Interaction of Arrestins with intracellular domains of muscarinic and α 2-adrenergic receptors

Guangyu Wu, Jason G. Krupnick, Jeffrey L. Benovic, Stephen M. Lanier

Research output: Contribution to journalArticlepeer-review

126 Scopus citations


The intracellular domains of G-protein-coupled receptors provide sites for interaction with key proteins involved in signal initiation and termination. As an initial approach to identify proteins interacting with these receptors and the receptor motifs required for such interactions, we used intracellular subdomains of G-protein-coupled receptors as probes to screen brain cytosol proteins. Peptides from the third intracellular loop (i3) of the M 2-muscarinic receptor (MR) (His 208-Arg 387), M 3-MR (Gly 308-Leu 497), or α(2A/D)-adrenergic receptor (AR) (Lys 224- Phe 374) were generated in bacteria as glutathione S-transferase (GST) fusion proteins, bound to glutathione-Sepharose and used as affinity matrices to detect interacting proteins in fractionated bovine brain cytosol. Bound proteins were identified by immunoblotting following SDS-polyacrylamide gel electrophoresis. Brain arrestins bound to the GST-M 3 fusion protein, but not to the control GST peptide or i3 peptides derived from the α(2A/D)-AR and M 2-MR. However, each of the receptor subdomains bound purified β-arrestin and arrestin-3. The interaction of the M 3-MR and M 2-MR i3 peptides with arrestins was further investigated. The M 3-MR i3 peptide bound in vitro translated [ 3H]β-arrestin and [ 3H]arrestin-3, but did not interact with in vitro translated or purified visual arrestin. The properties and specificity of the interaction of in vitro translated [ 3H]β-arrestin, [ 3H]visual arrestin, and [ 3H]β-arrestin/visual arrestin chimeras with the M 2-MR i3 peptide were similar to those observed with the intact purified M 2-MR that was phosphorylated and/or activated by agonist. Subsequent binding site localization studies indicated that the interaction of β-arrestin with the M 3-MR peptide required both the amino (Gly 308-Leu 368) and carboxyl portions (Lys 425-Leu 497) of the receptor subdomain. In contrast, the carboxyl region of the M 3-MR i3 peptide was sufficient for its interaction with arrestin-3.

Original languageEnglish (US)
Pages (from-to)17836-17842
Number of pages7
JournalJournal of Biological Chemistry
Issue number28
StatePublished - 1997
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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