Amyloid β peptide oligomers directly activate NMDA receptors

Amyloid beta (Aβ) oligomers accumulate in the brain tissue of Alzheimer disease patients and are related to disease pathogenesis. The precise mechanisms by which Aβ oligomers cause neurotoxicity remain unknown. We recently reported that Aβ oligomers cause intracellular Ca(2+) overload and neuronal death that can be prevented by NMDA receptor antagonists. This study investigated whether Aβ oligomers directly activated NMDA receptors (NMDARs) using NR1/NR2A and NR1/NR2B receptors that were heterologously expressed in Xenopus laevis oocytes. Indeed, Aβ oligomers induced inward non-desensitizing currents that were blocked in the presence of the NMDA receptor antagonists memantine, APV, and MK-801. Intriguingly, the amplitude of the responses to Aβ oligomers was greater for NR1/NR2A heteromers than for NR1/NR2B heteromers expressed in oocytes. Consistent with these findings, we observed that the increase in the cytosolic concentration of Ca(2+) induced by Aβ oligomers in cortical neurons is prevented by AP5, a broad spectrum NMDA receptor antagonist, but slightly attenuated by ifenprodil which blocks receptors with the NR2B subunit. Together, these results indicate that Aβ oligomers directly activate NMDA receptors, particularly those with the NR2A subunit, and further suggest that drugs that attenuate the activity of such receptors may prevent Aβ damage to neurons in Alzheimer?s disease.     

Oligomerization of wild type and nonfunctional mutant angiotensin II type I receptors inhibits galphaq protein signaling but not ERK activation

The 7-transmembrane or G protein-coupled receptors relay signals from hormones and sensory stimuli to multiple signaling systems at the intracellular face of the plasma membrane including heterotrimeric G proteins, ERK1/2, and arrestins. It is an emerging concept that 7-transmembrane receptors form oligomers; however, it is not well understood which roles oligomerization plays in receptor activation of different signaling systems. To begin to address this question, we used the angiotensin II type 1 (AT(1)) receptor, a key regulator of blood pressure and fluid homeostasis that in specific context has been described to activate ERKs without activating G proteins. By using bioluminescence resonance energy transfer, we demonstrate that AT(1) receptors exist as oligomers in transfected COS-7 cells. AT(1) oligomerization was both constitutive and receptor-specific as neither agonist, antagonist, nor co-expression with three other receptors affected the bioluminescence resonance energy transfer 2 signal. Furthermore, the oligomerization occurs early in biosynthesis before surface expression, because we could control AT(1) receptor export from the endoplasmic reticulum or Golgi by using regulated secretion/aggregation technology (RPD trade mark ). Co-expression studies of wild type AT(1) and AT(1) receptor mutants, defective in either ligand binding or G protein and ERK activation, yielded an interesting result. The mutant receptors specifically exerted a dominant negative effect on Galpha(q) activation, whereas ERK activation was preserved. These data suggest that distinctly active conformations of AT(1) oligomers can couple to each of these signaling systems and imply that oligomerization plays an active role in supporting these distinctly active conformations of AT(1) receptors.     

Cross-inhibition of angiotensin AT1 receptors supports the concept of receptor oligomerization

