Receptor Homo Research Articles

Integration and Spatial Organization of Signaling by G Protein-Coupled Receptor Homo- and Heterodimers.

Information flow from a source to a receiver becomes informative when the recipient can process the signal into a meaningful form. Information exchange and interpretation is essential in biology and understanding how cells integrate signals from a variety of information-coding molecules into complex orchestrated responses is a major challenge for modern cell biology. In complex organisms, cell to cell communication occurs mostly through neurotransmitters and hormones, and receptors are responsible for signal recognition at the membrane level and information transduction inside the cell. The G protein-coupled receptors (GPCRs) are the largest family of membrane receptors, with nearly 800 genes coding for these proteins. The recognition that GPCRs may physically interact with each other has led to the hypothesis that their dimeric state can provide the framework for temporal coincidence in signaling pathways. Furthermore, the formation of GPCRs higher order oligomers provides the structural basis for organizing distinct cell compartments along the plasma membrane where confined increases in second messengers may be perceived and discriminated. Here, we summarize evidence that supports these conjectures, fostering new ideas about the physiological role played by receptor homo- and hetero-oligomerization in cell biology.

Selective recognition of Cr3+ in multivitamin formulations in aqueous medium by fluorescent organic–inorganic nanohybrids

An effective method for the detection of Cr3+ in multivitamin formulations has been developed by using fluorescent organic nanoparticles (FONPs) based on N,N′-bis(5-bromothiophen-2-ylmethyl)ethylenediimine (BTED). In the study, FONPs selectively recognize Cr3+ without inferring other coexisting metal ions in the medium. Since the FONPs enhance the fluorescence intensity with respect to the concentration of Cr ion, they were applied in real samples like multivitamin formulations to detect Cr3+. The stoichiometry of FONPs/Cr3+ analyzed by a Job’s plot was 2:1 (FONPs:metal). The results show that FONPs accurately measure the concentration of Cr3+ in the vitamin tablets in aqueous medium, agreeing with the reported chromium content in the vitamin formulations, and the detection limit was found to be 7.2 μM. Furthermore, the binding nature of the ligand with Cr3+ was studied through the molecular orbital analysis, where a lower energy HOMO was observed for [Cr(BTED)]3+ than that for the free ligand, meaning that the lowering of the receptor HOMO energy enhances the fluorescence emission in the photo-induced electron transfer.

Three novel input logic gates supported by fluorescence studies: Organic nanoparticles (ONPs) as chemo-sensor for detection of Zn2+ and Al3+ in aqueous medium

Organic nanoparticles (ONPs) of N,N′-ethylenebis(salicylimine) (salen) were synthesized and applied for specific recognition of Zn2+ and Al3+ ions in an aqueous medium. The results show that fluorescence intensity rises with the increasing concentration of Zn2+ in salen solution, proving that salen-ONPs detect Zn2+ efficiently in the aqueous medium as chemo-sensor. Furthermore, the salen-ONPs/Zn2+ system performs as an ON–OFF switch between pH 6.0 and 4.0. Amusingly, although salen-ONPs/Al3+ does not show any significant effect in the fluorescence spectra, highest fluorescence intensity was observed when Al3+ ion was added to salen-ONPs/Zn2+ in a sequential order (addition of Zn2+ to salen-ONPs, followed by Al3+). This system can be applied as a novel three inputs logic gate supported by the fluorescence for the detection of Zn2+ and Al3+ in biological and environmental samples. It appears that photo induced electron transfer (PET) occurs in the salen-ONPs when the fluorophore is excited. For salen/Zn2+ system, the PET is being inhibited considerably by lowering the receptor HOMO energy due to the formation of a bond between the metal ion and ligand, enhancing the fluorescence emission. This is consistent with the theoretical study that the energy of HOMO of the ligand is lower than that of Zn(salen)2+ complex.

