Quinacrine binds to the lipid-protein interface of the Torpedo acetylcholine receptor: a fluorescence study.

It has been argued both that there is a high affinity noncompetitive inhibitor binding site in the lumen of the acetylcholine receptor and that this lumen exists on the central axis of the receptor. Such a site would be expected to be 20-40 A from the membrane lipids. We tested whether, in fact, quinacrine, a potent fluorescent noncompetitive inhibitor, binds to such a site. We measured quenching of receptor-bound quinacrine fluorescence by fluorescence dipolar energy transfer to lipid probes, 5-(N-dodecanoylamino)eosin and N-(3-sulfopropyl)-4-(p-didecylaminostyryl)pyridinium, or by collision with paramagnetic lipid probes 2,2,6,6-tetramethylpiperidine-1-oxyl and 3-doxyl-17 beta-hydroxy-5 alpha-androstane (spin-labeled androstane). Initial control experiments established that in the presence of carbamylcholine, quinacrine binds to a phencyclidine-sensitive site on the Torpedo receptor with a Kd equal to 0.14 microM and with a quantum yield of 0.18. Fluorescence energy transfer from receptor-bound quinacrine had a magnitude consistent with quinacrine being less than 10 A from the lipid fluorescent probes. 2,2,6,6-Tetramethylpiperidine-1-oxyl and spin-labeled androstane were two to five times more effective at quenching receptor-bound quinacrine fluorescence than the fluorescence from membrane-partitioned 5-(dodecanoylamino)fluorescein. These results suggest that the quinacrine binding site is too close to the lipid domain to be in the lumen of the receptor, and therefore it is probably located on the outer surface of the membrane-spanning domain of the acetylcholine receptor.     

Application of the message-address concept to the docking of naltrexone and selective naltrexone-derived opioid antagonists into opioid receptor models

A binding site model for the opioid family of G-protein coupled receptors (GPCRs) is proposed based on the message-address concept of ligand recognition. Using ligand docking studies of the universal opioid antagonist, naltrexone, the structural basis for ‘message’ recognition is explored across all three receptor types, μ, δ, and κ. The binding mode proposed and basis for selectivity are also rationalized using the naltrexone-derived ligands, naltrindole (NTI) and norbinaltorphimine (nor BNI). These ligands are docked to the receptor according to the common naltrexone core or message. The resulting orientation places key ‘address’ elements in close proximity to amino acid residues critical to selectivity among receptor types. Selectivity is explained by sequence differences in the μ, δ, and κ receptors at these recognition points. Support for the model is derived from site directed mutagenesis studies and ligand binding data for the opioid receptors and other related GPCRs. Special issue dedicated to Dr. Eric J. Simon     

Mapping the molecular structure of the voltage-dependent sodium channel. Distances between the tetrodotoxin and Leiurus quinquestriatus quinquestriatus scorpion toxin receptors

The Leiurus quinquestriatus quinquestriatus receptor site of the voltage-dependent sodium channel has been characterized using several fluorescent scorpion toxins. The derivatives show fluorescence enhancements upon binding to the receptor site on the channel together with blue shifts. The fluorescence properties of the bound probes indicate a conformationally flexible, hydrophobic site. Binding of tetrodotoxin has no effect on the fluorescence spectra of the bound derivatives, whereas binding of the allosteric activator batrachotoxin enhances the fluorescence about 2-fold and causes a red shift in the emission spectra, suggesting a batrachotoxin-induced conformational change in the scorpion toxin receptor. The distance between the tetrodotoxin receptor and the Leiurus scorpion toxin receptor on the channel was measured by fluorescence resonance energy transfer. Five different chromophoric scorpion toxin derivatives were used as energy transfer acceptors or donors with anthraniloyltetrodotoxin or N-methylanthraniloylglycine-tetrodotoxin as the energy donor or acceptor. Because of the presence of three tetrodotoxin receptors for each Leiurus receptor, the positions of the donors and acceptors were exchanged. Efficiencies of transfer were measured by both donor quenching and sensitized emission. The average distance of separation between these sites is 35 A. Upon batrachotoxin addition, this distance changes to 42 A indicating a conformational change in one subunit of the channel or a change in the interaction between two subunits coupled to the batrachotoxin-binding site. On the basis of these studies, we present a model suggesting that tetrodotoxin binds to a subunit/site which is extracellularly placed and is 35 A from the Leiurus subunit/site which is located in a protein cleft of the channel which extends partly into the membrane, and undergoes a neurotoxin and voltage-dependent conformational change.     

