Changes in conformation at the cytoplasmic ends of the fifth and sixth transmembrane helices of a yeast G protein-coupled receptor in response to ligand binding

The third intracellular loop (IL3) of G protein-coupled receptors (GPCRs) is an important contact domain between GPCRs and their G proteins. Previously, the IL3 of Ste2p, a Saccharomyces cerevisiae GPCR, was suggested to undergo a conformational change upon activation as detected by differential protease susceptibility in the presence and absence of ligand. In this study using disulfide cross-linking experiments we show that the Ste2p cytoplasmic ends of helix 5 (TM5) and helix 6 (TM6) that flank the amino and carboxyl sides of IL3 undergo conformational changes upon ligand binding, whereas the center of the IL3 loop does not. Single Cys substitution of residues in the middle of IL3 led to receptors that formed high levels of cross-linked Ste2p, whereas Cys substitution at the interface of IL3 and the contiguous cytoplasmic ends of TM5 and TM6 resulted in minimal disulfide-mediated cross-linked receptor. The alternating pattern of residues involved in cross-linking suggested the presence of a 3(10) helix in the middle of IL3. Agonist (WHWLQLKPGQPNleY) induced Ste2p activation reduced cross-linking mediated by Cys substitutions at the cytoplasmic ends of TM5 and TM6 but not by residues in the middle of IL3. Thus, the cytoplasmic ends of TM5 and TM6 undergo conformational change upon ligand binding. An α-factor antagonist (des-Trp, des-His-α-factor) did not influence disulfide-mediated Ste2p cross-linking, suggesting that the interaction of the N-terminus of α-factor with Ste2p is critical for inducing conformational changes at TM5 and TM6. We propose that the changes in conformation revealed for residues at the ends of TM5 and TM6 are affected by the presence of G protein but not G protein activation. This study provides new information about role of specific residues of a GPCR in signal transduction and how peptide ligand binding activates the receptor.     

High and low affinity receptors for interleukin 2: implications of pronase, phorbol ester, and cell membrane studies upon the basis for differential ligand affinities

Cellular binding sites for IL 2 exist in two forms which differ with respect to their apparent affinity for the factor. The present studies were designed to evaluate various models for the difference. Receptor-mediated internalization and covalent receptor-ligand coupling were discounted as explanations on the basis of ligand binding and elution studies on permeabilized cells and cell membranes. Phosphorylation of the receptor during activation of protein kinase C failed to modulate the ratio of high and low affinity sites, demonstrating that it also did not provide a potential mechanism. Selective destruction of low affinity receptors with pronase, on the other hand, indicated that the two forms of binding sites differed significantly in their cell surface structure. Either the two types of receptor consist of distinct molecules or the conformation of the high affinity binding sites renders them more resistant to proteolysis. Antibody inhibition studies revealed that the high affinity receptors remaining after protease treatment and their low affinity counterparts both utilized the same ligand-binding component. Thus, this result ruled out the possibility of two totally distinct receptor structures. Together, the findings support the hypothesis that other membrane components modify the conformation of the ligand-binding polypeptide to confer a high affinity protease-resistant configuration.     

Identification of residue-to-residue contact between a peptide ligand and its G protein-coupled receptor using periodate-mediated dihydroxyphenylalanine cross-linking and mass spectrometry

Fundamental knowledge about how G protein-coupled receptors and their ligands interact is important for understanding receptor-ligand binding and the development of new drug discovery strategies. We have used cross-linking and tandem mass spectrometry analyses to investigate the interaction of the N terminus of the Saccharomyces cerevisiae tridecapeptide pheromone, α-factor (WHWLQLKPGQPMY), and Ste2p, its cognate G protein-coupled receptor. The Trp(1) residue of α-factor was replaced by 3,4-dihydroxyphenylalanine (DOPA) for periodate-mediated chemical cross-linking, and biotin was conjugated to Lys(7) for detection purposes to create the peptide [DOPA(1),Lys(7)(BioACA),Nle(12)]α-factor, called Bio-DOPA(1)-α-factor. This ligand analog was a potent agonist and bound to Ste2p with ～65 nanomolar affinity. Immunoblot analysis of purified Ste2p samples that were treated with Bio-DOPA(1)-α-factor showed that the peptide analog cross-linked efficiently to Ste2p. The cross-linking was inhibited by the presence of either native α-factor or an α-factor antagonist. MALDI-TOF and immunoblot analyses revealed that Bio-DOPA(1)-α-factor cross-linked to a fragment of Ste2p encompassing residues Ser(251)-Met(294). Fragmentation of the cross-linked fragment and Ste2p using tandem mass spectrometry pinpointed the cross-link point of the DOPA(1) of the α-factor analog to the Ste2p Lys(269) side chain near the extracellular surface of the TM6-TM7 bundle. This conclusion was confirmed by a greatly diminished cross-linking of Bio-DOPA(1)-α-factor into a Ste2p(K269A) mutant. Based on these and previously obtained binding contact data, a mechanism of α-factor binding to Ste2p is proposed. The model for bound α-factor shows how ligand binding leads to conformational changes resulting in receptor activation of the signal transduction pathway.     

