title: Binding of wild type and chimeric arrestins to the m2 muscarinic cholinergic receptor.
abstract: Arrestins play an important role in regulating the activity of the G protein-coupled receptors rhodopsin and the beta 2-adrenergic receptor. Recently, we described the expression and functional characterization of visual arrestin using an in vitro translation system. Here we report the expression of beta-arrestin and development of a direct binding assay to study the interaction of arrestins with a muscarinic cholinergic receptor. In vitro translated beta-arrestin was found to specifically bind to purified reconstituted human m2 muscarinic cholinergic receptor (hm2 mAChR) in an agonist- and phosphorylation-dependent manner. Visual arrestin also bound to the hm2 mAChR, albeit to a lesser extent and with lower affinity. In an attempt to dissect the major domains responsible for determining the receptor binding specificity of arrestin and beta-arrestin, we generated several chimeric arrestins. One contained the first 340 residues of beta-arrestin followed by residues 346-404 of arrestin (BRV4), another consisted of the first 207 residues of beta-arrestin and residues 214-404 of visual arrestin (BV3), and a third had residues 1-43 of beta-arrestin replaced by residues 1-47 of arrestin (VIN1). All of these arrestins were able to specifically bind to the activated and phosphorylated form of both the hm2 mAChR and rhodopsin, with a clear preference for the muscarinic receptor. The Kd values for beta-arrestin, BRV4, BV3, VIN1, and visual arrestin binding to the hm2 mAChR were 0.48 +/- 0.06, 0.51 +/- 0.19, 1.38 +/- 0.26, 1.13 +/- 0.26, and 7.2 +/- 1.2 nM, respectively. These data demonstrate that: 1) beta-arrestin binds to the hm2 mAChR in an activation- and phosphorylation-dependent fashion, 2) visual arrestin has 15-fold lower affinity for the hm2 mAChR as compared to beta-arrestin, and 3) the N-terminal half of beta-arrestin plays a key role in determining receptor binding specificity. The use of in vitro translated arrestins to directly assess receptor binding may serve as a viable approach for elucidating the specificity and molecular mechanisms involved in receptor-arrestin interaction.
authors.affiliation: Department of Pharmacology, Northwestern University Medical School, Chicago, Illinois 60611.
authors.forename: V V
authors.initials: VV
authors.lastname: Gurevich
authors.forename: R M
authors.initials: RM
authors.lastname: Richardson
authors.forename: C M
authors.initials: CM
authors.lastname: Kim
authors.forename: M M
authors.initials: MM
authors.lastname: Hosey
authors.forename: J L
authors.initials: JL
authors.lastname: Benovic
mesh_terms: D000818:Animals; D000941:Antigens / Q000378:metabolism*; D019393:Arrestin; D002417:Cattle; D003001:Cloning, Molecular; D005136:Eye Proteins / Q000378:metabolism*; D006801:Humans; D014176:Protein Biosynthesis; D011976:Receptors, Muscarinic / Q000378:metabolism*
chemical_list: D000941:Antigens; D019393:Arrestin; D005136:Eye Proteins; D011976:Receptors, Muscarinic