Identification in the rat neurotensin receptor of amino-acid residues critical for the binding of neurotensin

In order to identify charged amino-acid residues of the cloned rat brain neurotensin (NT) receptor (NTR) that are critical for NT binding, we performed site-directed mutagenesis on the cDNA encoding this protein, 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 receptor either did not affect I-125-Tyr(3)-NT binding or resulted in a decrease in binding affinity by a factor of 2-3. Mutations of amino acids 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 the native receptor. By contrast, replacement of the Asp(139) residue in the ist extracellular loop, or of Arg(143) or Arg(327)-Arg(328) residues at the top of TM III and in TM VI, respectively, completely abolished ligand binding. Confocal and EM immunocytochemical studies of the expression of these affected receptors, tagged with the C-terminal sequence of the vesicular stomatitis virus glycoprotein (VSV-G), indicated that this loss of binding was not due to altered receptor expression or to their improper insertion into the plasma membrane. When these mutated forms of neurotensin receptor were expressed into Xenopus oocytes, Asp(139)-Gly- and Arg(143)-Gly-modified receptors remained functional 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 insensibility to NT could be the result of a drastic conformational alteration of this mutant protein. By contrast, it would appear that Asp(139), and Arg(143) residues located in the first extracellular loop of the receptor may be directly involved in the interaction of the receptor with neurotensin. (C) 1997 Elsevier Science B.V.