The structure of α- and β-adrenergic receptors

doi:10.1016/0165-6147(83)90390-5

Trends in Pharmacological Sciences

Volume 4, 1983, Pages 256-258

https://doi.org/10.1016/0165-6147(83)90390-5 Get rights and content

Abstract

The use of specific affinity labels, monoclonal antibodies, autoantibodies, and radiation inactivation have provided considerable information concerning adrenergic receptor structure. Studies to date suggest that β₁- and β₂-receptors may have evolved as a result of gene duplication whereas α₁-adrenergic receptors may have more in common with muscarinic acetylcholine receptors.

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Cited by (21)

Multiscale modelling to understand the self-assembly mechanism of human β2-adrenergic receptor in lipid bilayer
2014, Computational Biology and Chemistry
Citation Excerpt :
The notion that all GPCRs function as independent monomeric units has long been challenged by various biochemical and biophysical studies (like fluorescence or bioluminescence resonance energy transfer, FRET and BRET, respectively) which suggest many GPCR molecules function either as homodimers or heterodimers (Bouvier, 2001; George et al., 2002; Issafras et al., 2002; Overton and Blumer, 2002; Terrillon et al., 2003; Floyd et al., 2003), the minimal oligomeric assemblies in the membrane environment and that these processes occur constitutively and are not modulated by ligand binding (Issafras et al., 2002; Terrillon et al., 2003; Floyd et al., 2003; Overton and Blumer, 2000; Ayoub et al., 2002; Jensen et al., 2002; Babcock et al., 2003; Canals et al., 2003; Dinger et al., 2003; Guo et al., 2003; Stanasila et al., 2003; Trettel et al., 2003). Various functions ranging from ontogeny to regulation of their pharmacological and signalling properties have been attributed to the dimerization of GPCRs (Terrillon and Bouvier, 2004; Gurevich and Gurevich, 2008) The notion that GPCRs form dimers, is supported by various structural studies including radiation inactivation of α- and β-AR (Venter and Fraser, 1983), hydrodynamic properties of cardiac muscarinic receptors (Peterson et al., 1986), photoaffinity labelling of muscarinic receptors (Avissar et al., 1983) and cross-linking of glucagon receptors (Herberg et al., 1985). Using atomic force microscopy, Palczewski and co-workers have shown that rhodopsin and opsin form constitutive dimers in dark-adapted native retinal membranes involving transmembrane (TM) segments TM4 and TM5 (Fotiadis et al., 2003; Liang et al., 2003).
The long perceived notion that G-Protein Coupled Receptors (GPCRs) function in monomeric form has recently been changed by the description of a number of GPCRs that are found in oligomeric states. The mechanism of GPCR oligomerization, and its effect on receptor function, is not well understood. In the present study, coarse grained molecular dynamics (CGMD) approach was adopted for studying the self-assembly process of the human GPCR, β2-adrenergic receptor (β2-AR), for which several experimental evidences of the dimerization process and its effect on cellular functions are available. Since the crystal structure of β2-AR lacks the third intracellular loop, initially it was modelled and simulated using restrained MD in order to get a stable starting conformation. This structure was then converted to CG representation and 16 copies of it, inserted into a hydrated lipid bilayer, were simulated for 10 μs using the MARTINI force field. At the end of 10 μs, oligomers of β2-AR were found to be formed through the self-assembly mechanism which were further validated through various analyses of the receptors. The lipid bilayer analysis also helped to quantify this assembly mechanism. In order to identify the domains which are responsible for this oligomerization, a reverse transformation of the CG system back to all-atom structure and simulated annealing run were carried out at the end of 10 μs CGMD run. Analysis of the all-atom dimers thus obtained, revealed that TM1/TM1, H8/H8, TM1/TM5 and TM6/TM6 regions formed most of the dimerization surfaces, which is in accordance with some of the experimental observations and recent simulation results.
Heterodimerization of Calcium Sensing Receptors with Metabotropic Glutamate Receptors in Neurons
2001, Journal of Biological Chemistry
Citation Excerpt :
Results from both Flag-CaR:mGluR1α or Flag-mGluR1α:CaR heterodimers indicate an increased sensitivity to desensitization, i.e.internalization, in response to exposure to the agonist which activates the non-tagged heterodimer partner. As early as the 1980s, oligomers of β2-adrenergic receptor (43) and muscarinic receptors (44) were observed, but their functional significance was uncertain. More recently, oligomerization has been suggested to account for the multiple agonist affinity states observed for GPCRs (45, 46).
