A Novel Form of Neurotensin Post-translationally Modified by Arginylation

Abstract

A novel bioactive form of neurotensin post-translationally modified at a Glu residue was isolated from porcine intestine. Purification of the peptide was guided by detection of intracellular Ca2+ release in SK-N-SH neuroblastoma cells. Using high resolution accurate mass analysis on an ion trap Fourier transform mass spectrometer, the post-translational modification was identified as arginine linked to the γ-carboxyl of Glu via an isopeptide bond, and we named the newly identified peptide “arginylated neurotensin” (R-NT, N-(neurotensin-C5–4-yl)arginine). Although arginylation is a known modification of N-terminal amino groups in proteins, its presence at a Glu side chain is unique. The finding places neurotensin among the few physiologically active peptides that occur both in post-translationally modified and unmodified forms. Pharmacologically, we characterized R-NT for its ligand activity on three known neurotensin receptors, NTR1, -2, and -3, and found that R-NT has similar pharmacological properties to those of neurotensin, however, with a slightly higher affinity to all three receptors. We expressed the intracellular receptor NTR3 as a soluble protein secreted into the cell culture medium, which allowed characterization of its R-NT and neurotensin binding properties. The creation of soluble NTR3 also provides a potential tool for neutralizing neurotensin action in vivo and in vitro. We have shown that SK-N-SH neuroblastoma cells express NTR1 and NTR3 but not NTR2, suggesting that the Ca2+ mobilization elicited by R-NT is via NTR1. A novel bioactive form of neurotensin post-translationally modified at a Glu residue was isolated from porcine intestine. Purification of the peptide was guided by detection of intracellular Ca2+ release in SK-N-SH neuroblastoma cells. Using high resolution accurate mass analysis on an ion trap Fourier transform mass spectrometer, the post-translational modification was identified as arginine linked to the γ-carboxyl of Glu via an isopeptide bond, and we named the newly identified peptide “arginylated neurotensin” (R-NT, N-(neurotensin-C5–4-yl)arginine). Although arginylation is a known modification of N-terminal amino groups in proteins, its presence at a Glu side chain is unique. The finding places neurotensin among the few physiologically active peptides that occur both in post-translationally modified and unmodified forms. Pharmacologically, we characterized R-NT for its ligand activity on three known neurotensin receptors, NTR1, -2, and -3, and found that R-NT has similar pharmacological properties to those of neurotensin, however, with a slightly higher affinity to all three receptors. We expressed the intracellular receptor NTR3 as a soluble protein secreted into the cell culture medium, which allowed characterization of its R-NT and neurotensin binding properties. The creation of soluble NTR3 also provides a potential tool for neutralizing neurotensin action in vivo and in vitro. We have shown that SK-N-SH neuroblastoma cells express NTR1 and NTR3 but not NTR2, suggesting that the Ca2+ mobilization elicited by R-NT is via NTR1. Orphan G-protein-coupled receptors are present in established cell lines and may serve as screening systems to find their endogenous ligands. In the present study, SK-N-SH neuroblastoma cells were utilized to screen for novel peptides in porcine duodenal extracts using intracellular Ca2+ release as a reporter system. However, SK-N-SH cells also express other G-protein-coupled receptors and may respond to a number of neuropeptides. The bioactivities were followed by peptide purification and subsequent structural characterization, yielding peptides of potential physiological significance, among them a unique form of neurotensin. Neurotensin is a 13-residue peptide that was first isolated from bovine hypothalamus during purification of substance P and was named from its neuronal localization and a vasodilatation after local administration (1Carraway R. Leeman S.E. J. Biol. Chem. 1973; 248: 6854-6861Abstract Full Text PDF PubMed Google Scholar). Neurotensin is localized to both the