Characterization of low density lipoprotein receptor ligand interactions by fluorescence resonance energy transfer

The low density lipoprotein receptor (LDLR) is the prototype of a family of cell surface receptors involved in a wide range of biological processes. A soluble low density lipoprotein receptor (sLDLR) and a tryptophan (Trp)-deficient variant human apolipoprotein E3 (apoE3) N-terminal domain (NT) were used in binding studies. The sole cysteine in apoE3-NT was covalently modified with an extrinsic fluorescence probe, N-(iodoacetyl)-N'-(5-sulfo-1-napthyl)ethylenediamine (AEDANS), and the protein was complexed with lipid. Incubation of sLDLR with AEDANS-Trp-null apoE3-NT dimyristoylphosphatidylcholine (DMPC) disks, but not lipid-free AEDANS-apoE, induced an enhancement in AEDANS fluorescence emission intensity (excitation, 280 nm) consistent with intermolecular energy transfer from excited Trp in sLDLR to receptor-bound apoE. Ligand binding to sLDLR required calcium and was saturable. In competition binding assays, unlabeled apoE3-NT DMPC inhibited AEDANS-apoE DMPC binding to sLDLR more effectively than low density lipoprotein. Fluorescence changes in this system reflected pH-dependent ligand binding and release from sLDLR consistent with models derived from the X-ray crystal structure of the receptor at endosomal pH. Intermolecular energy transfer from excited Trp in LDLR family members to fluorescently tagged ligands represents a sensitive and convenient assay for the characterization of the myriad molecular interactions ascribed to this family of receptor.     

Molecular modelling study of the role of cholesterol in the stimulation of the oxytocin receptor.

Cholesterol, an integral component of membranes in Eucaryota, is a modifier of membrane properties. In vivo studies have demonstrated that cholesterol can also modulate activities of some G protein-coupled receptors (GPCRs), which are integral membrane proteins. This can result either from an effect of cholesterol on the membrane fluidity or from specific interactions of the membrane cholesterol with the receptor, as recently demonstrated for the cholecystokinin type beta (CCKRbeta) or the oxytocin receptor (OTR). Using molecular modelling, we studied conformational preferences of cholesterol and several of its analogues. Subsequently, we simulated the distributions of their preferred conformations around the surface of OTR, CCKRbeta and a chimeric oxytocin/cholecystokinin receptor. Consequently, we suggest residues on the surface of OTR which are potentially significant in the OTR/cholesterol interaction.     

Ligand-stabilized conformational states of human beta(2) adrenergic receptor: insight into G-protein-coupled receptor activation