G protein-coupled receptors are cell surface receptors that mediate the effects of extracellular signals in the endocrine/paracrine and sensory systems. Experimental evidence is accumulating, which suggest that these receptors form dimers or higher order oligomers. The functional relevance of G protein-coupled receptor dimerization or oligomerization has been raised in a number of different processes, including ontogeny, internalization, ligand-induced regulation, pharmacological diversity and signal transduction of these receptors. Agonist-independent homo- and hetero-oligomerization of the angiotensin AT1 receptor has been reported, and it has been suggested that hetero-oligomerization with beta-adrenergic receptors leads to cross-inhibition of these receptors. Much less is known about the functional interactions between AT1 receptor homo-oligomers. The aim of the present study was to analyze the functional interactions between these homo-oligomers by determining the functions of normal, AT1 receptor blocker (candesartan) resistant (S109Y) and G protein coupling deficient (DRY/AAY) AT1 receptors (co-)expressed in COS-7 cells. Although we have found no evidence that stimulation of a G protein coupling deficient receptor could cross-activate co-expressed normal receptors, candesartan binding to a signaling deficient receptor caused cross-inhibition of co-expressed candesartan resistant AT1 receptors. Since the studied mutations were in the third intracellular helix of the receptor, the observed effects cannot be explained with domain swapping. These data suggest that AT1 receptor blockers cause cross-inhibition of homo-oligomerized AT1 receptors, and support the concept that receptor dimers/oligomers serve as the functional unit of G protein-coupled receptors.     

Metabotropic glutamate type 5, dopamine D2 and adenosine A2a receptors form higher-order oligomers in living cells

G protein-coupled receptors are known to form homo- and heteromers at the plasma membrane, but the stoichiometry of these receptor oligomers are relatively unknown. Here, by using bimolecular fluorescence complementation, we visualized for the first time the occurrence of heterodimers of metabotropic glutamate mGlu₅ receptors (mGlu₅R) and dopamine D₂ receptors (D₂R) in living cells. Furthermore, the combination of bimolecular fluorescence complementation and bioluminescence resonance energy transfer techniques, as well as the sequential resonance energy transfer technique, allowed us to detect the occurrence receptor oligomers containing more than two protomers, mGlu₅R, D₂R and adenosine A_2A receptor (A_2AR). Interestingly, by using high-resolution immunoelectron microscopy we could confirm that the three receptors co-distribute within the extrasynaptic plasma membrane of the same dendritic spines of asymmetrical, putative glutamatergic, striatal synapses. Also, co-immunoprecipitation experiments in native tissue demonstrated the existence of an association of mGlu₅R, D₂R and A_2AR in rat striatum homogenates. Overall, these results provide new insights into the molecular composition of G protein-coupled receptor oligomers in general and the mGlu₅R/D₂R/A_2AR oligomer in particular, a receptor oligomer that might constitute an important target for the treatment of some neuropsychiatric disorders.     

Chemokine oligomerization and interactions with receptors and glycosaminoglycans: the role of structural dynamics in function

The first chemokine structure, that of IL-8/CXCL8, was determined in 1990. Since then, many chemokine structures have emerged. To the initial disappointment of structural biologists, the tertiary structures of these small proteins were found to be highly conserved. However, they have since proven to be much more interesting and diverse than originally expected. Somewhat like lego blocks, many chemokines oligomerize and there is significant diversity in their oligomeric forms and propensity to oligomerize. Chemokines not only interact with receptors where different oligomeric forms can induce different signaling responses, they also interact with glycosaminoglycans which can stabilize oligomers and other structures that would not otherwise form in solution. Although chemokine monomers and dimers yielded quickly to structure determination, structural information about larger chemokine oligomers, chemokines receptors, and complexes of chemokines with glycosaminoglycans and receptors has been more difficult to obtain, but recent breakthroughs suggest that this information will be forthcoming, especially with receptor structures. Equally important and challenging, will be efforts to correlate the structural information with function.     

Regulation of heptaspanning-membrane-receptor function by dimerization and clustering

G-protein-coupled receptors form homomers and heteromers; agonist-induced conformational changes within interacting receptors of the oligomer modify their pharmacology, signalling and/or trafficking. When these receptors are activated, the oligomers rearrange and cluster and a novel mechanism of receptor-operation regulation by oligomer intercommunication is possible. This intercommunication would be assisted by components of the plasma membrane and by scaffolding proteins. Receptor cross-sensitization, cross-desensitization and novel, integrated receptor responses can then develop between oligomeric receptor complexes of the cluster without direct contact between them. This concept gives a new perspective to the understanding of neurotransmission and neuronal plasticity.     