central nervous system and peripheral tissues mainly in the gastrointestinal tract (2Kitabgi P. Carraway R. Leeman S.E. J. Biol. Chem. 1976; 251: 7053-7058Abstract Full Text PDF PubMed Google Scholar). In the central nervous system, neurotensin plays the role of a neurotransmitter or neuromodulator of dopamine transmission and of anterior pituitary hormone secretion (3Vincent J.P. Mazella J. Kitabgi P. Trends Pharmacol. Sci. 1999; 20: 302-309Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar). It also shows potent hypothermic and analgesic effects in the brain (4Bissette G. Nemeroff C.B. Loosen P.T. Prange Jr., A.J. Lipton M.A. Nature. 1976; 262: 607-609Crossref PubMed Scopus (268) Google Scholar). Neurotensin has been implicated in the pathophysiology of several brain diseases, such as schizophrenia and Huntington and Parkinson diseases (5Garver D.L. Bissette G. Yao J.K. Nemeroff C.B. Am. J. Psychiatry. 1991; 148: 484-488Crossref PubMed Scopus (104) Google Scholar). In the periphery, neurotensin acts as a local hormone exerting a paracrine and endocrine modulation of the digestive tract (3Vincent J.P. Mazella J. Kitabgi P. Trends Pharmacol. Sci. 1999; 20: 302-309Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar). It also stimulates the growth of various gastrointestinal tissues, including the pancreas and the gastric antrum (6Feurle G.E. Muller B. Rix E. Gut. 1987; 28: 19-23Crossref PubMed Scopus (77) Google Scholar). Three subtypes of neurotensin receptors (NTRs) 2The abbreviations used are: NTRneurotensin receptorCPYcarboxypeptidase YCTIPconcentrate of thermostable intestinal peptidesCVcolumn volume(s)MALDImatrix-assisted laser desorption ionizationTOFtime-of-flightMSmass spectrometryR-NTarginylated neurotensin (N-(neurotensin-C5–4-yl)arginine)HPLChigh performance liquid chromatographyTFAtrifluoroacetic acidFTFourier transform. have been reported. Neurotensin receptor 1 (NTR1) (7Tanaka K. Masu M. Nakanishi S. Neuron. 1990; 4: 847-854Abstract Full Text PDF PubMed Scopus (487) Google Scholar) and receptor 2 (NTR2) (8Chalon P. Vita N. Kaghad M. Guillemot M. Bonnin J. Delpech B. Le Fur G. Ferrara P. Caput D. FEBS Lett. 1996; 386: 91-94Crossref PubMed Scopus (242) Google Scholar, 9Mazella J. Zsurger N. Navarro V. Chabry J. Kaghad M. Caput D. Ferrara P. Vita N. Gully D. Maffrand J.P. Vincent J.P. J. Biol. Chem. 1998; 273: 26273-26276Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar, 10Vita N. Oury-Donat F. Chalon P. Guillemot M. Kaghad M. Bachy A. Thurneyssen O. Garcia S. Poinot-Chazel C. Casellas P. Keane P. Le Fur G. Maffrand J.P. Soubrie P. Caput D. Ferrara P. Eur. J. Pharmacol. 1998; 360: 265-272Crossref PubMed Scopus (119) Google Scholar) are two related G-protein-coupled receptors sharing 43% amino acid sequence identity and 64% structural identity. Neurotensin receptor 3 (NTR3) (9Mazella J. Zsurger N. Navarro V. Chabry J. Kaghad M. Caput D. Ferrara P. Vita N. Gully D. Maffrand J.P. Vincent J.P. J. Biol. Chem. 1998; 273: 26273-26276Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar) is a single transmembrane receptor previously characterized as sortilin (11Petersen C.M. Nielsen M.S. Nykjaer A. Jacobsen L. Tommerup N. Rasmussen H.H. Roigaard H. Gliemann J. Madsen P. Moestrup S.K. J. Biol. Chem. 1997; 272: 3599-3605Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar). NTR1 has been reported to regulate the turning behavior in mice (12Dubuc I. Sarret P. Labbe-Jullie C. Botto J.M. Honore E. Bourdel E. Martinez J. Costentin J. Vincent J.P. Kitabgi P. Mazella J. J. Neurosci. 1999; 19: 503-510Crossref PubMed Google Scholar). For signal transduction, NTR1 is coupled with Ca2+ signaling. Activation of NTR1 by neurotensin leads to intracellular Ca2+ mobilization. NTR2 was discovered based on its homology to NTR1. Recent studies show that NTR2 is involved in neurotensin-induced analgesia (12Dubuc I. Sarret P. Labbe-Jullie C. Botto J.M. Honore E. Bourdel E. Martinez J. Costentin J. Vincent J.P. Kitabgi P. Mazella J. J. Neurosci. 