Neuropeptide Y (NPY) is a 36-amino acid neuropeptide that exerts its activity by at least five different receptor subtypes that belong to the family of G-protein-coupled receptors. We isolated an aptamer directed against NPY from a nuclease-resistant RNA library. Mapping experiments with N-terminally, C-terminally, and centrally truncated analogues of NPY revealed that the aptamer recognizes the C terminus of NPY. Individual replacement of the four arginine residues at positions 19, 25, 33, and 35 byl-alanine showed that arginine 33 is essential for binding. The aptamer does not recognize pancreatic polypeptide, a highly homologous Y4 receptor-specific peptide of the gut. Furthermore, the affinity of the aptamer to the Y5 receptor-selective agonist [Ala31,Aib32]NPY and the Y1/Y5 receptor-binding peptide [Leu31,Pro34]NPY was considerably reduced, whereas Y2 receptor-specific NPY mutants were bound well by the aptamer. Accordingly, the NPY epitope was recognized by the Y2 receptor, and the aptamer was highly similar. This Y2 receptor mimicking effect was further confirmed by competition binding studies. Whereas the aptamer competed with the Y2 receptor for binding of [3H]NPY with high affinity, a low affinity displacement of [3H]NPY was observed at the Y1 and the Y5 receptors. Consequently, competition at the Y2 receptor occurred with a considerably lower Ki value compared with the Y1 and Y5 receptors. These results indicate that the aptamer mimics the binding of NPY to the Y2 receptor more closely than to the Y1 and Y5 receptors. Neuropeptide Y (NPY) is a 36-amino acid neuropeptide that exerts its activity by at least five different receptor subtypes that belong to the family of G-protein-coupled receptors. We isolated an aptamer directed against NPY from a nuclease-resistant RNA library. Mapping experiments with N-terminally, C-terminally, and centrally truncated analogues of NPY revealed that the aptamer recognizes the C terminus of NPY. Individual replacement of the four arginine residues at positions 19, 25, 33, and 35 byl-alanine showed that arginine 33 is essential for binding. The aptamer does not recognize pancreatic polypeptide, a highly homologous Y4 receptor-specific peptide of the gut. Furthermore, the affinity of the aptamer to the Y5 receptor-selective agonist [Ala31,Aib32]NPY and the Y1/Y5 receptor-binding peptide [Leu31,Pro34]NPY was considerably reduced, whereas Y2 receptor-specific NPY mutants were bound well by the aptamer. Accordingly, the NPY epitope was recognized by the Y2 receptor, and the aptamer was highly similar. This Y2 receptor mimicking effect was further confirmed by competition binding studies. Whereas the aptamer competed with the Y2 receptor for binding of [3H]NPY with high affinity, a low affinity displacement of [3H]NPY was observed at the Y1 and the Y5 receptors. Consequently, competition at the Y2 receptor occurred with a considerably lower Ki value compared with the Y1 and Y5 receptors. These results indicate that the aptamer mimics the binding of NPY to the Y2 receptor more closely than to the Y1 and Y5 receptors. Neuropeptide Y (NPY) 1The abbreviations used are: NPYneuropeptide YGPCRG-protein-coupled receptorFCSfetal calf serumPBSphosphate-buffered salineDMFdimethylformamideMe2SODMSO, dimethyl sulfoxideFmocfluorenylmethoxycarbonylhPPhuman pancreatic polypeptideAhxaminohexoylAibamino isobutyric acid 1The abbreviations used are: NPYneuropeptide YGPCRG-protein-coupled receptorFCSfetal calf serumPBSphosphate-buffered salineDMFdimethylformamideMe2SODMSO, dimethyl sulfoxideFmocfluorenylmethoxycarbonylhPPhuman pancreatic polypeptideAhxaminohexoylAibamino isobutyric acid is a 36-amino acid neuropeptide and is one of the most conserved peptides during evolution (1.Larhammar D. Regul. Pept. 1996; 65: 165-174Crossref PubMed Scopus (215) Google Scholar, 2.Cerda-Reverter J.M. Larhammar D. Biochem. Cell Biol. 2000; 78: 371-392Crossref PubMed Scopus (189) Google Scholar). It shows high homology to the other members of the pancreatic polypeptide hormone family, namely pancreatic polypeptide and peptide YY (3.Glover I.D. Barlow D.J. Pitts J.E. Wood S.P. Tickle I.J. Blundell T.L. Tatemoto K. Kimmel J.R. Wollmer A. Strassburger W. Zhang Y.S. Eur. J. Biochem. 