The oligomeric state sets GABA(B) receptor signalling efficacy

G protein-coupled receptors (GPCRs) have key roles in cell-cell communication. Recent data suggest that these receptors can form large complexes, a possibility expected to expand the complexity of this regulatory system. Among the brain GPCRs, the heterodimeric GABA(B) receptor is one of the most abundant, being distributed in most brain regions, on either pre- or post-synaptic elements. Here, using specific antibodies labelled with time-resolved FRET compatible fluorophores, we provide evidence that the heterodimeric GABA(B) receptor can form higher-ordered oligomers in the brain, as suggested by the close proximity of the GABA(B1) subunits. Destabilizing the oligomers using a competitor or a GABA(B1) mutant revealed different G protein coupling efficiencies depending on the oligomeric state of the receptor. By examining, in heterologous system, the G protein coupling properties of such GABA(B) receptor oligomers composed of a wild-type and a non-functional mutant heterodimer, we provide evidence for a negative functional cooperativity between the GABA(B) heterodimers.     

T-cell activation by soluble MHC oligomers can be described by a two-parameter binding model

T-cell activation is essential for initiation and control of immune system function. T cells are activated by interaction of cell-surface antigen receptors with major histocompatibility complex (MHC) proteins on the surface of other cells. Studies using soluble oligomers of MHC-peptide complexes and other types of receptor cross-linking agents have supported an activation mechanism that involves T cell receptor clustering. Receptor clustering induced by incubation of T cells with MHC-peptide oligomers leads to the induction of T-cell activation processes, including downregulation of engaged receptors and upregulation of the cell-surface proteins CD69 and CD25. Dose-response curves for these T-cell activation markers are bell-shaped, with different maxima and midpoints, depending on the valency of the soluble oligomer used. In this study, we have analyzed the activation behavior using a mathematical model that describes the binding of multivalent ligands to cell-surface receptors. We show that a simple equilibrium binding model accurately describes the activation data for CD4(+) T cells treated with MHC-peptide oligomers of varying valency. The model can be used to predict activation and binding behavior for T cells and MHC oligomers with different properties.     

Biochemical and biophysical demonstration of GPCR oligomerization in mammalian cells

In contrast to other families of cell surface receptors, like tyrosine kinase receptors, for which dimerization is an integral part of the activation process, G-protein-coupled receptors (GPCRs) were thought, until recently, to function as monomeric units. However, a growing body of evidence indicates that GPCRs could exist and be active as oligomeric complexes. Because they are major pharmacological targets, their existence as homo- or hetero- oligomers could have important implications for the development and screening of new drugs. The major evidences supporting the idea of GPCR oligomerization come from indirect biochemical or pharmacological experiments. Here we report, using traditional co-immunoprecipitation methods, the existence of differentially epitope-tagged beta2-adrenergic receptor (beta2AR) oligomers in mammalian HEK-293 cells. Moreover, we validate the existence of receptor oligomers in living cells by a new Bioluminescence Resonance Energy Transfer (BRET) technique. Our results clearly demonstrate the presence of constitutive beta2AR oligomers in living cells that can be modulated by the selective adrenergic agonist isoproterenol, suggesting a pertinent physiological role for GPCR oligomerization.     

Oligomers of ERBB3 have two distinct interfaces that differ in their sensitivity to disruption by heregulin