1999; 19: 503-510Crossref PubMed Google Scholar). NTR2 has been shown to bind neurotensin and can be activated by neurotensin when NTR2 is expressed in Xenopus oocyte. However, unlike NTR1, which stimulates Ca2+ in mammalian cells, recombinant NTR2, when expressed in HEK293 cells, does not activate Ca2+ signaling in response to neurotensin stimulation. NTR3 normally exists in intracellular compartments and not on the cell surface. NTR3 expression has been reported in rat adipocytes in vesicles containing the glucose transporter GLUT4 and mouse neurons (13Morris N.J. Ross S.A. Lane W.S. Moestrup S.K. Petersen C.M. Keller S.R. Lienhard G.E. J. Biol. Chem. 1998; 273: 3582-3587Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Insulin or neurotensin stimulation of rat adipocytes or mouse neurons, respectively, has been reported to trigger the translocation of NTR3 from the intracellular compartment to the cell surface. So far, very little is known about the physiological function and the signal transduction pathway of NTR3. neurotensin receptor carboxypeptidase Y concentrate of thermostable intestinal peptides column volume(s) matrix-assisted laser desorption ionization time-of-flight mass spectrometry arginylated neurotensin (N-(neurotensin-C5–4-yl)arginine) high performance liquid chromatography trifluoroacetic acid Fourier transform. Earlier structural analyses of hormonal peptides have shown that several peptides are post-translationally modified. The most frequently occurring post-translational modification is C-terminal amidation found in peptide hormones such as secretin (14Jorpes J.E. Mutt V. Magnusson S. Steele B.B. Biochem. Biophys. Res. Commun. 1962; 9: 275-279Crossref PubMed Scopus (44) Google Scholar, 15Mutt V. Jorpes J.E. Magnusson S. Eur. J. Biochem. 1970; 15: 513-519Crossref PubMed Scopus (140) Google Scholar), cholecystokinin (16Mutt V. Jorpes J.E. Eur. J. 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We in from porcine the structural and pharmacological of neurotensin post-translationally with arginine We have shown that has a potent two neurotensin and both were also isolated and recombinant and acid were from acid was from and in at Y was in at and in at the and used R-NT was by Ca2+ cells were and at in The culture was and the cells were for 1 at with in modified were and in modified and receptor were used as and the were by an and to the with cells. were during 2 and as a of The ligand and intracellular Ca2+ were on a Purification of is shown by the in The was as a side from the purification of secretin and its V. Jörnvall H. Mutt V. Sillard R. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) after chromatography on with and a of were on a column using acid and on and systems The from 1 and 2 was in of after which was on a column using a system of A containing and 2 with a of in column of were were with to to and on the column with a system of A at and with a with of The was by three of were with 2 of and on using a system of and and a of with of This chromatography was three and two active were and of and is in the and were with and on the column with the system as in the The was in and were was with and on using the system as in the and a of was to structural Purification of and from the chromatography 1 and with the activity as were and with to The was on the column using the system as A of was and were was with of and on column 3 using a system of and A of was and were was to structural Purification of for was as previously E. Norberg Å. V. Nepomuceno D. Lovenberg T.W. Sillard R. Jörnvall H. Sci. 1999; PubMed Scopus Google Scholar) The from the and at E. Norberg Å. V. Nepomuceno D. Lovenberg T.W. Sillard R. Jörnvall H. Sci. 1999; PubMed Scopus Google Scholar) was in of the was to with 2 and a The liquid was into and the with of peptides were with a of in the in and in were The activity was in and for the The active were with and the was to The was the column and with a system of The peptides were with a of in the The active were were the The used were and The peptides were with a of The active from 2 were for the The was with and the The system was the as in the were with a of The active were by matrix-assisted laser desorption ionization mass spectrometry and was by for The from after was and The peptides were 3 in were with a of in the was in The most active and were for the purification on a as with the system. The was with a in was and mass were on a from the in were on a with of and of and allowed to at A was in yielding at and at The were using the for of and of mass was ionization time-of-flight were on an ionization time-of-flight mass were on a mass with a as previously V. Eriste E. Jonsson A.P. Efendic S. Jörnvall H. Sillard R. Biochem. Biophys. 