The rat neurotensin receptor expressed in Chinese hamster ovary cells mediates the release of inositol phosphates.
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 (KD) 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/10(6) cells (approximately 4.4 x 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 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.
Watson
M A
MA
Department of Psychiatry and Psychology, Mayo Foundation, Jacksonville, Florida 32224.
Yamada
M
M
Yamada
M
M
Cusack
B
B
Veverka
K
K
Bolden-Watson
C
C
Richelson
E
E
D000818:Animals; D016466:CHO Cells / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D005822:Genetic Vectors; D007295:Inositol Phosphates / Q000378:metabolism*; D009496:Neurotensin / Q000235:genetics*; D051381:Rats; D018028:Receptors, Neurotensin; D017981:Receptors, Neurotransmitter / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D013329:Structure-Activity Relationship; D014162:Transfection
D007295:Inositol Phosphates; D018028:Receptors, Neurotensin; D017981:Receptors, Neurotransmitter; D009496:Neurotensin