ErbB receptors associate in a ligand-dependent or -independent manner, and overexpression of epidermal growth factor receptor (ErbB1) or ErbB2 results in ligand-independent activation. Ligand-independent activation is poorly understood, and dimerization alone is not sufficient for activation. ErbB receptors also form higher order oligomers, but the mechanism of oligomer formation and their contribution to signaling are not known. The kinase-deficient ErbB3 as well as its extracellular domains are particularly prone to ligand-independent oligomerization, and oligomers are destabilized by binding of the ligand heregulin. In contrast, ligand binding facilitates heterodimerization with ErbB2 and is expected to stabilize an extended conformation of the ErbB3 extracellular domain (ECD) in which the dimerization interface is exposed. In the absence of ligand, ErbB3 can adopt a closed conformation that is held together by an intramolecular tether. We used a constitutively extended form of the ErbB3-ECD to analyze the conformation of the ECD in oligomers and the mechanism of oligomer disruption by heregulin. The extended conformation of the ECD forms oligomers more readily, suggesting the crystallographically defined dimer interface is one of the interfaces involved in oligomerization. Heregulin destabilizes oligomeric complexes but not dimers, which are neither stabilized nor disrupted by ligand binding, indicating a distinct second interface in oligomers of ErbB3. Cross-linking and activation studies on membrane-embedded ErbB3/ErbB2 chimeras confirm this dual effect of heregulin. Most of the ErbB3-ECD on the cell surface is apparently kept in an open conformation through oligomerization, and the resulting oligomers adopt a conformation representing a state of reduced activity.     

Internalization dissociates β2-adrenergic receptors

G protein-coupled receptors (GPCRs) self-associate as dimers or higher-order oligomers in living cells. The stability of associated GPCRs has not been extensively studied, but it is generally thought that these receptors move between the plasma membrane and intracellular compartments as intact dimers or oligomers. Here we show that β(2)-adrenergic receptors (β(2)ARs) that self-associate at the plasma membrane can dissociate during agonist-induced internalization. We use bioluminescence-resonance energy transfer (BRET) to monitor movement of β(2)ARs between subcellular compartments. BRET between β(2)ARs and plasma membrane markers decreases in response to agonist activation, while at the same time BRET between β(2)ARs and endosome markers increases. Energy transfer between β(2)ARs is decreased in a similar manner if either the donor- or acceptor-labeled receptor is mutated to impair agonist binding and internalization. These changes take place over the course of 30 minutes, persist after agonist is removed, and are sensitive to several inhibitors of arrestin- and clathrin-mediated endocytosis. The magnitude of the decrease in BRET between donor- and acceptor-labeled β(2)ARs suggests that at least half of the receptors that contribute to the BRET signal are physically segregated by internalization. These results are consistent with the possibility that β(2)ARs associate transiently with each other in the plasma membrane, or that β(2)AR dimers or oligomers are actively disrupted during internalization.     

The extracellular N-terminal domain and transmembrane domains 1 and 2 mediate oligomerization of a yeast G protein-coupled receptor

G protein-coupled receptors (GPCRs) can form homodimers/oligomers and/or heterodimers/oligomers. The mechanisms used to form specific GPCR oligomers are poorly understood because the domains that mediate such interactions and the step(s) in the secretory pathway where oligomerization occurs have not been well characterized. Here we have used subcellular fractionation and fluorescence resonance energy transfer (FRET) experiments to show that oligomerization of a GPCR (alpha-factor receptor; STE2 gene product) of the yeast Saccharomyces cerevisiae occurs in the endoplasmic reticulum. To identify domains of this receptor that mediate oligomerization, we used FRET and endocytosis assays of oligomerization in vivo to analyze receptor deletion mutants. A mutant lacking the N-terminal extracellular domain and transmembrane (TM) domain 1 was expressed at the cell surface but did not self-associate. In contrast, a receptor fragment containing only the N-terminal extracellular domain and TM1 could self-associate and heterodimerize with wild type receptors. Analysis of other mutants suggested that oligomerization is facilitated by the N-terminal extracellular domain and TM2. Therefore, the N-terminal extracellular domain, TM1, and TM2 appear to stabilize alpha-factor receptor oligomers. These domains may form an interface in contact or domain-swapped oligomers. Similar domains may mediate dimerization of certain mammalian GPCRs.     

