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W2170174733 abstract "The conformational freedom of single-chain peptide hormones, such as the 41-amino acid hormone corticotropin releasing factor (CRF), is a major obstacle to the determination of their biologically relevant conformation, and thus hampers insights into the mechanism of ligand-receptor interaction. Since N- and C-terminal truncations of CRF lead to loss of biological activity, it has been thought that almost the entire peptide is essential for receptor activation. Here we show the existence of two segregated receptor binding sites at the N and C termini of CRF, connection of which is essential for receptor binding and activation. Connection of the two binding sites by highly flexible ε-aminocaproic acid residues resulted in CRF analogues that remained full, although weak agonists (EC50: 100–300 nm) independent of linker length. Connection of the two sites by an appropriate helical peptide led to a very potent analogue, which adopted, in contrast to CRF itself, a stable, monomer conformation in aqueous solution. Analogues in which the two sites were connected by helical linkers of different lengths were potent agonists; their significantly different biopotencies (EC50: 0.6–50 nm), however, suggest the relative orientation between the two binding sites rather than the maintenance of a distinct distance between them to be essential for a high potency. The conformational freedom of single-chain peptide hormones, such as the 41-amino acid hormone corticotropin releasing factor (CRF), is a major obstacle to the determination of their biologically relevant conformation, and thus hampers insights into the mechanism of ligand-receptor interaction. Since N- and C-terminal truncations of CRF lead to loss of biological activity, it has been thought that almost the entire peptide is essential for receptor activation. Here we show the existence of two segregated receptor binding sites at the N and C termini of CRF, connection of which is essential for receptor binding and activation. Connection of the two binding sites by highly flexible ε-aminocaproic acid residues resulted in CRF analogues that remained full, although weak agonists (EC50: 100–300 nm) independent of linker length. Connection of the two sites by an appropriate helical peptide led to a very potent analogue, which adopted, in contrast to CRF itself, a stable, monomer conformation in aqueous solution. Analogues in which the two sites were connected by helical linkers of different lengths were potent agonists; their significantly different biopotencies (EC50: 0.6–50 nm), however, suggest the relative orientation between the two binding sites rather than the maintenance of a distinct distance between them to be essential for a high potency. corticotropin releasing factor growth hormone releasing factor parathyroid hormone calcitonin gene-related peptide ε-aminocaproic acid urocortin urocortin-EK G protein-coupled receptor double quantum-filtered total correlation spectroscopy adrenocorticotropic hormone nuclear Overhauser effect high performance liquid chromatography nuclear Overhauser effect spectroscopy The biologically important peptide hormones corticotropin releasing factor (CRF)1, glucagon, secretin, vasoactive intestinal polypeptide, growth hormone releasing factor (GRF), calcitonin, parathyroid hormone (PTH), calcitonin gene-related peptide (CGRP), etc. have significant features in common. All exert their activity via binding to and activation of class 2 G protein-coupled receptors (GPCRs). They are polypeptides