1984; 142: 379-385Crossref PubMed Scopus (138) Google Scholar, 4.Conlon J.M. Bjornholm B. Jorgensen F.S. Youson J.H. Schwartz T.W. Eur. J. Biochem. 1991; 199: 293-298Crossref PubMed Scopus (46) Google Scholar). NPY is widely distributed in the central and peripheral nervous system (5.Chronwall B.M. DiMaggio D.A. Massari V.J. Pickel V.M. Ruggiero D.A. O'Donohue T.L. Neuroscience. 1985; 15: 1159-1181Crossref PubMed Scopus (1024) Google Scholar). It modulates a variety of physiological processes such as the central regulation of food intake (6.Morley J.E. Hernandez E.N. Flood J.F. Am. J. Physiol. 1987; 253: R516-R522Crossref PubMed Google Scholar, 7.Loftus T.M. Jaworsky D.E. Frehywot G.L. Townsend C.A. Ronnett G.V. Lane M.D. Kuhajda F.P. Science. 2000; 288: 2379-2381Crossref PubMed Scopus (826) Google Scholar), vasoconstriction, memory retention (8.Flood J.F. Hernandez E.N. Morley J.E. Brain Res. 1987; 421: 280-290Crossref PubMed Scopus (207) Google Scholar, 9.Heilig M. Murison R. Regul. Pept. 1987; 19: 221-231Crossref PubMed Scopus (113) Google Scholar), and regulation of circadian rhythm (10.Albers H.E. Ferris C.F. Leeman S.E. Goldman B.D. Science. 1984; 223: 833-835Crossref PubMed Scopus (143) Google Scholar, 11.Hall A.C. Earle-Cruikshanks G. Harrington M.E. Eur. J. Neurosci. 1999; 11: 3424-3432Crossref PubMed Scopus (25) Google Scholar, 12.Calza L. Giardino L. Zanni M. Velardo A. Parchi P. Marrama P. Regul. Pept. 1990; 27: 127-137Crossref PubMed Scopus (50) Google Scholar). NPY transmits its activity by at least three receptor subtypes (Y1, Y2, and Y5), which all belong to the large family of G-protein-coupled receptors (GPCR). They are coupled to Gi proteins and accordingly inhibit adenylate cyclase (13.Bischoff A. Michel M.C. Trends Pharmacol. Sci. 1999; 20: 104-106Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 14.Ingenhoven N. Beck-Sickinger A.G. Curr. Med. Chem. 1999; 6: 1055-1066PubMed Google Scholar). The different receptor subtypes are distributed heterogeneously in various tissues in the central nervous system and the periphery and so far it is not understood how NPY selectively activates a particular receptor pathway. neuropeptide Y G-protein-coupled receptor fetal calf serum phosphate-buffered saline dimethylformamide DMSO, dimethyl sulfoxide fluorenylmethoxycarbonyl human pancreatic polypeptide aminohexoyl amino isobutyric acid neuropeptide Y G-protein-coupled receptor fetal calf serum phosphate-buffered saline dimethylformamide DMSO, dimethyl sulfoxide fluorenylmethoxycarbonyl human pancreatic polypeptide aminohexoyl amino isobutyric acid In general, structural insight how GPCRs are activated is scarce. Only recently the first crystal structure of a G-protein-coupled receptor, rhodopsin, has been solved and provided insight into the molecular mechanism of GPCR activation (15.Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le Trong I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Crossref PubMed Scopus (5004) Google Scholar). However, these structural analyses are highly advanced and cannot be considered routine. Furthermore, because the ligand is covalently bound in rhodopsin, the process of ligand approaching is still unknown. Accordingly, biochemical methods that facilitate our understanding of GPCRs are urgently required. All current information on structure/activity or structure/affinity relationships of NPY and its G-protein-coupled receptor subtypes have been obtained by indirect methods such as site-directed mutagenesis in transmembrane regions and extracellular loops of the receptors (16.Sautel M. Rudolf K. Wittneben H. Herzog H. Martinez R. Munoz M. Eberlein W. Engel W. Walker P. Beck-Sickinger A.G. Mol. Pharmacol. 1996; 50: 285-292PubMed Google Scholar) and replacement of amino acid residues in NPY (17.Beck-Sickinger A.G. Wieland H.A. Wittneben H. Willim K.D. Rudolf K. Jung G. Eur. J. Biochem. 