Identification and functional characterization of a 5-transmembrane domain variant isoform of the NTS2 neurotensin receptor in rat central nervous system.
The present study demonstrated that alternative splicing of the rat nts2 receptor gene generates a 5-transmembrane domain variant isoform (vNTS2) that is co-expressed with the full-length NTS2 receptor throughout the brain and spinal cord, as evidenced by reverse transcription-PCR. The vNTS2 polypeptide is 281 amino acids in length, which is 135 amino acids shorter than the full-length isoform. Immunohistochemical and radioligand binding studies revealed that the HA-tagged recombinant vNTS2 receptor is poorly targeted to plasma membranes in transfected COS-7 cells. Binding studies also showed that the truncated receptor displayed a 5000-fold lower affinity for neurotensin (NT) than its full-length counterpart (IC(50) of 10 mum and 2 nm, respectively). Yet NT binding induced efficient internalization of receptor-ligand complexes in vNTS2-transfected cells. Furthermore, it produced a rapid (<5 min) activation of the mitogen-activated protein kinases (ERK1/2) pathway, indicating functional coupling of the variant receptor. This activation is sustained (>1 h) and is also produced by the NTS2 agonist levocabastine. Western blotting experiments suggested that vNTS2 is not expressed in monomeric form in the rat central nervous system. However, it does appear to form a variety of multimeric complexes, including homodimers and heterodimers, with the full-length NTS2. Indeed, co-immunoprecipitation studies in dually transfected cells demonstrated that the two receptor isoforms can form stable associations. Taken together, the present results indicated that the rat vNTS2 is a functional receptor that may play a role in NT signaling in mammalian central nervous system.
Perron
Amélie
A
Montreal Neurological Institute, Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec H3A 2B4, Canada.
Sarret
Philippe
P
Gendron
Louis
L
Stroh
Thomas
T
Beaudet
Alain
A
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D016466:CHO Cells; D019556:COS Cells; D002460:Cell Line; D002490:Central Nervous System / Q000378:metabolism*; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D003720:Densitometry; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D000939:Epitopes / Q000737:chemistry; D015151:Immunoblotting; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D020128:Inhibitory Concentration 50; D008024:Ligands; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D008969:Molecular Sequence Data; D009496:Neurotensin / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D010880:Piperidines / Q000494:pharmacology; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D018028:Receptors, Neurotensin / Q000737:chemistry* / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013116:Spinal Cord / Q000378:metabolism; D013997:Time Factors; D014018:Tissue Distribution; D014162:Transfection
D018076:DNA, Complementary; D000939:Epitopes; D008024:Ligands; C099727:Ntsr2 protein, rat; D010455:Peptides; D010880:Piperidines; D020033:Protein Isoforms; D012333:RNA, Messenger; D018028:Receptors, Neurotensin; D009496:Neurotensin; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; C047340:levocabastine