The kyotorphin (tyrosine-arginine) receptor and a selective reconstitution with purified G(i), measured with GTPase and phospholipase C assays

H. Ueda, Y. Yoshihara, H. Misawa, N. Fukushima, T. Katada, M. Ui, H. Takagi, M. Satoh

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66 Citations (Scopus)

Abstract

We attempted to identify the kyotorphin receptor and the post receptor mechanisms mediated by GTP-binding proteins (G-proteins), using reconstitution techniques. The specific binding of [3H]kyotorphin in rat brain membranes was composed of high affinity (K(d) = 0.34 nM) and low affinity (K(d) = 9.07 nM) binding. As the high affinity binding disappeared in the presence of guanosine 5'-O-(3-thiotriphosphate) and MgCl2, we investigated the kyotorphin receptor-mediated changes in membrane G-protein activity by measuring low K(m) GTPase activity. Kyotorphin produced a stimulation of low K(m) GTPase, and this stimulation was antagonized by Leu-Arg, a synthetic dipeptide which showed a potent displacement of [3H]kyotorphin binding, yet in itself had no effect on the low K(m) GTPase. The kyotorphin stimulation of low K(m) GTPase was abolished by pretreating membranes with islet-activating protein, pertussis toxin, and was recovered by reconstitution with purified G-protein, G(i), but not with G(o). Similar evidence of selective coupling of kyotorphin receptor to G(i) was obtained with the phospholipase C assay. Kyotorphin-induced stimulation of phospholipase C was also abolished by islet-activating protein-treatment and recovered by reconstitution with G(i) but not with G(o). These findings indicate that specific high and low affinity kyotorphin receptors exist in the rat brain and that the kyotorphin receptor is functionally coupled to stimulation of phospholipase C, through G(i). This study provides the first evidence of a selective involvement of G(i) in the receptor-mediated activation of phospholipase C.

Original languageEnglish
Pages (from-to)3732-3741
Number of pages10
JournalJournal of Biological Chemistry
Volume264
Issue number7
Publication statusPublished - 1989 Jan 1
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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