Molecular determinants of glucocorticoid receptor mobility in living cells: the importance of ligand affinity

The actions of glucocorticoids are mediated by the glucocorticoid receptor (GR), which is activated upon ligand binding, and can alter the expression of target genes either by transrepression or transactivation. We have applied FRAP (fluorescence recovery after photobleaching) to quantitatively assess the mobility of the yellow fluorescent protein (YFP)-tagged human GR alpha-isoform (hGRalpha) in the nucleus of transiently transfected COS-1 cells and to elucidate determinants of its mobility. Addition of the high-affinity agonist dexamethasone markedly decreases the mobility of the receptor in a concentration-dependent manner, whereas low-affinity ligands like corticosterone decrease the mobility to a much lesser extent. Analysis of other hGRalpha ligands differing in affinity suggests that it is the affinity of the ligand that is a major determinant of the decrease in mobility. Similar results were observed for two hGRalpha antagonists, the low-affinity antagonist ZK98299 and the high-affinity antagonist RU486. The effect of ligand affinity on mobility was confirmed with the hGRalpha mutant Q642V, which has an altered affinity for triamcinolone acetonide, dexamethasone, and corticosterone. Analysis of hGRalpha deletion mutants indicates that both the DNA-binding domain and the ligand-binding domain of the receptor are required for a maximal ligand-induced decrease in receptor mobility. Interestingly, the mobility of transfected hGRalpha differs among cell types. Finally, the proteasome inhibitor MG132 immobilizes a subpopulation of unliganded receptors, via a mechanism requiring the DNA-binding domain and the N-terminal part of the ligand-binding domain. Ligand binding makes the GR resistant to the immobilizing effect of MG132, and this effect depends on the affinity of the ligand. Our data suggest that ligand binding induces a conformational change of the receptor which is dependent on the affinity of the ligand. This altered conformation decreases the mobility of the receptor, probably by targeting the receptor to relatively immobile nuclear domains with which it transiently associates. In addition, this conformational change blocks immobilization of the receptor by MG132.     

Substitutions in the periplasmic domain of low-abundance chemoreceptor trg that induce or reduce transmembrane signaling: kinase activation and context effects

We extended characterization of mutational substitutions in the ligand-binding region of Trg, a low-abundance chemoreceptor of Escherichia coli. Previous investigations using patterns of adaptational methylation in vivo led to the suggestion that one class of substitutions made the receptor insensitive, reducing ligand-induced signaling, and another mimicked ligand occupancy, inducing signaling in the absence of ligand. We tested these deductions with in vitro assays of kinase activation and found that insensitive receptors activated the kinase as effectively as wild-type receptors and that induced-signaling receptors exhibited the low level of kinase activation characteristic of occupied receptors. Differential activation by the two mutant classes was not dependent on high-abundance receptors. Cellular context can affect the function of low-abundance receptors. Assays of chemotactic response and adaptational modification in vivo showed that increasing cellular dosage of mutant forms of Trg to a high-abundance level did not significantly alter phenotypes, nor did the presence of high-abundance receptors significantly correct phenotypic defects of reduced-signaling receptors. In contrast, defects of induced-signaling receptors were suppressed by the presence of high-abundance receptors. Grafting the interaction site for the adaptational-modification enzymes to the carboxyl terminus of induced-signaling receptors resulted in a similar suppression of phenotypic defects of induced-signaling receptors, implying that high-abundance receptors could suppress defects in induced-signaling receptors by providing their natural enzyme interaction sites in trans in clusters of suppressing and suppressed receptors. As in the case of cluster-related functional assistance provided by high-abundance receptors for wild-type low-abundance receptors, suppression by high-abundance receptors of phenotypic defects in induced-signaling forms of Trg involved assistance in adaptation, not signaling.     