G-protein-coupled receptors (GPCRs) are known to exist in dynamic equilibrium between inactive- and several active-state conformations, even in the absence of a ligand. Recent experimental studies on the β₂ adrenergic receptor (β₂AR) indicate that structurally different ligands with varying efficacies trigger distinct conformational changes and stabilize different receptor conformations. We have developed a computational method to study the ligand-induced rotational orientation changes in the transmembrane helices of GPCRs. This method involves a systematic spanning of the rotational orientation of the transmembrane helices (TMs) that are in the vicinity of the ligand for predicting the helical rotations that occur on ligand binding. The predicted ligand-stabilized receptor conformations are characterized by a simultaneous lowering of the ligand binding energy and a significant gain in interhelical and receptor-ligand hydrogen bonds. Using the β₂AR as a model, we show that the receptor conformational state depends on the structure and efficacy of the ligand for a given signaling pathway. We have studied the ligand-stabilized receptor conformations of five different ligands, a full agonist, norepinephrine; a partial agonist, salbutamol; a weak partial agonist, dopamine; a very weak agonist, catechol; and an inverse agonist, ICI-115881. The predicted ligand-stabilized receptor models correlate well with the experimentally observed conformational switches in β₂AR, namely, the breaking of the ionic lock between R131^3.50 at the intracellular end of TM3 (part of the DRY motif) and E268^6.30 on TM6, and the rotamer toggle switch on W286^6.48 on TM6. In agreement with trp-bimane quenching experiments, we found that norepinephrine and dopamine break the ionic lock and engage the rotamer toggle switch, whereas salbutamol, a noncatechol partial agonist only breaks the ionic lock, and the weak agonist catechol only engages the rotamer toggle switch. Norepinephrine and dopamine occupy the same binding region, between TM3, TM5, and TM6, whereas the binding site of salbutamol is shifted toward TM4. Catechol binds deeper into the protein cavity compared to the other ligands, making contact with TM5 and TM6. A part of the catechol binding site overlaps with those of dopamine and norepinephrine but not with that of salbutamol. Virtual ligand screening on 10,060 ligands on the norepinephrine-stabilized receptor conformation shows an enrichment of 38% compared to ligand unbound receptor conformation. These results show that ligand-induced conformational changes are important for developing functionally specific drugs that will stabilize a particular receptor conformation. These studies represent the first step toward a more universally applicable computational method for studying ligand efficacy and GPCR activation.     

Peptide interactions with G-protein coupled receptors.

Peptide recognition by G-protein coupled receptors (GPCRs) is reviewed with an emphasis on the indirect approach used to determine the receptor-bound conformation of peptide ligands. This approach was developed in response to the lack of detailed structural information available for these receptors. Recent advances in the structural determination of rhodopsin (the GPCR of the visual system) by crystallography have provided a scaffold for homology modeling of the inactive state of a wide variety of GPCRs that interact with peptide messages. Additionally, the ability to mutate GPCRs and assay compounds of similar chemical structure to test a common binding site on the receptor provides a firm experimental basis for structure-activity studies. Recognition motifs, common in other well-studied systems such as proteolytic enzymes and major histocompatibility class receptors (MHC) are reviewed briefly to provide a basis of comparison. Finally, the development of true peptidomimetics is contrasted with nonpeptide ligands, discovered through combinatorial chemistry. In many systems, the evidence suggests that the peptide ligands bind at the interface between the transmembrane segments and the extracellular loops, while nonpeptide antagonists bind within the transmembrane segments. Plausible models of GPCRs and the mechanism by which they activate G-proteins on binding peptides are beginning to emerge.     

Live-cell microscopy or fluorescence anisotropy with budded baculoviruses—which way to go with measuring ligand binding to M4 muscarinic receptors?

M₄ muscarinic acetylcholine receptor is a G protein-coupled receptor (GPCR) that has been associated with alcohol and cocaine abuse, Alzheimer''s disease, and schizophrenia which makes it an interesting drug target. For many GPCRs, the high-affinity fluorescence ligands have expanded the options for high-throughput screening of drug candidates and serve as useful tools in fundamental receptor research. Here, we explored two TAMRA-labelled fluorescence ligands, UR-MK342 and UR-CG072, for development of assays for studying ligand-binding properties to M₄ receptor. Using budded baculovirus particles as M₄ receptor preparation and fluorescence anisotropy method, we measured the affinities and binding kinetics of both fluorescence ligands. Using the fluorescence ligands as reporter probes, the binding affinities of unlabelled ligands could be determined. Based on these results, we took a step towards a more natural system and developed a method using live CHO-K1-hM₄R cells and automated fluorescence microscopy suitable for the routine determination of unlabelled ligand affinities. For quantitative image analysis, we developed random forest and deep learning-based pipelines for cell segmentation. The pipelines were integrated into the user-friendly open-source Aparecium software. Both image analysis methods were suitable for measuring fluorescence ligand saturation binding and kinetics as well as for screening binding affinities of unlabelled ligands.     