1994; 225: 947-958Crossref PubMed Scopus (172) Google Scholar, 18.Cabrele C. Langer M. Bader R. Wieland H.A. Doods H.N. Zerbe O. Beck-Sickinger A.G. J. Biol. Chem. 2000; 275: 36043-36048Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 19.Cabrele C. Beck-Sickinger A.G. J. Pept. Sci. 2000; 6: 97-122Crossref PubMed Scopus (167) Google Scholar). Modified analogues and recombinant receptors are excellent tools to study ligand-receptor interaction in vitro or in cell lines. However, they cannot be applied to characterize interactions in vivo because the in vivo expression of each modification would require an individual transgenic animal. To circumvent these problems, selective low molecular weight antagonists have been developed (20.Rudolf K. Eberlein W. Engel W. Wieland H.A. Willim K.D. Entzeroth M. Wienen W. Beck-Sickinger A.G. Doods H.N. Eur. J. Pharmacol. 1994; 271: R11-R13Crossref PubMed Scopus (362) Google Scholar, 21.Söll R.M. Dinger M.C. Lundell I. Larhammer D. Beck-Sickinger A.G. Eur. J. Biochem. 2001; 268: 2828-2837Crossref PubMed Scopus (79) Google Scholar). These small organic molecules, however, do not necessarily bind to the receptor in a way similar to the endogenous ligand. For example, only partial overlapping of the binding site has been shown for BIBP3226, and in the substance P and angiotensin systems completely different binding sites for agonist and antagonist were identified (22.Elling C.E. Nielsen S.M. Schwartz T.W. Nature. 1995; 374: 74-77Crossref PubMed Scopus (160) Google Scholar). Mapping of the Y1 receptor with anti-receptor antibodies has provided some insight into the hypothetical binding topology of NPY (23.Wieland H.A. Eckard C.P. Doods H.N. Beck-Sickinger A.G. Eur. J. Biochem. 1998; 255: 595-603Crossref PubMed Scopus (21) Google Scholar); however, the use of polyclonal antibodies directed against the 16–20-mer receptor segments only provided limited structural information. We therefore chose an alternative approach to characterize the interaction of NPY with its receptors. Here we describe the selection of that recognize NPY with The binding of the aptamer to NPY in to a of NPY or antagonists was we the of the aptamer as a and selective NPY by NPY from its receptors Y2, and The amino were obtained from and The are as for and for for and and for The was obtained from were by peptide on a peptide amino were by a with of amino in The was with in for for and for acid of the peptides are in The of the were by of the amino of NPY. to of the truncated NPY from the they were The was as during was by of and were in of to the of were The was by the with and at The peptides were from the with a of from by with and from of the peptides was by and high of aptamer was with a of peptide of NPY and the amino acid residues of and truncated NPY are on the centrally truncated amino acid residues were by an acid compared with the NPY are in are in to the interaction with the NPY of the NPY of the NPY compared with the NPY The amino acid residues which are essential for the RNA are and in in a In and the amino acid residues of and truncated NPY are on the centrally truncated amino acid residues were by an acid compared with the NPY are in are in to the interaction with the NPY of the NPY of the NPY compared with the NPY The amino acid residues which are essential for the RNA are and in The RNA was by in vitro from a as indicate the and the the as A. M. M. Sci. A. PubMed Scopus Google Scholar). The in vitro was in a by the with and and at for at in and The NPY was on in at The was In the first of the RNA were in binding for at and for on in the of to structure To with affinity for a selection was in The of the were with the NPY for at by The was with of binding RNA was with and by and was as In the NPY was from to and were in the of and fetal calf the was with and and as M. J. Am. Chem. 1994; Scopus Google Scholar). were with of each were with peptide from to in of binding of for at was to the retention of the peptide on the and were as The of RNA on each was by For RNA binding were D. C. N. 