comprising about 25–40 amino acid residues without preferred conformation in aqueous solution and exhibit no documented biologically relevant secondary and tertiary structure. Under structure-inducing conditions (e.g. in the presence of trifluoroethanol or membrane mimicking lipids), however, these peptide hormones (CRF (Ref.1.Romier C. Bernassau J.M. Cambillau C. Darbon H. Protein Eng. 1993; 6: 149-156Crossref PubMed Scopus (50) Google Scholar), PTH (Refs. 2.Pellegrini M. Royo M. Rosenblatt M. Chorev M. Mierke D.F. J. Biol. Chem. 1998; 273: 10420-10427Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar and 3.Barden J.A. Kemp B.E. Biochemistry. 1993; 32: 7126-7132Crossref PubMed Scopus (61) Google Scholar), calcitonin (Ref. 4.Motta A. Andreotti G. Amodeo P. Strazullo G. Castiglione Morelli M.A. Proteins. 1998; 32: 314-323Crossref PubMed Scopus (59) Google Scholar), glucagon (Ref. 5.Braun W. Wider G. Lee K.H. Wüthrich K. J. Mol. Biol. 1983; 169: 921-948Crossref PubMed Scopus (296) Google Scholar), GRF (Ref. 6.Clore G.M. Martin S.R. Gronenborn A.M. J. Mol. Biol. 1986; 191: 553-561Crossref PubMed Scopus (94) Google Scholar)) exhibit strong helix formation, suggesting that a helical ligand conformation is essential for receptor interaction. Furthermore, N-terminal truncations of the hormones convert them from agonists to antagonists (CRF (Ref. 7.Rivier J. Rivier C. Vale W. Science. 1984; 224: 889-891Crossref PubMed Scopus (445) Google Scholar), GRF (Ref. 8.Coy D.H. Murphy W.A. Sueiras-Diaz J. Coy E.J. Lance V.A. J. Med. Chem. 1985; 28: 181-185Crossref PubMed Scopus (64) Google Scholar), calcitonin (Refs. 9.Feyen J.H. Cardinaux F. Gamse R. Bruns C. Azria M. Trechsel U. Biochem. Biophys. Res. Commun. 1992; 187: 8-13Crossref PubMed Scopus (42) Google Scholar and 19.Rivier J. Rivier C. Galyean R. Miranda A. Miller Ch. Craig A.G. Yamamoto G. Brown M. Vale W. J. Med. Chem. 1993; 36: 2851-2859Crossref PubMed Scopus (56) Google Scholar), PTH (Ref. 11.Segre G.V. Rosenblatt M. Reiner B.L. Mahaffey J.E. Potts Jr., J.T J. Biol. Chem. 1979; 254: 6980-6986Abstract Full Text PDF PubMed Google Scholar), CGRP (Ref. 12.Maton P.N. Pradhan T. Zhou Z.-C. Gardner J.D. Jensen R.T. Peptides. 1990; 11: 485-489Crossref PubMed Scopus (34) Google Scholar), glucagon (Ref. 13.Unson C.G. Gurzender E.M. Iwasa K. Merrifield R.B. J. Biol. Chem. 1989; 264: 789-794Abstract Full Text PDF PubMed Google Scholar)), proving the existence of an important receptor binding/activation site within the peptide N terminus. C-terminal truncations result in a drastic decrease in receptor binding, indicating an essential function for C-terminal residues (CRF (Refs. 9.Feyen J.H. Cardinaux F. Gamse R. Bruns C. Azria M. Trechsel U. Biochem. Biophys. Res. Commun. 1992; 187: 8-13Crossref PubMed Scopus (42) Google Scholar and 14.Vale W. Spiess J. Rivier C. Rivier J. Science. 1981; 213: 1394-1397Crossref PubMed Scopus (3993) Google Scholar), GRF (Ref. 15.Coy D.H. Murphy W.A. Lance V.A. Heiman M.L. J. Med. Chem. 1987; 30: 219-222Crossref PubMed Scopus (24) Google Scholar), CGRP (Ref. 16.Smith D.D. Wang Q. Murphy R.F. Adrian T.E. Elias Y. Bockman C.S. Abel P.W. J. Med. Chem. 1993; 36: 2536-2541Crossref PubMed Scopus (18) Google Scholar)). Based on these results, it has been assumed that almost the entire peptide is necessary for binding to and activation of the corresponding receptors.In the case of CRF, which is the principal neuroregulator of the basal and stress-induced secretion of ACTH, β-endorphin and other proopiomelanocortin-related peptides from the anterior pituitary (see Ref. 17.Chalmers D.T. Lovenberg T.W. Grigoriades D.E. Behan D.P. De Souza E.B. Trends Pharmacol. Sci. 1996; 17: 166-172Abstract Full Text PDF PubMed Scopus (373) Google Scholar for review), previously published structure-activity relationship studies of single-point substituted (18.Kornreich W.D. Galyean R. Hernandez J.