Studies on the electrogenic action of acetylcholine with Torpedo marmorata electric organ: IV. Quinacrine: a fluorescent probe for the conformational transitions of the cholinergic receptor protein in its membrane-bound state

Quinacrine, like a typical local anaesthetic, blocks the response of Electrophorus electricus electroplaque in vivo in a non-competitive manner and enhances, in vitro, the affinity of the cholinergic receptor present in Torpedo marmorata membrane fragments for acetylcholine. The interaction of quinacrine with T. marmorata membrane fragments can be followed by differential fluorescence spectroscopy either upon direct illumination (λ_Ex = 350 nm) or by energy transfer from membrane proteins (λ_Ex = 290 nm). Carbamylcholine and most of the cholinergic ligands tested cause an increase of the light intensity emitted by membrane-bound quinacrine under conditions of direct excitation; all these effects are blocked by a preincubation of the membrane fragments with the α-toxin from Naja nigricollis. When quinacrine is excited by energy transfer, carbamylcholine, phenyltrimethylammonium and hexamethonium cause an increase of fluorescence but flaxedil, tetraethylammonium and the α-toxin give a much smaller fluorescence increase or none.Local anaesthetics like prilocaine or quotane cause a decrease of fluorescence intensity of membrane-bound quinacrine in both the presence and absence of carbamylcholine. Quantitative studies on quinacrine binding and fluorescence as a function of quinacrine concentration reveal at least two populations (saturable and non-saturable) of binding sites, the saturable one being identical or closely related to the specific site of action of local anaesthetics. It is concluded that binding of cholinergic ligands primarily increases the quantum yield of a fraction of bound quinacrine.The curves of variation of fluorescence intensity with agonist and antagonist concentrations determined under conditions of direct illumination, closely resemble the binding curves determined at equilibrium with radioactive ligands. Under these conditions quinacrine therefore enables us to determine the occupancy of the receptor site by cholinergic ligands. On the other hand, the change of quinacrine fluorescence observed by energy transfer, which takes place with some of the cholinergic ligands but not with others, and does not correlate with any variation of the intrinsic fluorescence of membrane proteins, most likely reflects a change of structure bearing a qualitative relationship to the pharmacological activity of the tested ligands.     

Elucidating the molecular interaction of serum albumin with nizatidine and the role of β-cyclodextrin: multi-spectroscopic and computational approach

Abstract

Nizatidine is a histamine H2 receptor antagonist which act by inhibiting the production of stomach acid, thereby, finds its application in treating various diseases related to the gastrointestinal tract. Studying albumin–drug interaction is important for understanding the pharmacokinetics and pharmacodynamics of therapeutic candidates. In the present work, the interaction of nizatidine with BSA was investigated by employing multi-spectroscopic and computational studies. The formation of BSA–nizatidine complex was characterised by UV-visible and fluorescence based-spectroscopic studies. Steady-state fluorescence demonstrated the static mode of quenching of BSA by nizatidine. The interaction was spontaneous and nizatidine binds to BSA with a stoichiometry of 1:1. Forster resonance energy transfer calculations revealed that there was a high possibility of energy transfer between nizatidine and BSA. The resultant secondary structural change in BSA on the addition of nizatidine was studied by circular dichroism spectroscopy. Moreover, synchronous and three-dimensional fluorescence spectroscopy was used to determine the conformational changes occurred in the structure of albumin on the binding of nizatidine. Competitive-site marker experiments suggested that nizatidine binds in the Sudlow site II of BSA. Additionally, the effect of β-cyclodextrin as an inclusion compound on the interaction was studied. Furthermore, molecular modelling and simulation studies were performed to corroborate the results obtained above.

