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The Rat Neurotensin Receptor Expressed in Chinese Hamster Ovary Cells Mediates the Release of Inositol Phosphates
Author(s) -
Watson Michael A.,
Yamada Mitsuhiko,
Yamada Misa,
Cusack Bernadette,
Veverka Karen,
BoldenWatson Carolyn,
Richelson Elliott
Publication year - 1992
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1992.tb11035.x
Subject(s) - neurotensin , chinese hamster ovary cell , biology , neurotensin receptor , inositol phosphate , receptor , microbiology and biotechnology , transfection , cell culture , clone (java method) , inositol , biochemistry , neuropeptide , gene , genetics
To study second messenger synthesis mediated by the cloned rat neurotensin receptor, we derived a cell line stably expressing this receptor. The cDNA clone of this receptor was subcloned into the pcDNA 1 neo expression vector. This construct was then used to transfect Chinese hamster ovary (CHO)‐K1 cells. Colony clones, selected for resistance to antibiotic G‐418 sulfate, were isolated and grown separately. Nineteen individual clones were screened for total [ 3 H]neurotensin binding as an indication of neurotensin receptor expression. The clone (CHO‐rNTR‐10) showing the highest level of specific [ 3 H]neurotensin binding was characterized further. With intact cells, the equilibrium dissociation constant (K D ) for specific [ 3 H]neurotensin binding was 18 n M , and the maximal number of binding sites ( B max ) was 900 fmol/mg of protein or 740 fmol/10 6 cells (sim4.4 × 10 5 sites on the cellular surface). Whereas the KD was similar to that found in other cellular systems, for example, the murine neuroblastoma clone N1E‐115, the B max exceeded previously reported values. Incubation of intact CHO‐rNTR‐10 cells with neurotensin caused the release of inositol phosphates in a dose‐dependent manner (EC 50 = 3 n M ), results indicating that the expressed transfected receptor was functional. Neurotensin did not inhibit cyclic AMP levels stimulated by forskolin. As with other systems, neurotensin (8‐13) was more potent than neurotensin. Neurotensin‐mediated inositol phosphate release is the first report of second messenger synthesis for this receptor expressed in a transfected cell line. These results suggest that the relation between structure and function of the neurotensin receptor can be readily studied in transfected cell lines.