Calcium sensing (CaR) and Group I metabotropic glutamate receptors exhibit overlapping expression patterns in brain, and share common signal transduction pathways. To determine whether CaR and Group I metabotropic glutamate receptors (mGluRs) (mGluR1α and mGluR5) can form heterodimers, we immunoprecipitated CaR from bovine brain and observed co-precipitation of mGluR1α. CaR and mGluR1α co-localize in hippocampal and cerebellar neurons, but are expressed separately in other brain regions. In vitro transfection studies in HEK-293 cells established the specificity and disulfide-linked nature of the CaR:mGluR1α (CaR:mGluR5) interactions. CaR:mGluR1α (CaR:mGluR5) heterodimers exhibit altered trafficking via Homer 1c when compared with CaR:CaR homodimers. CaR becomes sensitive to glutamate-mediated internalization when present in CaR:mGluR1α heterodimers. These results demonstrate cross-family covalent heterodimerization of CaR with Group I mGluRs, and increase the potential role(s) for CaR in modulating neuronal function.
Dimerization of the calcium-sensing receptor occurs within the extracellular domain and is eliminated by Cys → Ser mutations at Cys<sup>101</sup> and Cys<sup>236</sup>
1999, Journal of Biological Chemistry
Calcium-sensing receptors are present in membranes as dimers that can be reduced to monomers with sufhydryl reagents. All studies were carried out on the human calcium-sensing receptor tagged at the carboxyl terminus with green fluorescent protein (hCaR-GFP) to permit identification and localization of expressed proteins. Truncations containing either the extracellular agonist binding domain plus transmembrane helix 1 (ECD/TMH1-GFP) or the transmembrane domain plus the intracellular carboxyl terminus (TMD/carboxyl terminus-GFP) were used to identify the dimerization domain. ECD/TMH1-GFP was a dimer in the absence of reducing reagents, whereas TMD/carboxyl-terminal GFP was a monomer in the absence or presence of reducing agents, suggesting that dimerization occurs via the ECD. To identify the residue(s) involved in dimerization within the ECD, cysteine → serine point mutations were made in residues that are conserved between hCaR and metabotropic glutamate receptors. Mutations at positions 60 and 131 were expressed at levels comparable to wild type in HEK 293 cells, had minimal effects on hCaR function, and did not eliminate dimerization, whereas mutations at positions 101 and 236 greatly decreased receptor expression and resulted in significant amounts of monomer in the absence of reducing agents. The double point mutant hCaR(C101S/C236S)-GFP was expressed more robustly than either C101S or C236S and covalent dimerization was eliminated. hCaR(C101S/C236S)-GFP had a decreased affinity for extracellular Ca²⁺ and slower response kinetics upon increases or decreases in agonist concentration. These results suggest that covalent, disulfide bond-mediated dimerization of the calcium-sensing receptor contributes to stabilization of the ECD and to acceleration of the transitions between inactive and active receptor conformations.
Synthetic rat V(1a) vasopressin receptor fragments interfere with vasopressin binding via specific interaction with the receptor
1997, Journal of Biological Chemistry
To study the vasopressin receptor domains involved in the hormonal binding, we synthesized natural and modified fragments of V_1a vasopressin receptor and tested their abilities to affect hormone-receptor interactions. Natural fragments mimicking the external loops one, two, and three were able to inhibit specific vasopressin binding to V_1a receptor. In contrast, the natural N-terminal part of the V_1avasopressin receptor was found inactive. One fragment, derived from the external second loop and containing an additional C-terminal cysteine amide, was able to fully inhibit the specific binding of both labeled vasopressin agonist and antagonist to rat liver V_1avasopressin receptor and the vasopressin-sensitive phospholipase C of WRK1 cells. The peptide-mediated inhibition involved specific interactions between the V_1a receptor and synthetic V_1a vasopressin receptor fragment since 1) it was dependent upon the vasopressin receptor subtype tested (K_i_(app) for the peptide: 3.7, 14.6, and 64.5 μm for displacing [³H]vasopressin from rat V_1a, V_1b, and V₂receptors, respectively; 2) it was specific and did not affect sarcosin 