Communicated by Ramaswamy H. Sarma     

3D modeling, ligand binding and activation studies of the cloned mouse delta, mu; and kappa opioid receptors

Refined 3D models of the transmembrane domains of the cloned delta, mu and kappa opioid receptors belonging to the superfamily of G-protein coupled receptors (GPCRs) were constructed from a multiple sequence alignment using the alpha carbon template of rhodopsin recently reported. Other key steps in the procedure were relaxation of the 3D helix bundle by unconstrained energy optimization and assessment of the stability of the structure by performing unconstrained molecular dynamics simulations of the energy optimized structure. The results were stable ligand-free models of the TM domains of the three opioid receptors. The ligand-free delta receptor was then used to develop a systematic and reliable procedure to identify and assess putative binding sites that would be suitable for similar investigation of the other two receptors and GPCRs in general. To this end, a non-selective, 'universal' antagonist, naltrexone, and agonist, etorphine, were used as probes. These ligands were first docked in all sites of the model delta opioid receptor which were sterically accessible and to which the protonated amine of the ligands could be anchored to a complementary proton-accepting residue. Using these criteria, nine ligand-receptor complexes with different binding pockets were identified and refined by energy minimization. The properties of all these possible ligand-substrate complexes were then examined for consistency with known experimental results of mutations in both opioid and other GPCRs. Using this procedure, the lowest energy agonist-receptor and antagonist-receptor complexes consistent with these experimental results were identified. These complexes were then used to probe the mechanism of receptor activation by identifying differences in receptor conformation between the agonist and the antagonist complex during unconstrained dynamics simulation. The results lent support to a possible activation mechanism of the mouse delta opioid receptor similar to that recently proposed for several other GPCRs. They also allowed the selection of candidate sites for future mutagenesis experiments.     

Fluorescence Characteristics of Hydrophobic Partial Agonist Probes of the Cholecystokinin Receptor

Fluorescence spectroscopic studies are powerful tools for the evaluation of receptor structure and the dynamic changes associated with receptor activation. Here, we have developed two chemically distinct fluorescent probes of the cholecystokinin (CCK) receptor by attaching acrylodan or a nitrobenzoxadiazole moiety to the amino terminus of a partial agonist CCK analogue. These two probes were able to bind to the CCK receptor specifically and with high affinity, and were able to elicit only submaximal intracellular calcium responses typical of partial agonists. The fluorescence characteristics of these probes were compared with those previously reported for structurally-related full agonist and antagonist probes. Like the previous probes, the partial agonist probes exhibited longer fluorescence lifetimes and increased anisotropy when bound to the receptor than when free in solution. The receptor-bound probes were not easily quenched by potassium iodide, suggesting that the fluorophores were protected from the extracellular aqueous milieu. The fluorescence characteristics of the partial agonist probes were quite similar to those of the analogous full agonist probes and quite distinct from the analogous antagonist probes. These data suggest that the partially activated conformational state of this receptor is more closely related to its fully active state than to its inactive state.     

Mapping the binding sites of peptide and non-peptide molecules to G protein-coupled receptors by fluorescence

Novel fluorescence approaches to investigate ligand recognition and structure of G protein-coupled receptors in native membranes have been developed. These methods combine the biosynthetic incorporation of unnatural fluorescent amino acids at known sites in receptors with the technique of fluorescence energy transfer for distance measurement. This permits one to fix the ligand in space and to define the structure of the receptor in a model of ligand–receptor interactions. Subdomains of ligand binding sites on NK1 and NK2 receptors were also characterized using environment-sensitive fluorophores and the techniques of collisional quenching and anisotropy. Antagonists and agonists have different binding sites on NK1 and NK2.     

Ligands differentially modulate the protein interactions of the human estrogen receptors alpha and beta