1994; PubMed Scopus Google Scholar), The various NPY were on The RNA was with the coupled peptides of at the was into a and the were by with of binding and the of the RNA was by For competition of interaction on cell the receptor, cell and with the receptor were as C.P. C. Wieland H.A. Beck-Sickinger A.G. 2001; 6: Scopus Google Scholar, B. Wieland H.A. Willim K.D. Beck-Sickinger A.G. J. Pept. Sci. 1995; PubMed Scopus Google Scholar). were at in phosphate-buffered saline and or in the of in essential serum and and for the was in the and to of cell were with of in and a of to a of at was by for at at The were with in and with and the was To for and to the binding of NPY to the the NPY a of NPY was were three to four in of were with the the Y whereas at and to with of of aptamer. is as the of Ki were to the of and Biochem. Pharmacol. PubMed Scopus Google Scholar) the is the of competed [3H]NPY which was for Y1 and Y5 and for Y2 receptors. In vitro selection of has been used to or RNA with high affinity for L. B. O. M. Biochem. 1995; PubMed Scopus Google Scholar, Biochem. 1999; PubMed Scopus Google Scholar, M. G. Curr. 1999; Google Scholar, M. G. M. Chem. Res. 2000; PubMed Scopus Google Scholar). To be in cell we nuclease-resistant by in which all residues were by C. T. D. C. 15: PubMed Scopus Google Scholar, B.A. 15: PubMed Scopus Google Scholar, J. J. L. N. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, D. C. B.D. N. Chem. Biol. 1995; Full Text PDF PubMed Scopus Google Scholar, D. C. N. C. G. P. R. D. N. 1995; PubMed Scopus (189) Google Scholar). This modification is to against the of serum so that such be applied in the of in which RNA would Biochem. 1995; PubMed Scopus Google O. W. J. PubMed Scopus Google Scholar). the of NPY has recently been shown C. Beck-Sickinger A.G. J. Pept. Sci. 2000; 6: 97-122Crossref PubMed Scopus (167) Google Scholar) to be essential for its interaction with all receptor we NPY on to of the C terminus an RNA with a of different in which all residues were by were in of selection and shows the binding of NPY to the and the from by Whereas the at an NPY of retention of the was the affinity of the for NPY at least of compared with the and of from revealed one and a family of and three that the was considerably of RNA from the For each only the is the of The and residues are shown as C or in an on the in or in to the most We of from each family for binding to NPY by experiments shown in from a of the NPY binding aptamer. a of the most bound with showed only or affinity and and not we the RNA for binding to NPY and the binding of to a of NPY with We affinity in that NPY with high for the RNA and the and experiments were with of and the to truncated of and human pancreatic peptide of the were were were by an acid of were in the of and by binding and as of the were were were by an acid of in a were in the of and by binding and as We to the epitope on NPY recognized by the aptamer by its binding to a variety of truncated analogues of NPY with that of the we the binding on NPY for with the aptamer. The results are in and NPY central of the and C do not interaction with the which the amino acid and shows interaction with and and which is to is recognized by with the of as NPY. The is for and In binding to NPY with the of NPY by all of its receptor subtypes which do not bind NPY the amino acid C. Beck-Sickinger A.G. J. Pept. Sci. 2000; 6: 97-122Crossref PubMed Scopus (167) Google Scholar). not affinity to peptide only NPY bound to receptor not In the well centrally truncated Y2 receptor-selective in which amino acid residues are by an shows of the binding to in the of as NPY. We a of analogues in which arginine residues that are to an for interaction were in to the of individual amino acid residues for All four of NPY were by These residues were because we that arginine are to to the binding affinity and the NPY C The not aptamer binding whereas a of binding was observed for the and the and In arginine 33 was for the peptide completely its affinity to the aptamer To for of the aptamer the pancreatic polypeptide family, its binding to the human pancreatic polypeptide was to it does not with the neuropeptide Y receptors of the Y4 for which it is the endogenous ligand Walker J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, I. A.G. D.A. D. Larhammar D. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). Whereas Y4 with to Y5 receptors with considerably lower affinity, whereas the Y1 and Y2 receptors are not recognized at all Walker J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, I. A.G. D.A. D. Larhammar D. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). affinity of to the aptamer was that different peptide To further the receptor and aptamer we binding to other analogues that have been shown to selectively with some Y receptor For example, the Y5 receptor selective [Ala31,Aib32]NPY C. Langer M. Bader R. Wieland H.A. Doods H.N. Zerbe O. Beck-Sickinger A.G. J. Biol. Chem. 2000; 275: 36043-36048Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar) was considerably bound by whereas the Y1/Y5 receptor selective agonist [Leu31,Pro34]NPY was not recognized by the aptamer at all the replacement of by to of affinity at the Y2 the binding of the aptamer to all NPY that of the Y2 to NPY in vitro and shows for NPY analogues that are bound by the Y2 We therefore and how the in vitro binding of is in a in which the aptamer with various receptor subtypes for NPY binding. We used cell that the different receptor subtypes as the cell and the and the For competition at the Y5 receptor, with the receptor were experiments were by and of NPY was by in the of of a the was in the of of the RNA which not bind NPY. The results are in In general, shows of binding of NPY to all receptor subtypes However, the binding of the Y1 and Y5 receptors from of the Y2 The competition and Y2 receptors for NPY a competition that with the Y2 receptor for NPY binding a of of the competition is observed In the competition at the Y1 and Y5 receptors a completely different and of receptor affinity is only obtained at the we of NPY competition by the aptamer for the various receptor subtypes and Ki the of receptor interaction with an Ki of which is at a lower than for the Y1 and Y5 Y2 shows the affinity to NPY C. Beck-Sickinger A.G. J. Pept. Sci. 2000; 6: 97-122Crossref PubMed Scopus (167) Google and Ki from the competition in cell of were from the in with the the of for NPY binding to individual receptor subtypes are obtained from the Y1 receptor Y2 receptor Y5 receptor of to the of and of were from the in with the the of for NPY binding to individual receptor subtypes are obtained from the Y1 receptor N. Beck-Sickinger A.G. J. Res. PubMed Scopus (25) Google Scholar), Y2 receptor N. Eckard C.P. Beck-Sickinger A.G. 1999; PubMed Scopus Google Scholar), Y5 receptor C. G. Michel J. H. A. J. Physiol. Pharmacol. 2000; 78: PubMed Google of to the of and Biochem. Pharmacol. PubMed Scopus Google Scholar). in a We describe of RNA that bind to neuropeptide Y with and These binding well with other in with peptide For example, the aptamer its with a of the substance with the with and the aptamer with W. Sci. A. 1996; PubMed Scopus Google Scholar, D. M. L. 1995; PubMed Scopus Google Scholar, D.A. Sci. A. PubMed Scopus Google Scholar, M. W. M. Sci. A. 1999; PubMed Scopus Google Scholar). We have applied a of peptides shown to as selective NPY receptor and we binding to the aptamer. with high affinity to peptide This peptide is bound by the Y2 receptor with an of whereas the Y1 receptor only binding affinity to of C. Beck-Sickinger A.G. J. Pept. Sci. 2000; 6: 97-122Crossref PubMed Scopus (167) Google Scholar). The in was in which four amino are to the NPY an amino acid a of NPY that with an of to Y2 whereas Y1 and Y5 receptors are bound with considerably affinity with an and to of and and the does not a effect the (23.Wieland H.A. Eckard C.P. Doods H.N. Beck-Sickinger A.G. Eur. J. Biochem. 