C5a receptor oligomerization. I. Disulfide trapping reveals oligomers and potential contact surfaces in a G protein-coupled receptor

G protein-coupled receptors (GPCRs), stimulated by hormones and sensory stimuli, act as molecular switches to relay activation to heterotrimeric G proteins. Recent studies suggest that GPCRs form dimeric or oligomeric structures, a phenomenon that has long been established for growth factor receptors. The elucidation of the domains of GPCRs that mediate receptor association is of critical importance for understanding the function of GPCR oligomers. Using a disulfide-trapping strategy to probe the intermolecular contact surfaces, we demonstrate cross-linking of C5a receptors in membranes prepared from both human neutrophils and stably transfected mammalian cells that is mediated by a cysteine in the second intracellular loop. To explore other surfaces that might be involved in the oligomerization of C5a receptors, we constructed receptors with individual cysteines in other intracellular regions. C5a receptors with a cysteine in the first intracellular loop or the carboxyl terminus displayed the fastest kinetics of dimer formation, whereas an intracellular loop 3 cysteine displayed minimal cross-linking. Since the rate of disulfide trapping reflects the proximity of sulfhydryl groups, assuming similar accessibility and flexibility, these results imply a symmetric dimer interface that may involve either transmembrane helices 1 and 2 or helix 4. However, neither model can account for the ability of the native cysteine in the second intracellular loop to mediate efficient crosslinking. Based on these observations, we propose that C5a receptors form higher order oligomers (i.e. tetramers) or clusters in the membrane.     

Cutting edge: detection of antigen-specific CD4+ T cells by HLA-DR1 oligomers is dependent on the T cell activation state

Class I MHC tetramers have proven to be invaluable tools for following and deciphering the CD8(+) T cell response, but the development of similar reagents for detection of CD4(+) T cells based on class II MHC proteins has been more difficult. We evaluated fluorescent streptavidin-based oligomers of HLA-DR1 for use as reagents to analyze Ag-specific human CD4(+) T cells. Staining was blocked at low temperatures and by drugs that disrupt microfilament formation and endocytosis. Cell-associated MHC oligomers were resistant to a surface stripping protocol and were observed by microscopy in intracellular compartments. This behavior indicates that detection of CD4(+) T cells using class II MHC oligomers can depend on an active cellular process in which T cells cluster and/or endocytose their Ag receptors. T cells of identical specificity but in different activation states varied greatly in their ability to be detected by class II MHC oligomers.     

Drp1-dependent mitochondrial fission via MiD49/51 is essential for apoptotic cristae remodeling

Mitochondrial fission facilitates cytochrome c release from the intracristae space into the cytoplasm during intrinsic apoptosis, although how the mitochondrial fission factor Drp1 and its mitochondrial receptors Mff, MiD49, and MiD51 are involved in this reaction remains elusive. Here, we analyzed the functional division of these receptors with their knockout (KO) cell lines. In marked contrast to Mff-KO cells, MiD49/MiD51-KO and Drp1-KO cells completely resisted cristae remodeling and cytochrome c release during apoptosis. This phenotype in MiD49/51-KO cells, but not Drp1-KO cells, was completely abolished by treatments disrupting cristae structure such as OPA1 depletion. Unexpectedly, OPA1 oligomers generally thought to resist cytochrome c release by stabilizing the cristae structure were similarly disassembled in Drp1-KO and MiD49/51-KO cells, indicating that disassembly of OPA1 oligomers is not directly linked to cristae remodeling for cytochrome c release. Together, these results indicate that Drp1-dependent mitochondrial fission through MiD49/MiD51 regulates cristae remodeling during intrinsic apoptosis.     

Methods to monitor the quaternary structure of G protein-coupled receptors

A wide range of approaches has been applied to examine the quaternary structure of G protein-coupled receptors, the basis of such protein-protein interactions and how such interactions might modulate the pharmacology and function of these receptors. These include co-immunoprecipitation, various adaptations of resonance energy transfer techniques, functional complementation studies and the analysis of ligand-binding data. Each of the available techniques has limitations that restrict interpretation of the data. However, taken together, they provide a coherent body of evidence indicating that many, if not all, G protein-coupled receptors exist and function as dimer/oligomers. Herein we assess the widely applied techniques and discuss the relative benefits and limitations of these approaches.     