The interactions of human estrogen receptor subtypes ERalpha and ERbeta with DNA and a 210 amino acid residue fragment of the coactivator protein SRC-1 bearing three nuclear receptor interaction motifs were investigated quantitatively using fluorescence anisotropy in the presence of agonist and antagonist ligands. ERalpha and ERbeta were found to bind in a similar manner to DNA, and both salt and temperature affected the affinity and/or stoichiometry of these interactions. The agonist ligands estradiol, estrone and estriol did not modify the binding of ERalpha to the fluorescein-labeled target estrogen response element. However, in the case of ERbeta, these ligands led to the formation of some higher-order protein-DNA complexes and a small decrease in affinity. The partial agonist 4-hydroxytamoxifen had little effect on either ER subtype, whereas the pure antagonist ICI 182,780 led to the cooperative formation of protein-DNA complexes of higher order than dimer, as further demonstrated by competition experiments and gel mobility-shift assays. In addition to DNA binding, the interaction of both ER subtypes with the Alexa488-labeled SRC-1 coactivator fragment was investigated by fluorescence anisotropy. The agonist ligands estrone, estradiol, estriol, genistein and ethynyl estradiol exhibited distinct capacities for inducing the recruitment of SRC-1 that were not correlated with their affinity for the receptor. Moreover, estrone and genistein exhibited subtype specificity in that they induced SRC-1 recruitment to ERbeta with much higher efficiency than in the case of ERalpha. The differential coactivator recruitment capacities of the ER agonists and their receptor subtype coactivator recruitment specificity may be linked to the molecular structure of the agonists with respect to their interactions with a specific histidine residue located at the back of the ligand-binding pocket. Altogether, these quantitative in vitro studies of ER interactions reveal the complex energetic and stoichiometric consequences of changes in the chemical structures of these proteins and their ligands.     

Differences in nucleotide-binding site of isoapyrases deduced from tryptophan fluorescence

Comparative studies of intrinsic and extrinsic fluorescence of apyrases purified from two potato tuber varieties (Pimpernel and Desirée) were performed to determine differences in the microenvironment of the nucleotide binding site. The dissociation constants (K(d)) of Pimpernel apyrase for the binding of different fluorescent substrate analogs: methylanthranoyl (MANT-), trinitrophenyl (TNP-), and epsilon -derivatives of ATP and ADP were determined from the quenching of Trp fluorescence, and compared with K(d) values previously reported for Desirée enzyme. Binding of non-fluorescent substrate analogues decreased the Trp emission of both isoapyrases, indicating conformational changes in the vicinity of these residues. Similar effect was observed with fluorescent derivatives where, in the quenching effect, the transfer of energy from tryptophan residues to the fluorophore moiety could be additionally involved. The existence of energy transfer between Trp residues in the Pimpernel enzyme was demonstrated with epsilon -analogues, similar to our previous observations with the Desirée. From these results we deduced that tryptophan residues are close to or in the nucleotide binding site in both enzymes. Experiments with quenchers like acrylamide, Cs(+) and I(-), both in the presence and absence of nucleotide analogues, suggest the existence of differences in the nucleotide binding site of the two enzymes. From the results obtained in this work, we can conclude that the differences found in the microenvironment of the nucleotide binding site can explain, at least in part, the kinetic behaviour of both isoenzymes.     

Fluorescence imaging of two-photon linear dichroism: cholesterol depletion disrupts molecular orientation in cell membranes

The plasma membrane of cells is an ordered environment, giving rise to anisotropic orientation and restricted motion of molecules and proteins residing in the membrane. At the same time as being an organized matrix of defined structure, the cell membrane is heterogeneous and dynamic. Here we present a method where we use fluorescence imaging of linear dichroism to measure the orientation of molecules relative to the cell membrane. By detecting linear dichroism as well as fluorescence anisotropy, the orientation parameters are separated from dynamic properties such as rotational diffusion and homo energy transfer (energy migration). The sensitivity of the technique is enhanced by using two-photon excitation for higher photo-selection compared to single photon excitation. We show here that we can accurately image lipid organization in whole cell membranes and in delicate structures such as membrane nanotubes connecting two cells. The speed of our wide-field imaging system makes it possible to image changes in orientation and anisotropy occurring on a subsecond timescale. This is demonstrated by time-lapse studies showing that cholesterol depletion rapidly disrupts the orientation of a fluorophore located within the hydrophobic region of the cell membrane but not of a surface bound probe. This is consistent with cholesterol having an important role in stabilizing and ordering the lipid tails within the plasma membrane.     

Are specific nonannular cholesterol binding sites present in G-protein coupled receptors?

