Deletion mutation in the putative third intracellular loop of the rat neurotensin receptor abolishes polyphosphoinositide hydrolysis but not cyclic AMP formation in CHO-K1 cells.
The tridecapeptide neurotensin is a putative neurotransmitter in the central nervous system. Previously, we showed that the rat neurotensin receptor expressed in CHO-K1 cells mediates polyphosphoinositide hydrolysis and cAMP formation. To further investigate these neurotensin receptor-mediated signal transduction systems, we constructed three deletion mutations in the putative third intracellular loop of this receptor and transfected these mutated genes into CHO-K1 cells. The equilibrium dissociation constants for specific [3H]neurotensin binding to these mutants were not different from that for the wild-type receptor. However, one mutant, which lacked 27 amino acids (amino acids 270-296), did not stimulate polyphosphoinositide hydrolysis, whereas it retained its ability to stimulate cAMP formation. In addition, as found for the wild-type receptor, sequestration occurred with this mutant. We demonstrated here that the putative third intracellular loop of this receptor plays a role in coupling to certain G proteins that induce polyphosphoinositide hydrolysis but not in coupling to cAMP formation or in ligand binding. The two different signal transduction systems may be induced by different G proteins that couple at different sites of the neurotensin receptor protein in CHO-K1 cells. Furthermore, our data show that neurotensin receptor sequestration is independent of agonist-induced polyphosphoinositide hydrolysis.
Yamada
M
M
Department of Psychiatry and Psychology, Mayo Foundation, Jacksonville, Florida 32224.
Yamada
M
M
Watson
M A
MA
Richelson
E
E
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D016466:CHO Cells; D006224:Cricetinae; D000242:Cyclic AMP / Q000096:biosynthesis*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009496:Neurotensin / Q000494:pharmacology; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011869:Radioligand Assay; D051381:Rats; D018028:Receptors, Neurotensin / Q000819:agonists / Q000737:chemistry / Q000187:drug effects / Q000235:genetics*; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D014162:Transfection
D018129:Phosphatidylinositol Phosphates; D018028:Receptors, Neurotensin; D009496:Neurotensin; D000242:Cyclic AMP