1-angiotensin II binding to rat liver membranes; 3) it was not mimicked by vasopressin receptor unrelated peptides exhibiting putative detergent properties; and 4) no direct interaction between [³H]vasopressin and synthetic peptide linked to an affinity chromatography column could be observed. Such an inhibition affected both the maximal binding capacity of the V_1avasopressin receptor and its affinity for the labeled hormone, depending upon the dose of synthetic peptide used and was partially irreversible. Structure-activity studies using a serie of synthetic fragments revealed the importance of their size and cysteinyl composition. These data indicate that some peptides mimicking extracellular loops of the V_1a vasopressin receptor may interact with the vasopressin receptor itself and modify its coupling with phospholipase C.
A peptide derived from a β <inf>2</inf>-adrenergic receptor transmembrane domain inhibits both receptor dimerization and activation
1996, Journal of Biological Chemistry
One of the assumptions of the mobile receptor hypothesis as it relates to G protein-coupled receptors is that the stoichiometry of receptor, G protein, and effector is 1:1:1 (Bourne, H. R., Sanders, D. A., and McCormick, F. (1990) Nature 348, 125–132). Many studies on the cooperativity of agonist binding are incompatible with this notion and have suggested that both G proteins and their associated receptors can be oligomeric. However, a clear physical demonstration that G protein-coupled receptors can indeed interact as dimers and that such interactions may have functional consequences was lacking. Here, using differential epitope tagging we demonstrate that β₂-adrenergic receptors do form SDS-resistant homodimers and that transmembrane domain VI of the receptor may represent part of an interface for receptor dimerization. The functional importance of dimerization is supported by the observation that a peptide derived from this domain that inhibits dimerization also inhibits β-adrenergic agonist-promoted stimulation of adenylyl cyclase activity. Moreover, agonist stimulation was found to stabilize the dimeric state of the receptor, while inverse agonists favored the monomeric species, which suggests that interconversion between monomeric and dimeric forms may be important for biological activity.
Cooperativity manifest in the binding properties of purified cardiac muscarinic receptors
1995, Journal of Biological Chemistry
Muscarinic receptors were solubilized from porcine atria in digitonin-cholate and were purified by chromatography on DEAE-Sepharose and 3-(2′-aminobenzhydryloxy)tropane-Sepharose. The product identified on Western blots migrated with an apparent molecular mass of 60-75 kDa, with additional bands indicative of homotrimers (190 kDa) and homotetramers (240 kDa). Receptor eluted from the affinity column was accompanied by a mixture of guanyl nucleotide-binding proteins (G-proteins) identified on Western blots as G_i1/2, G_o, G_q/11, and G_s (preparation M2G); the G-proteins were largely removed by further processing on hydroxyapatite (preparation M2). Solubilized purified receptors bound muscarinic ligands in an apparently cooperative manner. In studies at equilibrium, the antagonists [³H]AF-DX 384, N-[³H]methylscopolamine (NMS), and quinuclidinylbenzilate (QNB) revealed Hill coefficients between about 0.8 and 1.2. Also, the apparent capacity for [³H]QNB exceeded that for [³H]AF-DX 384 and [³H]NMS by about 1.5-fold in M2 and by 2-fold in M2G. Binding to M2G at high concentrations of [³H]QNB was fully inhibited by unlabeled NMS, which therefore affected sites not labeled at similar concentrations of [³H]NMS. Oxotremorine-M displayed a biphasic inhibitory effect on the binding of [³H]AF-DX 384 in M2 and M2G, suggesting that multiple states of affinity are intrinsic to the receptor; 5′-guanylylimidodiphosphate was without appreciable effect in M2 but resulted in a bell-shaped binding profile for the agonist in M2G. All of the data can be described in terms of cooperative interactions within a receptor that is at least tetravalent and presumably an oligomer. In the context of the model, copurifying G-proteins and guanyl nucleotides serve to regulate the degree of cooperativity between successive equivalents of muscarinic ligands.

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Trends in Pharmacological Sciences

TIPS reviewThe structure of α- and β-adrenergic receptors

Abstract

Arch. Biochem. Biophys.

Biochem. Biophys. Res. Comm.

J. Biol. Chem.

Am. J. Physiol.

Nature (London)

Science

TIPS review
The structure of α- and β-adrenergic receptors