Agonist occupancy of a single monomeric element is sufficient to cause internalization of the dimeric β2-adrenoceptor
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.
References (24)
- et al.
J. Mol. Biol.
(2003) - et al.
FEBS Lett.
(2004) Trends Pharmacol. Sci.
(2004)- et al.
J. Biol. Chem.
(2004) - et al.
J. Biol. Chem.
(1992) - et al.
Life Sci.
(1998) - et al.
J. Biol. Chem.
(2002) - et al.
J. Biol. Chem.
(1991) - et al.
J. Biol. Chem.
(2006) - et al.
Annu. Rev. Pharmacol. Toxicol.
(2002)
Mol. Pharmacol.
J. Biol. Chem.
Cited by (36)
Differential Modulation of Adrenergic Receptor Signaling by Octopamine, Tyramine, Phenylethylamine, and 3-Iodothyronamine
2016, Trace Amines and Neurological Disorders: Potential Mechanisms and Risk FactorsDistinct agonist regulation of muscarinic acetylcholine M<inf>2</inf>-M<inf>3</inf> heteromers and their corresponding homomers
2015, Journal of Biological ChemistryCitation Excerpt :Beyond possible differences in efficacy, one further observation that is difficult to provide a clear explanation for was the marked difference in the effects of carbachol and CNO on hM2WT-hM3RASSL interactions and, thus, on hM2-hM3 heteromers. Although difficult to demonstrate without making further alterations in the ligand binding pocket to alter ligand pharmacology, as has been done for the β2-adrenoreceptor (38) and the leukotriene B(4) receptor (39), which, to some extent invalidates the basis of the experiment, it is anticipated that a ligand effect across the interface of a receptor homo-dimer/oligomer should be symmetric. Therefore, an effect of ligand binding to one protomer is anticipated to be reciprocated by (the same) agonist occupancy of the other protomer.
Homo- and hetero-oligomerization of β<inf>2</inf>-adrenergic receptor in receptor trafficking, signaling pathways and receptor pharmacology
2014, Cellular SignallingCitation Excerpt :It is well established that once at the plasma membrane β2AR undergoes robust agonist-induced internalization to endosomes [34]. Experiments of Sartania and colleagues demonstrated that co-expression of isoproterenol-insensitive Asp113Ser β2AR mutant together with the wild-type receptor resulted in internalization of both the wild-type and the mutated β2AR after challenge with either isoproterenol or Asp113Ser mutant-specific agonist [35]. The authors concluded that agonist binding to the single protomer is sufficient to induce internalization of the whole β2AR oligomer [35].
C5L2 and C5aR interaction in adipocytes and macrophages: Insights into adipoimmunology
2013, Cellular SignallingCitation Excerpt :With co-internalization, cross phosphorylation can occur and un-liganded receptor can be activated as a result of dimerization [36]. Dimerization may affect receptor stability, and heterodimerization may, although it does not necessarily, modulate ligand binding, G protein signalling phenotypes, β-arrestin recruitment/internalization, and down regulation [37,38]. C5aR has been shown to homodimerize and also heterodimerize with the CCR5 chemokine receptor [39,36].
GPCR oligomerization and receptor trafficking
2013, Methods in EnzymologyCitation Excerpt :Synthetic ligands which only have affinity for a mutationally modified GPCR that has lost affinity for its endogenous ligand have also been used to examine the interrelationship between homo-oligomerization and receptor trafficking. In particular, this approach has been used to access if a GPCR may be internalized from the cell surface as an oligomeric complex (Sartania et al., 2007). In these studies, the β2-adrenoceptor was modified to lose affinity for the agonist drug isoprenaline, which binds to and promotes internalization of the wild-type β2-adrenoceptor.