1998; 255: 595-603Crossref PubMed Scopus (21) Google A.G. H. W. Jung G. J. Res. PubMed Scopus Google Scholar). shown in and centrally truncated is well bound by the aptamer and Y2, to bind the This is a that the aptamer recognizes a of NPY. It has been that the of NPY that is bound selectively by Y2 a structure in which the and C are in to each the C terminus a whereas the terminus a structural for of the of the C terminus C. Beck-Sickinger A.G. J. Pept. Sci. 2000; 6: 97-122Crossref PubMed Scopus (167) Google Scholar). more is that [Leu31,Pro34]NPY is not recognized by the Y2 receptor and by the aptamer This is widely used to Y2 and of NPY. The Y5 receptor-selective ligand [Ala31,Aib32]NPY and the Y4 peptide bind only or not at all to the aptamer and the Y2 These indicate that shows only affinity to analogues that are bound well by the NPY receptors or Accordingly, shows similar binding as for the Y2 receptor for a of with the binding of NPY to its however, is the that the arginine residues are essential for receptor and are essential for NPY binding by all receptors. of and by residues the affinity for Y1 receptor binding than and whereas the affinity of the Y5 receptor for the is C.P. C. Wieland H.A. Beck-Sickinger A.G. 2001; 6: Scopus Google Scholar). shown in of the aptamer the whereas mutants and to the C terminus showed binding and the most for binding to be one of the positions to be conserved the pancreatic polypeptide all to these In the binding that applied NPY that are for individual NPY receptor subtypes that NPY binding by the aptamer is similar to that of the Y2 This in the that NPY in different in the that the Y2 receptor-specific NPY from the that is for the activation of the Y1 or Y5 receptors. The were by analogues of NPY. We chose the because residues have been identified at the However, at the Y1 and Y5 of one or considerably receptor affinity, whereas at the Y2 receptor segments still are to Accordingly, the Y2 receptor of NPY have been by the of NPY for aptamer and the of the to that is for high receptor affinity at the Y1 and Y5 receptor The competition experiments in which NPY binding to the Y2, or Y5 receptor subtypes was at different aptamer showed a for the Y2 Y1 and Y5 and showed the Ki at the Y2 receptor, which was and lower than the Ki obtained at the Y1 and Y5 This competition was as the not to at receptor these be shown by NPY in different in In the of to effect is at of that the binding of NPY to the Y2 receptor is whereas only displacement is recognized at Y1 and Y5 receptors. the aptamer a similar of NPY as the Y2 receptor a of [3H]NPY does not bind to the Y2 receptors at whereas more than of binding is still at Y1 and Y5 receptors the affinity of NPY to Y1 and Y5 receptors. This be to a of the of the ligand in the Y2 structure or to the of at In the would bind to further NPY at RNA In however, the that the aptamer different of the peptide ligand. Whereas is for and receptors and has been for antibodies as it has so far not been for Accordingly, for the first we identified a that mimics the Y2 NPY at the Y2 receptor with the affinity and than at Y1 and Y5 receptors as by the different in affinity, competition was more at Y2 which to the high of with to However, the of NPY for Y2 the more of Y2 receptor-binding sites be we on the of the at the receptor, is the of It is that the aptamer recognizes NPY by an binding as observed for binding of for the different of of NPY binding to receptors Y2 and Consequently, different NPY recognized by different at the aptamer. structural of the are to these These with the of NPY analogues that the aptamer mimics the binding of the Y2 receptor more closely than that of the Y1 and Y5 receptors. the different of NPY are by the Y2 receptor and are recognized by the or they are or by the aptamer cannot be at the that neuropeptide Y different in vivo the for Y2 receptor from the one that is bound by the Y1 or Y5 the of the in in vivo experiments as well as in In the Y2 and Y5 receptors are selectively the interaction at the Y2 receptor of the the to the understanding of the Y2 receptor in that Y receptor We and for and for The of is
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