The rat neurotensin receptor expressed in Chinese hamster ovary cells mediates the release of inositol phosphates

Michael A. Watson, Mitsuhiko Yamada, Misa Yamada, Bernadette Cusack, Karen Veverka, Carolyn Bolden-Watson, Elliott Richelson

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Abstract

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 pcDNA1neo 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 [3H]neurotensin binding as an indication of neurotensin receptor expression. The clone (CHO-rNTR-10) showing the highest level of specific [3H]neurotensin binding was characterized further. With intact cells, the equilibrium dissociation constant (AD) for specific [3H]neurotensin binding was 18 nM, and the maximal number of binding sites (Bmax) was 900 fmol/mg of protein or 740 fmol/106 cells (∼4.4 × 105 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 Bmax exceeded previously reported values. Incubation of intact CHO-rNTR-10 cells with neurotensin caused the release of inositol phosphates in a dose-dependent manner (EC50 = 3 nM), 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.

Original languageEnglish (US)
Pages (from-to)1967-1970
Number of pages4
JournalJournal of Neurochemistry
Volume59
Issue number5
StatePublished - Nov 1992

Fingerprint

Neurotensin Receptors
Neurotensin
Inositol Phosphates
Cricetulus
Rats
Ovary
Cells
Clone Cells
neurotensin (8-13)
Second Messenger Systems
Cell Line
Colforsin
Neuroblastoma
Cyclic AMP
Complementary DNA
Binding Sites

Keywords

  • Cyclic AMP
  • N1E-115 cells

ASJC Scopus subject areas

  • Biochemistry
  • Cellular and Molecular Neuroscience

Cite this

Watson, M. A., Yamada, M., Yamada, M., Cusack, B., Veverka, K., Bolden-Watson, C., & Richelson, E. (1992). The rat neurotensin receptor expressed in Chinese hamster ovary cells mediates the release of inositol phosphates. Journal of Neurochemistry, 59(5), 1967-1970.

The rat neurotensin receptor expressed in Chinese hamster ovary cells mediates the release of inositol phosphates. / Watson, Michael A.; Yamada, Mitsuhiko; Yamada, Misa; Cusack, Bernadette; Veverka, Karen; Bolden-Watson, Carolyn; Richelson, Elliott.

In: Journal of Neurochemistry, Vol. 59, No. 5, 11.1992, p. 1967-1970.

Research output: Contribution to journalArticle

Watson, MA, Yamada, M, Yamada, M, Cusack, B, Veverka, K, Bolden-Watson, C & Richelson, E 1992, 'The rat neurotensin receptor expressed in Chinese hamster ovary cells mediates the release of inositol phosphates', Journal of Neurochemistry, vol. 59, no. 5, pp. 1967-1970.
Watson MA, Yamada M, Yamada M, Cusack B, Veverka K, Bolden-Watson C et al. The rat neurotensin receptor expressed in Chinese hamster ovary cells mediates the release of inositol phosphates. Journal of Neurochemistry. 1992 Nov;59(5):1967-1970.
Watson, Michael A. ; Yamada, Mitsuhiko ; Yamada, Misa ; Cusack, Bernadette ; Veverka, Karen ; Bolden-Watson, Carolyn ; Richelson, Elliott. / The rat neurotensin receptor expressed in Chinese hamster ovary cells mediates the release of inositol phosphates. In: Journal of Neurochemistry. 1992 ; Vol. 59, No. 5. pp. 1967-1970.
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