-F. Craig A.G. Donaldson C.J. Yamamoto G. Rivier C. Vale W. Rivier J. J. Med. Chem. 1992; 35: 1870-1876Crossref PubMed Scopus (93) Google Scholar, 19.Rivier J. Rivier C. Galyean R. Miranda A. Miller Ch. Craig A.G. Yamamoto G. Brown M. Vale W. J. Med. Chem. 1993; 36: 2851-2859Crossref PubMed Scopus (56) Google Scholar, 20.Beyermann M. Fechner K. Furkert J. Krause E. Bienert M. J. Med. Chem. 1996; 39: 3324-3330Crossref PubMed Scopus (51) Google Scholar) and terminally truncated CRF analogues (7.Rivier J. Rivier C. Vale W. Science. 1984; 224: 889-891Crossref PubMed Scopus (445) Google Scholar, 21.Rivier J. Lahrichi S.L. Gulyas J. Erchegyi J. Koerber S.C. Grey Craig A. Corrigan A. Rivier C. Vale W. J. Med. Chem. 1998; 41: 2614-2620Crossref PubMed Scopus (24) Google Scholar) showed that the N-terminal sequence (9.Feyen J.H. Cardinaux F. Gamse R. Bruns C. Azria M. Trechsel U. Biochem. Biophys. Res. Commun. 1992; 187: 8-13Crossref PubMed Scopus (42) Google Scholar, 10.Rittel W. Maier R. Brugger M. Kamber B. Riniker B. Sieber P. Experientia (Basel). 1976; 32: 246-248Crossref PubMed Scopus (52) Google Scholar, 11.Segre G.V. Rosenblatt M. Reiner B.L. Mahaffey J.E. Potts Jr., J.T J. Biol. Chem. 1979; 254: 6980-6986Abstract Full Text PDF PubMed Google Scholar, 12.Maton P.N. Pradhan T. Zhou Z.-C. Gardner J.D. Jensen R.T. Peptides. 1990; 11: 485-489Crossref PubMed Scopus (34) Google Scholar, 13.Unson C.G. Gurzender E.M. Iwasa K. Merrifield R.B. J. Biol. Chem. 1989; 264: 789-794Abstract Full Text PDF PubMed Google Scholar, 14.Vale W. Spiess J. Rivier C. Rivier J. Science. 1981; 213: 1394-1397Crossref PubMed Scopus (3993) Google Scholar, 15.Coy D.H. Murphy W.A. Lance V.A. Heiman M.L. J. Med. Chem. 1987; 30: 219-222Crossref PubMed Scopus (24) Google Scholar, 16.Smith D.D. Wang Q. Murphy R.F. Adrian T.E. Elias Y. Bockman C.S. Abel P.W. J. Med. Chem. 1993; 36: 2536-2541Crossref PubMed Scopus (18) Google Scholar, 17.Chalmers D.T. Lovenberg T.W. Grigoriades D.E. Behan D.P. De Souza E.B. Trends Pharmacol. Sci. 1996; 17: 166-172Abstract Full Text PDF PubMed Scopus (373) Google Scholar, 18.Kornreich W.D. Galyean R. Hernandez J.-F. Craig A.G. Donaldson C.J. Yamamoto G. Rivier C. Vale W. Rivier J. J. Med. Chem. 1992; 35: 1870-1876Crossref PubMed Scopus (93) Google Scholar, 19.Rivier J. Rivier C. Galyean R. Miranda A. Miller Ch. Craig A.G. Yamamoto G. Brown M. Vale W. J. Med. Chem. 1993; 36: 2851-2859Crossref PubMed Scopus (56) Google Scholar) represents a receptor binding site, since substitutions in this region resulted in a significant decrease in receptor binding. The most N-terminal amino acid residues are thought to be responsible for receptor activation (7.Rivier J. Rivier C. Vale W. Science. 1984; 224: 889-891Crossref PubMed Scopus (445) Google Scholar), since truncation of these residues produced antagonists. The N-terminal peptide sequence (6.Clore G.M. Martin S.R. Gronenborn A.M. J. Mol. Biol. 1986; 191: 553-561Crossref PubMed Scopus (94) Google Scholar, 7.Rivier J. Rivier C. Vale W. Science. 1984; 224: 889-891Crossref PubMed Scopus (445) Google Scholar, 8.Coy D.H. Murphy W.A. Sueiras-Diaz J. Coy E.J. Lance V.A. J. Med. Chem. 1985; 28: 181-185Crossref PubMed Scopus (64) Google Scholar, 9.Feyen J.H. Cardinaux F. Gamse R. Bruns C. Azria M. Trechsel U. Biochem. Biophys. Res. Commun. 