The G-protein coupled receptors (GPCRs) are the largest class of molecules involved in signal transduction across membranes, and represent major drug targets in all clinical areas. Membrane cholesterol has been reported to have a modulatory role in the function of a number of GPCRs. Interestingly, recently reported crystal structures of GPCRs have shown structural evidence of cholesterol binding sites. Two possible mechanisms have been previously suggested by which membrane cholesterol could influence the structure and function of GPCRs (i) through a direct/specific interaction with GPCRs, which could induce a conformational change in the receptor, or (ii) through an indirect way by altering the membrane physical properties in which the receptor is embedded or due to a combination of both. We discuss here a novel mechanism by which membrane cholesterol could affect structure and function of GPCRs and propose that cholesterol binding sites in GPCRs could represent ‘nonannular’ binding sites. Interestingly, previous work from our laboratory has demonstrated that membrane cholesterol is required for the function of the serotonin_1A receptor, which could be due to specific interaction of the receptor with cholesterol. Based on these results, we envisage that there could be specific/nonannular cholesterol binding site(s) in the serotonin_1A receptor. We have analyzed putative cholesterol binding sites from protein databases in the serotonin_1A receptor, a representative GPCR, for which we have previously demonstrated specific requirement of membrane cholesterol for receptor function. Our analysis shows that cholesterol binding sites are inherent characteristic features of serotonin_1A receptors and are conserved over evolution. Progress in deciphering molecular details of the nature of GPCR-cholesterol interaction in the membrane would lead to better insight into our overall understanding of GPCR function in health and disease, thereby enhancing our ability to design better therapeutic strategies to combat diseases related to malfunctioning of GPCRs.     

C5a receptor oligomerization. II. Fluorescence resonance energy transfer studies of a human G protein-coupled receptor expressed in yeast

Recent studies demonstrate that members of the superfamily of G protein-coupled receptors (GPCRs) form oligomers both in vitro and in vivo. The mechanisms by which GPCRs oligomerize and the roles of accessory proteins in this process are not well understood. We used disulfide-trapping experiments to show that C5a receptors, expressed in mammalian cells, reside in membranes as oligomers (Klco, J. M., Lassere, T. B., and Baranski, T. J. (2003) J. Biol. Chem. 278, 35345-35353). To begin to address how C5a receptors form oligomers, we now use fluorescence resonance energy transfer experiments on human C5a receptors expressed in the lower eukaryote Saccharomyces cerevisiae. C5a receptors tagged with variants of the green fluorescent protein display energy transfer in intact yeast, demonstrating that mammalian accessory proteins are not required for C5a receptor oligomerization. In both intact yeast cells and membrane preparations, agonist does not affect FRET efficiency, and little energy transfer is observed between the C5a receptor and a co-expressed yeast pheromone receptor (encoded by STE2), indicating that C5a receptor oligomerization is both receptor-specific and constitutive. FRET studies performed on fractionated membranes demonstrate similar levels of energy transfer between tagged C5a receptors in endoplasmic reticulum compared with plasma membrane, and urea washing of membranes has little effect on the extent of energy transfer. The oligomerization of C5a receptors expressed in yeast displays characteristics similar to those observed for other GPCRs studied in mammalian cells. This model system should prove useful for further studies to define mechanisms of oligomerization of mammalian GPCRs.     

Changes in the distribution of platelet membrane proteins revealed by energy transfer

The redistribution of platelet membrane proteins in response to platelet activation was studied. To investigate this process we prepared a variety of platelet ligands, including di- and tetrameric concanavalin A, IgG, thrombin, wheat-germ agglutinin and other lectins. These ligands were conjugated either with acceptor (rhodamine isothiocyanate) or donor (fluoresceine isothiocyanate) fluorophore. Platelets exposed to various combinations of ligand species were stimulated with different aggregating agents, and changes in sensitized fluorescence emission or donor quenching were recorded. Energy transfer was observed with thrombin, dimeric concanavalin A after addition of thrombin and various combinations of dimeric concanavalin A with other membrane ligands. The preincubation of platelets with colchicine prevented energy transfer between appropriate ligand pairs and platelet activator. Our studies show that nonradiative energy transfer can be used to analyze redistribution of membrane receptor sites in platelets.