Novobiocin and peptide analogs of α-factor are positive allosteric modulators of the yeast G protein-coupled receptor Ste2p

G protein-coupled receptors (GPCRs) are the target of many drugs prescribed for human medicine and are therefore the subject of intense study. It has been recognized that compounds called allosteric modulators can regulate GPCR activity by binding to the receptor at sites distinct from, or overlapping with, that occupied by the orthosteric ligand. The purpose of this study was to investigate the nature of the interaction between putative allosteric modulators and Ste2p, a model GPCR expressed in the yeast Saccharomyces cerevisiae that binds the tridecapeptide mating pheromone α-factor. Biological assays demonstrated that an eleven amino acid α-factor analog and the antibiotic novobiocin were positive allosteric modulators of Ste2p. Both compounds enhanced the biological activity of α-factor, but did not compete with α-factor binding to Ste2p. To determine if novobiocin and the 11-mer shared a common allosteric binding site, a biologically-active analog of the 11-mer peptide ([Bio-DOPA]11-mer) was chemically cross-linked to Ste2p in the presence and absence of novobiocin. Immunoblots probing for the Ste2p–[Bio-DOPA]11-mer complex revealed that novobiocin markedly decreased cross-linking of the [Bio-DOPA]11-mer to the receptor, but cross-linking of the α-factor analog [Bio-DOPA]13-mer, which interacts with the orthosteric binding site of the receptor, was minimally altered. This finding suggests that both novobiocin and [Bio-DOPA]11-mer compete for an allosteric binding site on the receptor. These results indicate that Ste2p may provide an excellent model system for studying allostery in a GPCR.     

Role of Cross-Cleft Contacts in NMDA Receptor Gating

In response to brief glutamate exposure, NMDA receptors produce excitatory currents that have sub-maximal amplitudes and characteristically slow kinetics. The activation sequence starts when glutamate binds to residues located on the upper lobe of extracellularly located ligand-binding domains (LBDs) and then contacts lower lobe residues to bridge the cleft between the two hinged lobes. This event stabilizes a narrow-cleft LBD conformation and may facilitate subsequent inter-lobe contacts that further stabilize the closed cleft. Agonist efficacy has been traced to the degree of agonist-induced cleft-closure and may also depend on the stability of the closed-cleft conformation. To investigate how cross-cleft contacts contribute to the amplitude and kinetics of NMDA receptor response, we examined the activation reaction of GluN1/GluN2A receptors that had single-residue substitutions at the interface between LBD lobes. We found that side-chain truncations at residues of putative contact between lobes increased glutamate efficacy through independent additive mechanisms in GluN1 and GluN2A subunits. In contrast, removing side-chain charge with isosteric substitutions at the same sites decreased glutamate efficacy. These results support the view that in GluN1/GluN2A receptors’ natural interactions between residues on opposing sides of the ligand-binding cleft encode the stability of the glutamate-bound closed-cleft conformations and limit the degree of cleft closure, thus contributing to the sub-maximal response and emblematically slow NMDA receptor deactivation after brief stimulation.     

Membrane-displayed peptide ligand activates the pheromone response pathway in Saccharomyces cerevisiae

The budding yeast, Saccharomyces cerevisiae, is an attractive host for studying G protein-coupled receptors (GPCRs). We developed a system in which a peptide ligand specific for GPCR is displayed on yeast plasma membrane. The model system described here is based on yeast plasma membrane display of an analogue of α-factor, which is a peptide ligand for Ste2p, the GPCR that activates the yeast pheromone response pathway. α-Factor analogues, containing linkers of varying lengths and produced in yeast cells, became attached to the cell plasma membrane by linking to the glycosylphosphatidylinositol (GPI)-anchored plasma membrane protein Yps1p. We were able to demonstrate that an optimized α-factor analogue activated the pheromone response pathway in S. cerevisiae, as assessed by a fluorescent reporter assay. Furthermore, it was shown that linker length strongly influenced signalling pathway activation. To our knowledge, this is the first report documenting functional signalling by a plasma membrane-displayed ligand in S. cerevisiae.     

