miyagi M. Misono K.S. J. Biol. Chem. 2004; 279: 6115-6123Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). These mutations at the dimer The the the interface of the receptor dimer to become in the of ANP and a structure that the receptor an effect be for the of GCase activity. the produced an uncoupled receptor that bound ANP not cause cGMP two residues each the of the monomer movement O. The the two the structure of the bound complex and signaling and GCase Thus, the results of mutagenesis studies performed with the full-length ANP receptor expressed on the COS cell surface are with the crystal structures of the and ANPR dimer and with the structural change from these This in turn, suggests that the hormone-induced structural change in likely that occurring in the native full-length ANP receptor in the and Binding of ANP is and the binding in one ANPR molecule from that in the A mainly with the of the ANP whereas B mainly with the (Supplemental surface at the two are equal and for A and of to hormone The surface structures of A and B have a with residues b and ANP closely into the at with the high of the ANP receptor. ANP residue with and These play a role in the dimer interface and the structure of the ANPR dimer The role of is with the that residue is essential for the hormonal activity of ANP Res. 1990; 10: PubMed Scopus Google Scholar). An bond occurring and also to the of the essential for hormonal activity, to and with a in A by residues and (Supplemental The of ANP with a of in the ANPR and a bond occurs and of a An bond occurs and These are with the that the residues of ANP are for receptor binding and hormonal activities Res. 1990; 10: PubMed Scopus Google Scholar). The found the ANP residues involved in binding and the for ANP Res. 1990; 10: PubMed Scopus Google Scholar) the ANP peptide structure in the complex at the same suggests that the crystal structure of complex closely the structure of the native ANP receptor bound with ANP. ANP residues to binding to the receptor are in BNP, including in and (Supplemental Fig. The of these residues suggests that the mechanism of BNP binding to receptor as well as the structural change in the receptor be similar to those described for ANP. The ANP receptor binds ANP and BNP with high affinity affinity to CNP (16Misono K.S. Sivasubramanian N. Berkner K. Zhang X. Biochemistry. 1999; 38: 516-523Crossref PubMed Scopus (40) Google Scholar). The affinity to CNP, which lacks residues (Supplemental Fig. be of its to form the The orientation of the bound ANP is with the results of affinity in which ANP peptide containing an group at and with and of the receptor, in In the crystal structure of the is to and is the structure for the bound ANP. for ANP the movement of the two ANPR molecules ANP ANPR molecule is by a ANP binding causes little intramolecular conformational change. ANP the two molecules undergo a twist motion on and the ANP. In at the juxtamembrane from the twist motion causes the juxtamembrane domains of the two molecules by to by an of with to from the complexed without change in the interdomain distance. This alters the relative orientation of the two juxtamembrane domains in the receptor This change in the orientation is equivalent to each of the two domains by counterclockwise and W. Science. 1999; PubMed Scopus Google Scholar) for transmembrane signaling mechanism that on the motion in the transmembrane and The motion of the juxtamembrane domains in the ANP receptor closely to the rotation mechanism by these in Fig. by single span transmembrane receptors is thought to by the mechanism. of the receptor molecules to a dimer an their intracellular domains The of the intracellular in turn, the actions of the domains the single span transmembrane such as the receptor, as a dimer in the of the In the crystal structure of the extracellular domain of the receptor, two in the juxtamembrane are by in the dimer O. D.L. Science. 1999; PubMed Scopus Google Scholar). binding causes a large conformational change in the dimer that the to O. D.L. Science. 1999; PubMed Scopus Google Scholar, S.W. C. Liu H. A.J. Zhang J. J. S. K. J. R.M. Nature. 1998; PubMed Scopus Google Scholar). This of the two is to the intracellular signaling It has also been by that binding the intracellular domains into S.W. Science. 1999; PubMed Scopus Google Scholar), the mechanism proposed for receptor signaling O. D.L. Science. 1999; PubMed Scopus Google Scholar). In the juxtamembrane domains of the ANP receptor dimer are to each in the of the hormone, and ANP binding causes no change in the interdomain and ANP binding causes a large rotation of each of the two juxtamembrane their relative orientations In the ANP receptor, hormone-induced rotation of the juxtamembrane through the transmembrane the relative of the intracellular domains to cause GCase The ANPR expressed in a soluble form lacks the transmembrane and intracellular domains for its and structures not those of the native full-length receptor in the cell However, we that the of mutations at the dimer interface in the full-length receptor on COS cells are with the crystal structures of the and ANPR and with the structural change from those structures (21Qiu Y. Ogawa H. Miyagi M. Misono K.S. J. Biol. Chem. 2004; 279: 6115-6123Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). binding found in the complex structure are in with the for ANP Res. 1990; 10: PubMed Scopus Google Scholar). These suggest strongly that the structural change in that occurring in the native receptor in the cell and the proposed rotation mechanism ANP receptor The structures of the receptor in the with and without bound ANP for the mechanism in the GCase, which has sequence identity with the GCase domain of the ANP receptor, is only as a dimer each monomer a GCase catalytic M. F. 26: PubMed Scopus Google Scholar). The GCase domain of ANP receptor, expressed in a soluble form by of the and a dimer and is M. Biochemistry. 1995; PubMed Scopus Google Scholar). It is possible that in the ANP receptor dimer the GCase catalytic activity is the two GCase domains are to form an dimer We ANP binding and in the of and for GCase by ANP H. Inagami T. M. 1987; PubMed Scopus Google Scholar, M. S. Garbers D.L. J. Biol. Chem. 1991; Full Text PDF PubMed Google rotation of the juxtamembrane domains the relative the of the intracellular domains such that the catalytic of the two GCase domains are to an and thereby to GCase catalytic activity The rotation mechanism transmembrane signaling by the ANP receptor in the present to the such mechanism for any known of single span transmembrane the same a new a transmembrane conformational change a or of the receptor mediates transmembrane We Y. Kim and M. for in data and M. and M. for of the x-ray of the Advanced Photon Source beamline at the National was by the of with