1992; 187: 8-13Crossref PubMed Scopus (42) Google Scholar, 10.Rittel W. Maier R. Brugger M. Kamber B. Riniker B. Sieber P. Experientia (Basel). 1976; 32: 246-248Crossref PubMed Scopus (52) Google Scholar, 11.Segre G.V. Rosenblatt M. Reiner B.L. Mahaffey J.E. Potts Jr., J.T J. Biol. Chem. 1979; 254: 6980-6986Abstract Full Text PDF PubMed Google Scholar, 12.Maton P.N. Pradhan T. Zhou Z.-C. Gardner J.D. Jensen R.T. Peptides. 1990; 11: 485-489Crossref PubMed Scopus (34) Google Scholar, 13.Unson C.G. Gurzender E.M. Iwasa K. Merrifield R.B. J. Biol. Chem. 1989; 264: 789-794Abstract Full Text PDF PubMed Google Scholar, 14.Vale W. Spiess J. Rivier C. Rivier J. Science. 1981; 213: 1394-1397Crossref PubMed Scopus (3993) Google Scholar, 15.Coy D.H. Murphy W.A. Lance V.A. Heiman M.L. J. Med. Chem. 1987; 30: 219-222Crossref PubMed Scopus (24) Google Scholar, 16.Smith D.D. Wang Q. Murphy R.F. Adrian T.E. Elias Y. Bockman C.S. Abel P.W. J. Med. Chem. 1993; 36: 2536-2541Crossref PubMed Scopus (18) Google Scholar, 17.Chalmers D.T. Lovenberg T.W. Grigoriades D.E. Behan D.P. De Souza E.B. Trends Pharmacol. Sci. 1996; 17: 166-172Abstract Full Text PDF PubMed Scopus (373) Google Scholar, 18.Kornreich W.D. Galyean R. Hernandez J.-F. Craig A.G. Donaldson C.J. Yamamoto G. Rivier C. Vale W. Rivier J. J. Med. Chem. 1992; 35: 1870-1876Crossref PubMed Scopus (93) Google Scholar, 19.Rivier J. Rivier C. Galyean R. Miranda A. Miller Ch. Craig A.G. Yamamoto G. Brown M. Vale W. J. Med. Chem. 1993; 36: 2851-2859Crossref PubMed Scopus (56) Google Scholar, 20.Beyermann M. Fechner K. Furkert J. Krause E. Bienert M. J. Med. Chem. 1996; 39: 3324-3330Crossref PubMed Scopus (51) Google Scholar) is highly conserved within the CRF family, peptides from different species that activate CRF receptors. In contrast, there is great sequence diversity within the C-terminal region (21.Rivier J. Lahrichi S.L. Gulyas J. Erchegyi J. Koerber S.C. Grey Craig A. Corrigan A. Rivier C. Vale W. J. Med. Chem. 1998; 41: 2614-2620Crossref PubMed Scopus (24) Google Scholar, 22.Bergwitz C. Gardella T.J. Flannery M.R. Potts Jr., J.T. Kronenberg H.M. Goldring S.R. Jüppner H. J. Biol. Chem. 1996; 271: 26469-26472Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 23.Stroop S.D. Kuestner R.E. Serwold T.F. Chen L. 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Winter R. Beyermann M. Schühmann M. Bienert M. Int. J. Pept. Protein Res. 1996; 47: 383-393Crossref PubMed Scopus (28) Google Scholar, 38.Rothemund S. Krause E. Beyermann M. Bienert M. J. Pept. Res. 1997; 50: 184-192Crossref PubMed Scopus (8) Google Scholar, 39.Rothemund S. Krause E. Beyermann M. Dathe M. Bienert M. Hodges R.S. Sykes B.D. Sönnichsen F.D. Pept. Res. 1996; 9: 79-87PubMed Google Scholar, 40.Blankenfeldt W. Nokihara K. Naruse S. Lessel U. Schomburg D. Wray V. Biochemistry. 1996; 35: 5955-5962Crossref PubMed Scopus (29) Google Scholar, 41.Vickery B.H. Avnur Z. Cheng Y. Chiou S.S. Leaffer D. Caulfield J.P. Kimmel D.B. Ho T. Krstenansky J.L. J. Bone Miner. Res. 1996; 11: 1943-1951Crossref PubMed Scopus (38) Google Scholar). Substitutions of Arg-35 or Leu-38 in oCRF by alanine (18.Kornreich W.D. Galyean R. Hernandez J.-F. Craig A.G. Donaldson C.J. Yamamoto G. Rivier C. Vale W. Rivier J. J. Med. Chem. 1992; 35: 1870-1876Crossref PubMed Scopus (93) Google Scholar), conversion of the C-terminal carboxamide to a carboxyl group or truncation of the C-terminal dipeptide from oCRF, however, reduced biopotency dramatically (14.Vale W. Spiess J. Rivier C. Rivier J. Science. 