2001; PubMed Scopus Google Scholar). For C-terminal sequence analysis V. Bergman Jörnvall H. Sillard R. Chem. 1997; Scopus Google Scholar), from the were on a with and allowed to at of and of was to the and allowed to at N-terminal sequence analysis was on an of mass were on a The is a and of two mass a ion and a Fourier transform ion The ion trap is characterized by high also in the of The Fourier transform ion a mass resolution of at with a of 1 mass of of Neurotensin in the from SK-N-SH cells was with to was from 2 of using with of to the The was at for and at for 3 and on The was and 2 of was by using the in The of 1 of 3 of and were from at for of for for and for the a analysis was were also on a The NTR1, and NTR2, and and sortilin and of NTR1, NTR2, and NTR1 was by using and and brain as the NTR2 was from the brain using two and the NTR3 was using and NTR1, -2, and were into the mammalian expression were to the sequence of Neurotensin Using NTR1 and NTR2, the binding were as previously C. J. S. B. C. N. Sillard R. Lovenberg T.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) with the and as and were expressed in cells using as the after the cell were and used for binding in the presence of of neurotensin or R-NT as the The used for NTR1 and NTR2 binding is For which was expressed as a soluble form with a at the was expressed in cells. Three after the was and the soluble binding was as previously C. G. N. 1997; PubMed Scopus Google Scholar). the from or NTR3 cells, were for at to the cell The were in at 1 was to at a of of neurotensin or R-NT were to the respectively, as the of was to to the The were on a at The receptor with the which was by was by The which the were and the containing the were in a of and and were expressed in HEK293 cells or in with Steele Proc. Natl. Acad. Sci. U. S. A. 1991; PubMed Scopus Google Scholar) using as the after cells were from the cell culture using of and in culture as Ca2+ was to the cells, of neurotensin or R-NT were used to the intracellular Ca2+ which was using a of was as and shown in 1 and The peptide from of the which at was active in the Ca2+ release the peptide was by and ionization time-of-flight which an of a higher mass that of neurotensin. N-terminal sequence analysis was several that was The peptide was to C-terminal sequence analysis with at and which mass and for the two C-terminal with for the two with higher of an amino acid with with the C-terminal sequence is and is in with the neurotensin The peptide was on the with after which analysis by two of and The is in with the mass of the C-terminal neurotensin but the is higher that for the that a modification is the N-terminal and that the peptide may arginine and to an accurate mass of the were on an a ion trap Fourier transform the mass of the peptide was with and were in the mass that the modified peptide is of for unmodified neurotensin, suggesting that the modification a of that may be mass of the peptide was from ion of a single and was from the of the of the peptide and a The were found to be very of the of ion of the of post-translationally modified neurotensin and the of neurotensin modified by arginylation the into A mass resolution of was at the the both the peptide and its N-terminal were to analysis on a and on a of the ion of the peptide in the and suggesting that the modification is at analysis of the N-terminal with the and the The and were found to be by the mass of the modification and that the modification was at the Glu residue the the the sequence of the A with the was on the at a higher mass A similar for was but most of the were from we that the post-translational modification is at For accurate from mass and were with the for unmodified neurotensin. and yielding an of for the at a mass of two and with mass at and The