Investigating the Relative Stability of Opioid Receptor Homo- and Hetero-Dimers Using Biased Molecular Dynamics Methods

The crucial role of opiates in the clinical management of pain places opioid receptors (ORs) among the most pharmacologically important members of the family A G protein-coupled receptors (GPCRs). A body of evidence, gathered over the last decade, supports the ability of all of the major subtypes of OR, the MOR, DOR and KOR, to form oligomers of homogeneous or heterogeneous composition at the plasma membrane. The discovery of unique functionality for some of these arrangements has shifted the focus of research towards the study of dimers/oligomers of ORs to aid understanding of the fundamental molecular factors governing opiate binding and selective activation of ORs. Using biased molecular dynamics (MD) methods, we aim to determine thermodynamic and kinetic details of the molecular mechanisms underlying homo- and heteromerization of ORs. We recently performed coarse-grained umbrella sampling MD simulations of DOR from Mus musculus in an explicit palmitoyl-oleoyl-phosphatidyl-choline (POPC):10% cholesterol-water environment, in order to estimate the free-energy difference between isolated and interacting protomers, from which, we were able to predict the lifetime of DOR homodimers. Our results indicated that this lifetime was of the order of a few seconds, in rough agreement with recent observations from single molecule fluorescence experiments on other rhodopsin-like GPCRs. Here, we extend this investigation to an experimentally supported, physiologically relevant heterodimer of DOR and KOR, which exhibits unique antinociceptive signaling properties that differ from those of the individual protomers. We investigated the relative stability of DOR/KOR dimers utilizing a combination of umbrella sampling and metadynamics methods, and compared the results with those obtained for homodimers. Our results suggest that the dimer stability varies depending on the protein sequence at the interface, and offer a route to testable prediction of dimerization-disrupting mutations.

Allostery at G Protein-Coupled Receptor Homo- and Heteromers: Uncharted Pharmacological Landscapes

For many years seven transmembrane domain G protein-coupled receptors (GPCRs) were thought to exist and function exclusively as monomeric units. However, evidence both from native cells and heterologous expression systems has demonstrated that GPCRs can both traffic and signal within higher-order complexes. As for other protein-protein interactions, conformational changes in one polypeptide, including those resulting from binding of pharmacological ligands, have the capacity to alter the conformation and therefore the response of the interacting protein(s), a process known as allosterism. For GPCRs, allosterism across homo- or heteromers, whether dimers or higher-order oligomers, represents an additional topographical landscape that must now be considered pharmacologically. Such effects may offer the opportunity for novel therapeutic approaches. Allosterism at GPCR heteromers is particularly exciting in that it offers additional scope to provide receptor subtype selectivity and tissue specificity as well as fine-tuning of receptor signal strength. Herein, we introduce the concept of allosterism at both GPCR homomers and heteromers and discuss the various questions that must be addressed before significant advances can be made in drug discovery at these GPCR complexes.

Basic Concepts in G-Protein-Coupled Receptor Homo- and Heterodimerization

Until recently, heptahelical G-protein-coupled receptors (GPCRs) were considered to be expressed as monomers on the cell surface of neuronal and non-neuronal cells. It is now becoming evident that this view must be overtly changed since these receptors can form homodimers, heterodimers, and higher-order oligomers on the plasma membrane. Here we discuss some of the basics and some new concepts of receptor homo- and heteromerization. Dimers-oligomers modify pharmacology, trafficking, and signaling of receptors. First of all, GPCR dimers must be considered as the main molecules that are targeted by neurotransmitters or by drugs. Thus, binding data must be fitted to dimer-based models. In these models, it is considered that the conformational changes transmitted within the dimer molecule lead to cooperativity. Cooperativity must be taken into account in the binding of agonists-antagonists-drugs and also in the binding of the so-called allosteric modulators. Cooperativity results from the intramolecular cross-talk in the homodimer. As an intramolecular cross-talk in the heterodimer, the binding of one neurotransmitter to one receptor often affects the binding of the second neurotransmitter to the partner receptor. Coactivation of the two receptors in a heterodimer can change completely the signaling pathway triggered by the neurotransmitter as well as the trafficking of the receptors. Heterodimer-specific drugs or dual drugs able to activate the two receptors in the heterodimer simultaneously emerge as novel and promising drugs for a variety of central nervous system (CNS) therapeutic applications.

