Basic–Alimentary TractNeurotensin receptor–1 and –3 complex modulates the cellular signaling of neurotensin in the HT29 cell line☆,☆☆,★
Section snippets
Materials
NT was purchased from Peninsula Laboratories (San Carlos, CA) and NT (2-13) was synthesized by Neosystem (Strasbourg, France). 125I-Tyr3-NT, α-azidobenzoyl-125I-Tyr3-NT(2–13) were prepared and purified as described previously.31, 32 Sulfo-NHS-(LC)-Biotine was from Pierce (Rockford, IL). Antibodies against the luminal domain or the propeptide domain of NTR3/sortilin were a generous gift from Dr. C. M. Petersen (University of Aarhus, Aarhus, Denmark). Antibodies against the NTR-1 or the
Molecular identification and heterodimerization of neurotensin receptors expressed in HT29 cells
The expression of the 3 currently known NTRs was assessed by reverse-transcription polymerase chain reaction and revealed that both the human NTR1 and NTR3 transcripts were endogenously expressed in the HT29 cell line (not shown).
Photoaffinity labeling experiments using α-azidobenzoyl-125I-Tyr3-NT(2–13) (azido-125INT) on whole HT29 cells showed that 3 bands with Mr of 50, 135, and 105 kilodaltons were specifically labeled because their labeling was totally abolished in the presence of 1 μmol/L
Discussion
In the present study, we have shown a functional interaction between the GPCR NTR1 and the type I NTR3 that are endogenously expressed in HT29 cells. By using a heterologous expression system, we have shown that the heterodimer leads to modulation of the intracellular events induced by NT. In an attempt to define the mechanism of dimerization, we observed that a large amount of the NTR1-NTR3 complex is present at the plasma membrane in the absence of ligand. Then, when cells are stimulated with
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Address requests for reprints to: Jean Mazella, Ph.D., Institut de Pharmacologie Moléculaire et Cellulaire, Unité Mixte de Recherche 6097 du Centre National de la Recherche Scientifique, 660 route des lucioles, Sophia Antipolis, 06560 Valbonne, France. e-mail: [email protected]; fax: (33) 4-93957708.
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S.M. and V.N. are fellowship recipients of the Association pour la Recherche contre le Cancer (ARC).
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The authors thank P. Kitabgi for complementary DNA encoding the human NTR1, M. S. Nielsen and C. M. Petersen (University of Aarhus, Denmark) for the gift of anti-sortilin/NTR3 and anti-propeptide antibodies and of sortilin-expressing CHO cells.