1981; 213: 1394-1397Crossref PubMed Scopus (3993) Google Scholar), indicating an essential binding site to be located at the extreme of the C terminus. The existence of two receptor binding sites in peptide ligands of class 2 GPCRs was also suggested by studies using chimeric receptors and peptide ligands, but nothing has been described concerning the structural organization of the ligands (22.Bergwitz C. Gardella T.J. Flannery M.R. Potts Jr., J.T. Kronenberg H.M. Goldring S.R. Jüppner H. J. Biol. Chem. 1996; 271: 26469-26472Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar,23.Stroop S.D. Kuestner R.E. Serwold T.F. Chen L. Moore E.E. Biochemistry. 1995; 34: 1050-1057Crossref PubMed Scopus (92) Google Scholar).We have investigated whether segregated receptor binding sites in CRF do exist and, if so, what role the connector unit between the two binding sites might play. CRF analogues with highly flexible, structurally simplified as well as conformationally stabilized connector units between the two sites were investigated to address these questions.RESULTSTwo CRF receptor subtypes (CRFR-1 and CRFR-2) have been identified in vertebrates; CRFR-1, in contrast to CRFR-2, appears non-selective for human/rat CRF (h/rCRF), ovine CRF (oCRF), and the structurally related CRF analogues, rat urocortin (Uct), carp urotensin, and frog sauvagine. All these peptides stimulate ACTH release in an in vitro pituitary cell assay with similar potency (30.Vaughan J. Donaldson C. Bittencourt J. Perrin M.H. Lewis K. Sutton S. Chan R. Turnbull A.V. Lovejoy D. Rivier C. Rivier J. Sawchenko P.E. Vale W. Nature. 1995; 378: 287-292Crossref PubMed Scopus (1393) Google Scholar). Analogous results were described for CRF-stimulated testosterone production via CRFR-1 from mouse Leydig cells (24.Heinrich N. Meyer M.R. Furkert J. Sasse A. Beyermann M. Bonigk W. Berger H. Endocrinology. 1998; 139: 651-658Crossref PubMed Scopus (35) Google Scholar) (Table I), which was used as the preferred biological assay in this work.Table IPrimary structure and biological potency (testosterone production) of members of the CRF family and chimeric analoguesEC50Ref.nmOvine CRF (oCRF)S Q E P P I S L D L T F H L L R E V L E M T K A D Q L A Q Q A H S N R K L L D I A-NH24.82(24.Heinrich N. Meyer M.R. Furkert J. Sasse A. Beyermann M. Bonigk W. Berger H. Endocrinology. 1998; 139: 651-658Crossref PubMed Scopus (35) Google Scholar)Carp urotensinN D D - - - - I - - - - - - - - N M I - - A R N E N Q R E - - G L - - - Y - - E V-NH22.67(24.Heinrich N. Meyer M.R. Furkert J. Sasse A. Beyermann M. Bonigk W. Berger H. Endocrinology. 1998; 139: 651-658Crossref PubMed Scopus (35) Google Scholar)Frog sauvagine<E G - - - - - - - S L E - - - K M I - I E K Q E K E K Q - - A N - - L - - - T I-NH22.14(24.Heinrich N. Meyer M.R. Furkert J. Sasse A. Beyermann M. Bonigk W. Berger H. Endocrinology. 1998; 139: 651-658Crossref PubMed Scopus (35) Google Scholar)Rat Uct D D - - L - I - - - - - - - - T L L - L A R T Q S Q R E R - E Q - - I I F - S V-NH20.79(24.Heinrich N. Meyer M.R. Furkert J. Sasse A. Beyermann M. Bonigk W. Berger H. Endocrinology. 1998; 139: 651-658Crossref PubMed Scopus (35) Google Scholar)h/rCRF- - E - - - - - - - - - - - - - - - - - - A R - E - - - - - - - - - - - - M E - I-NH22.84(24.Heinrich N. Meyer M.R. Furkert J. Sasse A. Beyermann M. Bonigk W. Berger H. Endocrinology. 