IDENTIFICATION IN THE RAT NEUROTENSIN RECEPTOR OF AMINO-ACID-RESIDUESCRITICAL FOR THE BINDING OF NEUROTENSIN

In order to identify charged amino-acid residues of the cloned rat bra in neurotensin (NT) receptor (NTR) that are critical for NT binding, w e performed site-directed mutagenesis on the cDNA encoding this protei n, followed by transient expression into mammalian COS-7 cells and in Xenopus laevis oocytes. Point substitutions of charged residues in the N-terminal part and in the 2nd and 3rd extracellular loop of the rece ptor either did not affect I-125-Tyr(3)-NT binding or resulted in a de crease in binding affinity by a factor of 2-3. Mutations of amino acid s Asp(113) in the second transmembrane domain (TM) and of Arg(149) Or Asp(150) in TM III yielded receptors that bound NT as efficiently as t he native receptor. By contrast, replacement of the Asp(139) residue i n the ist extracellular loop, or of Arg(143) or Arg(327)-Arg(328) resi dues at the top of TM III and in TM VI, respectively, completely aboli shed ligand binding. Confocal and EM immunocytochemical studies of the expression of these affected receptors, tagged with the C-terminal se quence of the vesicular stomatitis virus glycoprotein (VSV-G), indicat ed that this loss of binding was not due to altered receptor expressio n or to their improper insertion into the plasma membrane. When these mutated forms of neurotensin receptor were expressed into Xenopus oocy tes, Asp(139)-Gly- and Arg(143)-Gly-modified receptors remained functi onal in spite of a lowered response to NT whereas the Arg(327)-Arg(328 ) mutant form was totally insensitive to NT at concentrations up to 10 mu M. In the case of the Arg(327)-Arg(328) mutation, the observed ins ensibility to NT could be the result of a drastic conformational alter ation of this mutant protein. By contrast, it would appear that Asp(13 9), and Arg(143) residues located in the first extracellular loop of t he receptor may be directly involved in the interaction of the recepto r with neurotensin. (C) 1997 Elsevier Science B.V.