Nm23-H2 interacts with a G protein-coupled receptor to regulate its endocytosis through an Rac1-dependent mechanism

J Biol Chem. 2004 Apr 30;279(18):18981-9. doi: 10.1074/jbc.M312621200. Epub 2004 Feb 19.

Abstract

G protein-coupled receptors (GPCRs) represent a vast family of transmembrane proteins involved in the regulation of several physiological responses. The thromboxane A2 receptor (present as two isoforms: TP alpha and TP beta) is a GPCR displaying diverse pharmacological effects. As seen for many other GPCRs, TP beta is regulated by agonist-induced internalization. In the present study, we report the identification by yeast two-hybrid screening of Nm23-H2, a nucleoside diphosphate kinase, as a new interacting molecular partner with the C-terminal tail of TP beta. This interaction was confirmed in a cellular context when Nm23-H2 was co-immunoprecipitated with TP beta in HEK293 cells, a process dependent on agonist stimulation of the receptor. We observed that agonist-induced internalization of TP beta was regulated by Nm23-H2 through modulation of Rac1 signaling. Immunofluorescence microscopy in HEK293 cells revealed that Nm23-H2 had a cytoplasmic and nuclear localization but was induced to translocate to the plasma membrane upon stimulation of TP beta to show extensive co-localization with the receptor. Our findings represent the first demonstration of an interaction of an Nm23 protein with a membrane receptor and constitute a novel molecular regulatory mechanism of GPCR endocytosis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • Endocytosis*
  • Humans
  • Microscopy, Fluorescence
  • NM23 Nucleoside Diphosphate Kinases
  • Nucleoside-Diphosphate Kinase*
  • Peptide Library
  • Protein Isoforms
  • Protein Transport
  • Proteins / metabolism*
  • Proteins / physiology
  • Receptors, G-Protein-Coupled / metabolism
  • Receptors, G-Protein-Coupled / physiology
  • Receptors, Thromboxane A2, Prostaglandin H2 / metabolism*
  • Receptors, Thromboxane A2, Prostaglandin H2 / physiology
  • Two-Hybrid System Techniques
  • rac1 GTP-Binding Protein / metabolism*
  • rac1 GTP-Binding Protein / physiology

Substances

  • NM23 Nucleoside Diphosphate Kinases
  • Peptide Library
  • Protein Isoforms
  • Proteins
  • Receptors, G-Protein-Coupled
  • Receptors, Thromboxane A2, Prostaglandin H2
  • NME1 protein, human
  • Nucleoside-Diphosphate Kinase
  • rac1 GTP-Binding Protein