Interaction of arrestins with intracellular domains of muscarinic and alpha(2)-adrenergic receptors

The intracellular domains of G-protein-coupled receptors provide sites for interaction with key proteins involved in signal inititaion 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 alpha(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 from the alpha(2A/D)-AR and M-2-MR. However, each of the receptor subdomains bound purified beta-arrestin and arrestin-3. The interaction of the M-3-MR and M-2-Mr i3 peptides with arrestins was further investigated. the tin and [H-3]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 [H-3]beta-arrestin, [H-3]visual arrestin, and [H-3]beta-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 p-arrestin with the M-3-MR peptide required both the amino (Gly(308)-Leu(368)) and carbonyl 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.