Arrestin Interactions with G Protein-coupled Receptors

doi:10.1074/jbc.270.2.720

Arrestin Interactions with G Protein-coupled Receptors

DIRECT BINDING STUDIES OF WILD TYPE AND MUTANT ARRESTINS WITH RHODOPSIN, β₂-ADRENERGIC, AND m2 MUSCARINIC CHOLINERGIC RECEPTORS (*)

From the (1)From theDepartment of Pharmacology, Northwestern University Medical School, Chicago, Illinois 60611, the
(2)Department of Medicine, University of Wisconsin, Madison, Wisconsin 53792, and the
(3)Department of Pharmacology, Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

^§ Established Investigator of the American Heart Association. To whom correspondence should be addressed to:
Thomas Jefferson University, 233 South 10th St., Philadelphia, PA 19107.
Tel.: 215-955-4607; Fax: 215-923-1098.

Abstract

Arrestins play an important role in quenching signal transduction initiated by G protein-coupled receptors. To explore the specificity of arrestin-receptor interaction, we have characterized the ability of various wild-type arrestins to bind to rhodopsin, the β₂-adrenergic receptor (β₂AR), and the m2 muscarinic cholinergic receptor (m2 mAChR). Visual arrestin was found to be the most selective arrestin since it discriminated best between the three different receptors tested (highest binding to rhodopsin) as well as between the phosphorylation and activation state of the receptor (>10-fold higher binding to the phosphorylated light-activated form of rhodopsin compared to any other form of rhodopsin). While β-arrestin and arrestin 3 were also found to preferentially bind to the phosphorylated activated form of a given receptor, they only modestly discriminated among the three receptors tested. To explore the structural characteristics important in arrestin function, we constructed a series of truncated and chimeric arrestins. Analysis of the binding characteristics of the various mutant arrestins suggests a common molecular mechanism involved in determining receptor binding selectivity. Structural elements that contribute to arrestin binding include: 1) a C-terminal acidic region that serves a regulatory role in controlling arrestin binding selectivity toward the phosphorylated and activated form of a receptor, without directly participating in receptor interaction; 2) a basic N-terminal domain that directly participates in receptor interaction and appears to serve a regulatory role via intramolecular interaction with the C-terminal acidic region; and 3) two centrally localized domains that are directly involved in determining receptor binding specificity and selectivity. A comparative structure-function model of all arrestins and a kinetic model of β-arrestin and arrestin 3 interaction with receptors are proposed.

Footnotes

↵* This research was supported in part by Grants GM44944 (to J. L. B.), GM47417 (to J. L. B.), GM47120 (to J. J. O.), and HL31601 (to M. M. H.) from the National Institutes of Health, a grant-in-aid from the American Heart Association (to M. M. H.), and a grant from the National Kidney Foundation of Wisconsin (to J. J. O.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¹ The abbreviations used are:

β₂AR

β₂-adrenergic receptor

arrestin

a generic term that includes all arrestins

arr

visual arrestin

βarr

β-arrestin

arr3

arrestin 3

βARK

β-adrenergic receptor kinase

G protein

guanine nucleotide binding protein

m2 mAChR

m2 muscarinic cholinergic receptor

mAChR

carbachol-activated mAChR

P-mAChR

phosphorylated mAChR

P-mAChR

phosphorylated carbachol-activated mAChR

β₂AR

isoproterenol-activated β₂AR

P-β₂AR

phosphorylated β₂AR

P-β₂AR

isoproterenol-activated phosphorylated β₂AR

Rh

dark rhodopsin

Rh

light-activated rhodopsin

P-Rh

phosphorylated rhodopsin

P-Rh

phosphorylated light-activated rhodopsin

S

short

L

long.
↵²We use the term activated to refer to the agonist-occupied receptor while non-activated refers to the antagonist-occupied receptor. However, this does not necessarily imply that all of the receptors are in a completely activated or non-activated conformation. We use the term specificity to refer to the ability of a given arrestin to bind to different receptors, while the term selectivity refers to the ability of an arrestin to discriminate among the different functional forms of a given receptor.
- Received July 8, 1994.
- Revision received November 14, 1994.