1998; 139: 651-658Crossref PubMed Scopus (35) Google Scholar)Chimerae oCRF(1–20)-Uts(21–41)- - - - - - - - - - - - - - - - - - - - - A R N E N Q R E - - G L - - - Y - - E V-NH24.15 oCRF(1–20)-Svg(20–40)- - - - - - - - - - - - - - - - - - - - I E K Q E K E K Q - - A N - - L - - - T I-NH23.85 Uts(1–20)-Svg(20–40)N D D - - - - I - - - - - - - - N M I - I E K Q E K E K Q - - A N - - L - - - T I-NH21.03 Open table in a new tab Structural Simplification of CRFIntramolecular interactions between the N and C termini of long-chain peptide ligands,e.g. CRF (31.Gulyas J. Rivier C. Perrin M. Koerber S.C. Sutton S. Corrigan A. Lahrichi S.L. Craig A.G. Vale W. Rivier J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10575-10579Crossref PubMed Scopus (228) Google Scholar), have been suggested as stabilizing a biologically active conformation. Therefore, amino acid variations within the N and C termini (non-conserved amino acid residues) within the CRF family may function interdependently in such interactions. In order to address the question, we synthesized chimeric peptides, combining the N and C termini of oCRF, urotensine, and sauvagine. The chimeric peptides exhibited a high biological potency (Table I), showing that these amino acid variations are, in fact, not interdependent with respect to stabilization of the biologically active conformation.Assuming an α-helical conformation of CRF to be advantageous for receptor interaction, substitution of those amino acid residues of CRF that are not individually essential for receptor interaction, especially the non-conserved residues within the CRF family, by others with a high helical propensity, such as alanine, should be possible without loss of biopotency. Thus, while retaining arginine-35 and the hydrophobic residues at positions 36/37/38 (leucine), the remaining amino acid residues within the C-terminal portion (residues 22–41) of h/rCRF were replaced by alanine or glutamine residues. This modification yielded an analogue of high biological potency (TableII), demonstrating that the amino acid residues of the middle portion (residues 22–33) of CRF are not individually essential for receptor interaction. From these results, the question arose as to whether this middle portion is essential at all for receptor activation. Combination of the urocortin N terminus (residues 1–19) via Ile-Glu and a highly flexible ε-aminocaproic acid (acp) residue with a mixed C-terminal site (residues 34–41) from members of the CRF family resulted in an analogue that exhibited full receptor activation (full intrinsic activity), but only at increased concentration (reduced biopotency) (Fig.1). Surprisingly, connection of the N-terminal to the C-terminal site via 1, 2, 3, or 4 acp residues produced only a slight difference in biopotency (Table II), showing that the length of the flexible connectors has little effect on agonistic potency. Direct connection of the two sites to yield Uct (1.Romier C. Bernassau J.M. Cambillau C. Darbon H. Protein Eng. 1993; 6: 149-156Crossref PubMed Scopus (50) Google Scholar, 2.Pellegrini M. Royo M. Rosenblatt M. Chorev M. Mierke D.F. J. Biol. Chem. 1998; 273: 10420-10427Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 3.Barden J.A. Kemp B.E. Biochemistry. 1993; 32: 7126-7132Crossref PubMed Scopus (61) Google Scholar, 4.Motta A. Andreotti G. Amodeo P. Strazullo G. Castiglione Morelli M.A. Proteins. 1998; 32: 314-323Crossref PubMed Scopus (59) Google Scholar, 5.Braun W. Wider G. Lee K.H. Wüthrich K. J. Mol. Biol. 1983; 169: 921-948Crossref PubMed Scopus (296) Google Scholar, 6.Clore G.M. Martin S.R. Gronenborn A.M. J. Mol. 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W2170174733 title "A Role for a Helical Connector between Two Receptor Binding Sites of a Long-chain Peptide Hormone" @default.
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