title: 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 gamma-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.
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden.
authors.forename: Elo
authors.initials: E
authors.lastname: Eriste
authors.forename: Ake
authors.initials: A
authors.lastname: Norberg
authors.forename: Diane
authors.initials: D
authors.lastname: Nepomuceno
authors.forename: Chester
authors.initials: C
authors.lastname: Kuei
authors.forename: Fredrik
authors.initials: F
authors.lastname: Kamme
authors.forename: Da-Thao
authors.initials: DT
authors.lastname: Tran
authors.forename: Kerstin
authors.initials: K
authors.lastname: Strupat
authors.forename: Hans
authors.initials: H
authors.lastname: Jörnvall
authors.forename: Changlu
authors.initials: C
authors.lastname: Liu
authors.forename: Timothy W
authors.initials: TW
authors.lastname: Lovenberg
authors.forename: Rannar
authors.initials: R
authors.lastname: Sillard
mesh_terms: D000818:Animals; D001120:Arginine / Q000737:chemistry*; D002118:Calcium / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D005973:Glutamine / Q000737:chemistry; D006801:Humans; D007477:Ions; D008024:Ligands; D013058:Mass Spectrometry; D008956:Models, Chemical; D009496:Neurotensin / Q000737:chemistry* / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D018028:Receptors, Neurotensin / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D017550:Spectroscopy, Fourier Transform Infrared; D013552:Swine
chemical_list: D007477:Ions; D008024:Ligands; C490985:NTSR2 protein, human; D010455:Peptides; D012333:RNA, Messenger; D018028:Receptors, Neurotensin; C426065:neurotensin receptor NTR-3; C114036:neurotensin type 1 receptor; D005973:Glutamine; D009496:Neurotensin; D001120:Arginine; D002118:Calcium