G protein-coupled receptor dimerisation: molecular basis and relevance to function

The belief that G protein-coupled receptors exist and function as monomeric, non-interacting species has been largely supplanted in recent years by evidence, derived from a range of approaches, that indicate they can form dimers and/or higher-order oligomeric complexes. Key roles for receptor homo-dimerisation include effective quality control of protein folding prior to plasma membrane delivery and interactions with hetero-trimeric G proteins. Growing evidence has also indicated the potential for many co-expressed G protein-coupled receptors to form hetero-dimers/oligomers. The relevance of this to physiology and function is only beginning to be unravelled but may offer great potential for more selective therapeutic intervention.     

G protein-coupled receptor dimerisation: Molecular basis and relevance to function

The belief that G protein-coupled receptors exist and function as monomeric, non-interacting species has been largely supplanted in recent years by evidence, derived from a range of approaches, that indicate they can form dimers and/or higher-order oligomeric complexes. Key roles for receptor homo-dimerisation include effective quality control of protein folding prior to plasma membrane delivery and interactions with hetero-trimeric G proteins. Growing evidence has also indicated the potential for many co-expressed G protein-coupled receptors to form hetero-dimers/oligomers. The relevance of this to physiology and function is only beginning to be unravelled but may offer great potential for more selective therapeutic intervention.     

G protein-coupled receptor dimers: look like their parents,but act like teenagers!

Abstract

G protein-coupled receptors (GPCRs) represent the largest group of cell surface receptors and an important pharmacological target. Though originally thought to act in a one receptor–one effector fashion, it is now known that these receptors are capable of oligomerization and can function as dimers or higher order oligomers in native tissue. They do not only assemble with identical receptors as homodimers, but also associate with different GPCRs to form heterodimers. We discuss here how heterodimeric GPCRs can assemble, traffic and signal in a manner distinct from their individual receptor components or from homodimers. These receptor pairs are also demonstrated to be regulated by different chaperones, Rabs and scaffolding proteins, further emphasizing their potential as unique targets. We believe in the importance of investigating each GPCR heterodimer as an individual signaling complex, as they appear to act differently from each monomer constituting them. Just as teenagers may resemble their parents and share their genetic makeup, they can still act in a manner that is entirely unique!     

Nuclear localization drives α1-adrenergic receptor oligomerization and signaling in cardiac myocytes

Conventional models of G-protein coupled receptor (GPCR) signaling describe cell surface receptors binding to external ligands, such as hormones or circulating peptides, to induce intracellular signaling and a physiologic response. However, recent studies identify new paradigms indicating that GPCRs localize to and signal at the nucleus and that GPCR oligomers can influence receptor function. Previously, we reported that endogenous α1-adrenergic receptors (α1-ARs) localize to and signal at the nuclei in adult cardiac myocytes. In this study, we examined the mechanisms behind α1-AR nuclear localization and how nuclear localization impacted receptor function. We verified that endogenous α1-ARs localized to the nuclear membrane of intact nuclei isolated from wild-type adult cardiac myocytes. Next, we identified and disrupted putative nuclear localization sequences in both the α1A- and α1B-adrenergic receptors, which led to mis-localization of α1-ARs in cultured adult cardiac myocytes. Using these mutants, we demonstrated that nuclear localization was required for α1-signaling in adult cardiac myocytes. We also found that the nuclear export inhibitor leptomycin B inhibited α1-AR signaling, indicating α1-AR signaling must arise in the nucleus in adult cardiac myocytes. Finally, we found that co-localization of the α1-subtypes at the nuclei in adult cardiac myocytes facilitated the formation of receptor oligomers that could affect receptor signaling. In summary, our data indicate that α1-AR nuclear localization can drive the formation of receptor oligomers and regulate signaling in adult cardiac myocytes.