INTERACTION OF ARRESTINS WITH INTRACELLULAR DOMAINS OF MUSCARINIC ANDALPHA(2)-ADRENERGIC RECEPTORS

The intracellular domains of G-protein-coupled receptors provide sites for interaction with key proteins involved in signal inititaion and t ermination. As an initial approach to identify proteins interacting wi th these receptors and the receptor motifs required for such interacti ons, we used intracellular subdomains of G-protein-coupled receptors a s probes to screen brain cytosol proteins. Peptides from the third int racellular 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 fractionat ed bovine brain cytosol. Bound proteins were identified by immunoblott ing 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-ar restin and arrestin-3. The interaction of the M-3-MR and M-2-Mr i3 pep tides with arrestins was further investigated. the tin and [H-3]arrest in-3, but did not interact with in vitro translated or purified visual arrestin. The properties and specificity of the interaction of in vit ro 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 si milar to those observed with the intact purified M-2-MR that was phosp horylated and/or activated by agonist, Subsequent binding site localiz ation 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, t he carboxyl region of the M-3-MR i3 peptide was sufficient for its int eraction with arrestin-3.