Flanking Sequence Composition Differentially Affects the Binding and Functional Characteristics of Glucocorticoid Receptor Homo- and Heterodimers

The core binding sites for a multitude of transcription factors have been identified and characterized, but these sequences cannot fully account for the nuances of cell-specific and gene-specific control of gene transcription. Many factors may contribute to the precise responsiveness of a gene to a particular transcriptional regulatory protein, including the nucleotides in the proximity of the core binding site for that protein. Here, we examine two flanking sequences bordering a site in the gamma-fibrinogen gene regulatory region that binds a heterodimer of the Xenopus glucocorticoid receptor accessory factor (XGRAF) and the glucocorticoid receptor (GR). Mutation of the upstream flank results in a decrease in the level of XGRAF binding but little change in hormone induction. However, alteration of the downstream flank adjacent to the GR binding site causes a decrease in levels of both GR monomer binding and hormone induction. Conversion of the XGRAF-GR binding site to a full glucocorticoid response element (GRE) alters the role of the flanking sequences. A full GRE in this position requires the wild-type upstream flank to bind GR homodimer and induce transcription to maximal levels. In contrast, mutation of the downstream flank is not detrimental to either the binding or the function of the GR dimer. Thus, flanking sequence composition and dimer partner combine to influence GR function, underscoring the complexities involved in the identification of authentic transcription factor response elements.

Allosteric Modulation of Binding Properties between Units of Chemokine Receptor Homo- and Hetero-Oligomers

We have demonstrated previously that the chemokine receptors CCR2 and CCR5 form homo- and heterodimers and that dimers can only bind a single chemokine molecule with high affinity. We provide here evidence from bioluminescence resonance energy transfer experiments that stimulation by chemokines does not influence the CCR2/CCR5 heterodimerization status. In addition, we show that the rate of radioligand dissociation from one unit of the heterodimer in "infinite" tracer dilution conditions is strongly increased in the presence of an unlabeled chemokine ligand of the other unit. These results demonstrate unambiguously that the interaction between heterodimer units is of allosteric nature. Agonists, but also some monoclonal antibodies, could promote such negative binding cooperativity, indicating that this phenomenon does not require the full conformational change associated with receptor activation. Finally, we show that G protein coupling is required for high-affinity binding of macrophage inflammatory protein-1beta (CCL4) to CCR5 and that the dissociation from G proteins, after incubation with Gpp(NH)p, promotes the release of prebound radiolabeled chemokines with kinetics similar to those measured after the addition of an excess of unlabeled chemokines. These observations suggest that the association with G proteins probably participates in the negative cooperativity observed between receptor monomers. We propose that negative cooperativity within homo- and heterodimers of chemokine receptors and probably other G protein-coupled receptors will probably have major implications in their pharmacology in vivo and in the physiopathology of the diseases with which they are associated.

Opioid Receptor Homo- and Heterodimerization in Living Cells by Quantitative Bioluminescence Resonance Energy Transfer

Opioid receptors have been shown to dimerize or oligomerize among themselves and each other, affecting their functional properties. This study used bioluminescence resonance energy transfer (BRET) between the mu, delta, and kappa opioid receptors to study opioid receptor aggregation in transfected human embryonic kidney 293 cells. Titration of receptor levels indicated that all three opioid receptors have a similar affinity to form homo- or hetero-oligomers in combination with any other opioid receptor type. In contrast, none of the opioid receptors formed detectable oligomers with the muscarinic M2 receptor, indicating that interactions among opioid receptors are selective. The formation of opioid receptor dimers, rather than higher order oligomers, is supported by binding kinetics in competition experiments between labeled and unlabeled receptors. Opioid receptor dimerization occurred at physiological temperatures upon receptor biosynthesis, before trafficking to the plasma membrane. Moreover, using BRET, coimmunoprecipitation, receptor binding, and G protein coupling, we demonstrate for the first time functional mu opioid receptor-kappa opioid receptor heterodimerization. These combined results demonstrate that opioid receptors can undergo homo- and heterodimerization, a process with potential implications for opioid physiology and pharmacology.

