Differential Affinities of Visual Arrestin, βArrestin1, and βArrestin2 for G Protein-coupled Receptors Delineate Two Major Classes of Receptors

Robert H Oakley,Stéphane A Laporte,Jason A Holt,Marc G Caron,Larry S Barak

doi:10.1074/jbc.m910348199

Robert H Oakley, Stéphane A Laporte + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m910348199

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Abstract

Visual arrestin, betaarrestin1, and betaarrestin2 comprise a family of intracellular proteins that desensitize G protein-coupled receptors (GPCRs). In addition, betaarrestin1 and betaarrestin2 target desensitized receptors to clathrin-coated pits for endocytosis. Whether arrestins differ in their ability to interact with GPCRs in cells is not known. In this study, we visualize the interaction of arrestin family members with GPCRs in real time and in live cells using green fluorescent protein-tagged arrestins. In the absence of agonist, visual arrestin and betaarrestin1 were found in both the cytoplasm and nucleus of HEK-293 cells, whereas betaarrestin2 was found only in the cytoplasm. Analysis of agonist-mediated arrestin translocation to multiple GPCRs identified two major classes of receptors. Class A receptors (beta2 adrenergic receptor, mu opioid receptor, endothelin type A receptor, dopamine D1A receptor, and alpha1b adrenergic receptor) bound betaarrestin2 with higher affinity than betaarrestin1 and did not interact with visual arrestin. In contrast, class B receptors (angiotensin II type 1A receptor, neurotensin receptor 1, vasopressin V2 receptor, thyrotropin-releasing hormone receptor, and substance P receptor) bound both betaarrestin isoforms with similar high affinities and also interacted with visual arrestin. Switching the carboxyl-terminal tails of class A and class B receptors completely reversed the affinity of each receptor for the visual and non-visual arrestins. In addition, exchanging the betaarrestin1 and betaarrestin2 carboxyl termini reversed their extent of binding to class A receptors as well as their subcellular distribution. These results reveal for the first time marked differences in the ability of arrestin family members to bind GPCRs at the plasma membrane. Moreover, they show that visual arrestin can interact in cells with GPCRs other than rhodopsin. These findings suggest that GPCR signaling may be differentially regulated depending on the cellular complement of arrestin isoforms and the ability of arrestins to interact with other cellular proteins.

Highlights

G protein-coupled receptors (GPCRs)1 comprise a large gene family of more than 1000 members that mediate distinct physiological functions as diverse as phototransduction, olfaction, vascular tone, cardiac output, digestion, and pain
Translocation of ␤Arrestin1 and ␤Arrestin2 to the ␤2AR— green fluorescent protein (GFP)-labeled proteins provide a means for analyzing intracellular protein-protein interactions without having to disrupt the plasma membrane, but they allow for the assessment of the kinetics of interactions that occur in seconds [27, 28]
To compare the ability of ␤arrestin1 and ␤arrestin2 to interact with agonist-activated GPCRs in real time and in live cells, we fused the green fluorescent protein (GFP) to the carboxyl terminus of ␤arrestin1 (␤arr1-GFP) and ␤arrestin2 (␤arr2-GFP)

Summary

Visualization of Arrestin Translocation to GPCRs

Preference to rhodopsin [5, 8, 20, 21]. ␤arrestin binds the ␤2AR with a 2.5-fold greater affinity than ␤arrestin, and ␤arrestin binds the m2mAChR with a 1.5-fold greater affinity than ␤arrestin1 [21]. In the following study we investigate the dynamic interactions between arrestin family members and GPCRs in live cells by assessing the redistribution of fluorescent arrestins from the cytoplasm to agonist-activated receptors at the plasma membrane. Class A receptors, such as the ␤2AR, do not interact with visual arrestin and bind ␤arrestin with less affinity than ␤arrestin. Class B receptors, such as the angiotensin II type 1A receptor (AT1AR), interact with visual arrestin and bind both ␤arrestin and ␤arrestin with similar high affinities. The molecular determinants underlying this classification appear to reside in specific serine residues located in the receptor carboxyl-terminal tail. These findings reveal a potential role for visual arrestin in the regulation of GPCRs outside the visual system. They suggest that the particular cellular complement of arrestin isoforms and their distinct interactions with intracellular proteins will play a critical role regulating the pattern of GPCR desensitization, sequestration, and resensitization

EXPERIMENTAL PROCEDURES

Bt BT

RESULTS

DISCUSSION