of with the the as the which to the of an arginine all show that neurotensin is post-translationally modified by arginine coupled to the γ-carboxyl of by an isopeptide of from the purification shown in and were for on SK-N-SH cells. was found in two at The active was to N-terminal sequence analysis by and to C-terminal analysis by The a sequence to neurotensin by amino acid analysis for the two C-terminal and the peptide was identified as of from the purification were to N-terminal sequence and a sequence for was This sequence to that of neurotensin at The was also to which for the This is in with the of with the sequence The peptide was to C-terminal sequence analysis by with and subsequent which and for the two C-terminal the C-terminal for The of the peptides isolated in the present are in of the peptides acid neurotensin, in a we were for peptides that intracellular Ca2+ release in SK-N-SH cells. A number of active were in the and the were followed several peptides were and In the of was also and Ca2+ neurotensin activity in SK-N-SH cells R-NT Ca2+ in SK-N-SH finding that R-NT is of Ca2+ response in SK-N-SH cells, R-NT was and in Ca2+ mobilization in with neurotensin using SK-N-SH and HEK293 cells. The show that similar to neurotensin, stimulates Ca2+ mobilization in SK-N-SH cells, with an of but not in HEK293 cells In the neurotensin Ca2+ response in SK-N-SH cells with an of SK-N-SH NTR1 and NTR3 but NTR2 which neurotensin receptor SK-N-SH cells was from SK-N-SH cells and was to the neurotensin receptor The show that SK-N-SH cells express NTR1 and NTR3 In the NTR2 was found in SK-N-SH cells, suggesting that the Ca2+ mobilization by R-NT in SK-N-SH cells is via NTR1, NTR3 is a single transmembrane receptor and not known to with Ca2+ signaling. R-NT NTR1, NTR2, and NTR3 at R-NT NTR1, NTR2, or in with neurotensin, was used as the in binding using as the and recombinant neurotensin receptors. The show that R-NT and neurotensin bind NTR1 at of 1 and 2 R-NT and neurotensin bind NTR2 with of and with NTR1 and NTR2, R-NT and neurotensin bind NTR3 at affinity with of and R-NT and Neurotensin Ca2+ in but HEK293 which neurotensin receptor R-NT R-NT in with neurotensin was on HEK293 cells recombinant NTR1 or The that both R-NT and neurotensin Ca2+ mobilization in cells NTR1 with of and However, neurotensin R-NT Ca2+ response in HEK293 cells NTR2 In study, we have isolated and characterized a novel post-translationally modified neurotensin and shown that is The structural analysis that the peptide is modified by arginylation of the side chain of such post-translational modification of peptides or has not been previously However, arginylation of N-terminal of is a known occurring in all cells and a function in cell H. A. Biophys. 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R-NT has very similar on all three neurotensin receptors with neurotensin but with higher In the Ca2+ mobilization we higher for R-NT and neurotensin in HEK293 cells NTR1 in SK-N-SH cells NTR1 for R-NT and for The for R-NT and neurotensin from the Ca2+ in HEK293 cells NTR1 are also the and 2 for R-NT and from the binding in the binding the or not on the receptor expression The for the Ca2+ on the receptor expression Ca2+ is not an in For NTR2, we were to however, we were not to show Ca2+ mobilization in cells. for the of Ca2+ response in cells may be of the of NTR2 with We that can with G-protein-coupled receptors and the signal transduction into Ca2+ with NTR2 and to Ca2+ response in those cells. However, for signaling in recombinant system. Although the for the of signaling in the recombinant cell is not are with a J.M. Chabry J. Sarret P. Vincent J.P. Mazella J. Biochem. Biophys. Res. Commun. 1998; PubMed Scopus Google Scholar). we expressed NTR3 as a soluble receptor for binding NTR3 has been reported to be expressed in the intracellular compartments and not on the cell which the binding NTR3 binding have been reported using NTR3 from using In by expression of the the transmembrane and the intracellular and containing an signal we were to express the recombinant NTR3 secreted into the The C-terminal of the soluble NTR3 of the soluble the binding We and for