Separation of retinoid X receptor homo- and heterodimerization functions.

As a promiscuous dimerization partner the retinoid X receptor (RXR) can contribute to signaling by multiple nuclear receptors. However, the impact of RXR cosignaling and the possible existence of an RXR homodimer signaling pathway are largely unexplored. We report here on the separation of RXR homo- and heterodimerization as an essential step towards the elucidation of the roles of RXR homo- and heterodimers in retinoid-rexinoid signaling. RXR homodimerization was specifically disrupted by single mutations in the RXR dimerization interface. In contrast, even multiple mutations did not fully impair RXR heterodimerization with retinoic acid receptor (RAR). Importantly, the mutation of mouse RXRalpha (mRXRalpha) Tyr402 substantially weakened RAR heterodimerization while concomitantly increasing homodimerization. Not only did this lead to cooperatively enhanced RXR homodimer binding to DR1 or DR5 elements, but unexpectedly, the mutant acquired significant binding efficiency for noncognate DR3 or DR4 elements as well. The increased stability of RXR homodimers on DR1 correlated with increased transcriptional activity of mRXRalpha(Y402A) on DR1-based reporter genes. Weak, if any, heterodimerization was observed with thyroid, vitamin D(3), or peroxisome proliferator-activating receptors. A model accounting for the structural impact of the Tyr402 mutation on dimerization is discussed. These results provide the basis for a genetic replacement of wild-type RXRs by mutants like mRXRalpha(Y402A) to elucidate the physiological impact of RXR homo- and heterodimerization.

Quantitative Assessment of β1- and β2-Adrenergic Receptor Homo- and Heterodimerization by Bioluminescence Resonance Energy Transfer

Quantitative bioluminescence resonance energy transfer (BRET) analysis was applied to the study of beta(1)- and beta(2)-adrenergic receptor homo- and heterodimerization. To assess the relative affinity between each of the protomers, BRET saturation experiments were carried out in HEK-293T cells. beta(1)- and beta(2)-adrenergic receptors were found to have similar propensity to engage in homo- and heterotropic interactions suggesting that, at equivalent expression levels of the two receptor subtypes, an equal proportion of homo- and heterodimers would form. Analysis of the data also revealed that, at equimolar expression levels of energy donor and acceptor, more than 80% of the receptor molecules exist as dimers and that this high incidence of receptor dimerization is insensitive to receptor density for expression levels varying between 1.4 and 26.9 pmol of receptor/mg of membrane protein. Taken together, these results indicate that most of the receptors expressed in cells exist as constitutive dimers and that, at least in undifferentiated fibroblasts, the proportion of homo- and heterodimers between the closely related beta(1)- and beta(2)-adrenergic receptors is determined by their relative levels of expression.

Differential transcriptional regulation of the apoAI gene by retinoic acid receptor homo- and heterodimers in yeast.

Several members of the nuclear receptor superfamily including RXR (retinoid X receptor) bind to a specific retinoic acid response element (site A) of the apoAI promoter. However, transcriptional activation of the apoAI gene by different homo- and heterodimeric forms of RXR or RAR (retinoic acid receptors) cannot be evaluated in mammalian cells, which contain endogenous RXR or RAR. In order to circumvent this limitation, we assessed the DNA-binding activities and transcriptional activation of different homo- and heterodimers of these receptors in yeast. Electrophoretic mobility shift assays (EMSA) demonstrated that yeast expressed RARalpha does not bind to site A of the apoAl promoter, whereas binding of RARbeta to site A is ligand-dependent. Both RARalpha and RARbeta form heterodimers with RXRalpha and bind to site A with high affinity. These DNA-binding studies correlate with the transcriptional data, which indicated that RARbeta but not RARalpha activates transcription from site A in response equally well to 9-cis and all-trans retinoic acids. 9-cis RA is a more potent ligand than all-trans RA to activate transcription via RXR/RAR heterodimers. We conclude that this yeast expression system is a useful tool to elucidate the 'transactivation code' for apoAl site A via specific combinations of different homo and heterodimeric versions of RXR and RAR.