Elsevier

Cellular Signalling

Volume 19, Issue 9, September 2007, Pages 1928-1938
Cellular Signalling

Agonist occupancy of a single monomeric element is sufficient to cause internalization of the dimeric β2-adrenoceptor

https://doi.org/10.1016/j.cellsig.2007.05.002Get rights and content

Abstract

A range of studies have indicated that many rhodopsin-like, family A G protein-coupled receptors, including the β2-adrenoceptor, exist and probably function as dimers. It is less clear if receptors internalize as dimers and if agonist occupancy of only one element of a dimer is sufficient to cause internalization of a receptor dimer into the cell. We have used a chemogenomic approach to demonstrate that this is the case. Following expression of the wild type β2-adrenoceptor, isoprenaline but not 1-(3′'4′-dihydroxyphenyl)-3-methyl-1-butanone, which does not have significant affinity for the wild type receptor, caused receptor internalization. By contrast, 1-(3′4′-dihydroxyphenyl)-3-methyl-1-butanone, but not isoprenaline that does not have high affinity for the mutated receptor, caused internalization of Asp113Serβ2-adrenoceptor. Following co-expression of wild type and Asp113Serβ2-adrenoceptors each of isoprenaline and 1-(3′4′-dihydroxyphenyl)-3-methyl-1-butanone caused the co-internalization of both of these two forms of the receptor. Co-expressed wild type and Asp113Serβ2-adrenoceptors were able to be co-immunoprecipitated and 1-(3′4′-dihydroxyphenyl)-3-methyl-1-butanone produced internalization of the wild type receptor that was not prevented by the β-adrenoceptor antagonist propranolol that binds with high affinity only to the wild type receptor. These results demonstrate that agonist occupancy of either single binding site of the β2-adrenoceptor dimer is sufficient to cause internalization of the dimer and that antagonist occupation of one of the two ligand binding sites is unable to prevent agonist-mediated internalization.

Introduction

In recent years it has become increasingly clear that G protein-coupled receptors (GPCRs)1 can exist as dimers [1], [2] and a growing body of evidence suggests that the dimer is probably the configuration able to interact with high affinity with a hetero-trimeric G protein [3], [4]. As with many aspects of the mechanism of action and regulation of members of the GPCR superfamily the β2-adrenoceptor (β2-AR) has been a key model system [5]. Co-immunoprecipitation studies employing differentially epitope-tagged forms of this GPCR [6] were instrumental in providing compelling evidence of dimerization and the first application of bioluminescence resonance energy transfer to probe GPCR quaternary structure in intact cells utilized this GPCR [7]. In more recent studies the β2-AR has been shown to dimerize during protein maturation and prior to plasma membrane delivery [8] and been used to indicate that transmembrane domain VI of this receptor contains sequences important for protein-protein dimer contacts [8].

Following agonist occupancy, the vast majority of GPCRs, including the β2-AR [9] internalize into cells, and frequently then recycle back to the cell surface, as part of the complex series of process that are generically described as desensitization and resensitization [10]. Whether a class A GPCR homo-dimer can be activated and internalize in response to agonist-occupancy of only one of the two monomers is currently unclear and there is a highly variable literature on whether activated GPCRs internalize as dimers or dissociate into the corresponding monomers during this process [11], [12], [13], [14].

The original demonstration that Asp113 in transmembrane domain III of the β2-AR is the charge partner that allows high affinity interactions with catecholamine ligands [15] was the prototypic exemplar of the application of chemogenomics to GPCR function. Conversion of Asp113 to Ser results in substantial loss of affinity for catecholamines and related ligands but synthesis of 1-(3′4′-dihydroxyphenyl)-3-methyl-1-butanone (also known as L-158,870) [15], which has no significant affinity for the wild type β2-AR but is capable of accepting hydrogen bonds from the β-hydroxymethyl side chain of Ser113, demonstrated conclusively that the loss of affinity for catecholamines produced by this mutation was not simply a reflection of lack of expression or misfolding of the mutant receptor [15]. Herein, we take advantage of these observations to demonstrate that internalization of an Asp113β2-AR-Ser113β2-AR ‘hetero-dimer’ requires agonist occupancy of only one monomer within the dimer, that it does not matter which monomer is agonist-occupied, that agonist occupancy of one monomer is dominant when the other monomer is occupied by an antagonist and, as a consequence, that the β2-AR is activated and internalizes as a dimeric complex.

Section snippets

Materials

[3H]dihydroalprenolol (94 Ci/mmol) was from GE Healthcare. 1-(3′4′-dihydroxyphenyl)-3-methyl-1-butanone (also known as L-158,870) was the kind gift of M. Candelore, Merck Research Laboratories (Rahway, NJ). Flp-In T-REx HEK293 cells were from Invitrogen (Paisley, U.K.). The anti-VSV-G antiserum was produced in house and the anti HA-antibody 12CA5 was from (Roche Molecular Biochemicals, Nutley NJ).

Molecular constructs

N-terminally HA-and VSV-G tagged forms of the human β2-AR were generated using standard molecular

Results

The human β2-AR was modified to introduce the VSV-G epitope tag sequence at the N-terminus to generate VSV-G-β2-AR. Following expression of VSV-G-β2-AR in HEK293 cells isoprenaline produced a robust, concentration-dependent increase in cyclic AMP production with EC50 = 1.3 × 10 7 M (Fig. 1a). When equivalent experiments were performed following expression of the point mutant VSV-G-Asp113Serβ2-AR, the potency of isoprenaline to stimulate adenylyl cyclase activity was reduced by almost 1000 fold (EC50

Discussion

The β2-AR is one of the most actively studied GPCRs [5] and key experiments that have contributed greatly to the now widely held view that members of the class A, rhodopsin-like family of GPCRs can exist as homo-dimers [1], [2] have utilized this receptor as a model [6], [7], [8]. The β2-AR has also been a key model system employed to understand processes and mechanisms involved in GPCR internalization and in agonist-mediated desensitization [5], [10]. Two major questions in relation to the

Acknowledgements

These studies were supported by the Biotechnology and Biosciences Research Council.

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