Temperature-sensitive mutations of bacteriophage T4 lysozyme occur at sites with low mobility and low solvent accessibility in the folded protein

Twenty-five different temperature-sensitive point mutations at 20 sites in the lysozyme gene of bacteriophage T4 have been identified. All of the mutations alter amino acid side chains that have lower than average crystallographic thermal factors and reduced solvent accessibility in the folded protein. This suggests that the amino acids with well-defined conformations can form specific intramolecular interactions that make relatively large contributions to the thermal stability of the protein. Residues with high mobility or high solvent accessibility are much less susceptible to destabilizing substitutions, suggesting that, in general, such amino acids contribute less to protein stability. The pattern of the sites of ts substitutions observed in the folded conformation of T4 lysozyme suggests that severe destabilizing mutations that primarily affect the free energy of the unfolded state are rare. These results indicate that proteins can be stabilized by adding new interactions to regions that are rigid or buried in the folded conformation.     

Inhibition and Reversal of Microbial Attachment by an Antibody with Parasteric Activity against the FimH Adhesin of Uropathogenic E. coli

Attachment proteins from the surface of eukaryotic cells, bacteria and viruses are critical receptors in cell adhesion or signaling and are primary targets for the development of vaccines and therapeutic antibodies. It is proposed that the ligand-binding pocket in receptor proteins can shift between inactive and active conformations with weak and strong ligand-binding capability, respectively. Here, using monoclonal antibodies against a vaccine target protein - fimbrial adhesin FimH of uropathogenic Escherichia coli, we demonstrate that unusually strong receptor inhibition can be achieved by antibody that binds within the binding pocket and displaces the ligand in a non-competitive way. The non-competitive antibody binds to a loop that interacts with the ligand in the active conformation of the pocket but is shifted away from ligand in the inactive conformation. We refer to this as a parasteric inhibition, where the inhibitor binds adjacent to the ligand in the binding pocket. We showed that the receptor-blocking mechanism of parasteric antibody differs from that of orthosteric inhibition, where the inhibitor replaces the ligand or allosteric inhibition where the inhibitor binds at a site distant from the ligand, and is very potent in blocking bacterial adhesion, dissolving surface-adherent biofilms and protecting mice from urinary bladder infection.     

Genetic analysis of the first and third extracellular loops of the C5a receptor reveals an essential WXFG motif in the first loop

The extracellular loops of G protein-coupled receptors (GPCRs) frequently contain binding sites for peptide ligands. However, the mechanism of receptor activation following ligand binding and the influence of the extracellular loops in other aspects of receptor function are poorly understood. Here we report a structure-function analysis of the first and third extracellular loops of the human C5a receptor, a GPCR that binds a 74-amino acid peptide ligand. Amino acid substitutions were randomly incorporated into each loop, and functional receptors were identified in yeast. The first extracellular loop contains a large number of positions that cannot tolerate amino acid substitutions, especially residues within the WXFG motif found in many rhodopsin-like GPCRs, yet disruption of these residues does not alter C5a binding affinity. These results demonstrate an unanticipated role for the first extracellular loop, and the WXFG motif in particular, in ligand-mediated activation of the C5a receptor. This motif likely serves a similar role in other GPCRs. The third extracellular loop, in contrast, contains far fewer preserved residues and appears to play a less essential role in receptor activation.     

Structural features of the ligand-binding domain of the serotonin 5HT3 receptor

The nicotinic acetylcholine receptor (AChR) and the serotonin type 3 receptor (5HT3R) are members of the ligand-gated ion channel gene family. Both receptors are inhibited by nanomolar concentrations of d-tubocurarine (curare) in a competitive fashion. Chemical labeling studies on the AChR have identified tryptophan residues on the gamma (gammaTrp-55) and delta (deltaTrp-57) subunits that interact with curare. Comparison of the sequences of these two subunits with the 5HT3R shows that a tryptophan residue is found in the homologous position in the 5HT3R (Trp-89), suggesting that this residue may be involved in curare-5HT3R interactions. Site-directed mutagenesis at position Trp-89 markedly reduces the affinity of the 5HT3R for the antagonists curare and granisetron but has little effect on the affinity for the agonist serotonin. To further examine the role of this region of the receptor in ligand-receptor interactions, alanine-scanning mutagenesis analysis of the region centered on Trp-89 (Thr-85 to Trp-94) was carried out, and the ligand binding properties of the mutant receptors were determined. Within this region of the receptor, curare affinity is reduced by substitution only at Trp-89, whereas serotonin affinity is reduced only by substitution at Arg-91. On the other hand, granisetron affinity is reduced by substitutions at Trp-89, Arg-91, and Tyr-93. This differential effect of substitutions on ligand affinity suggests that different ligands may have different points of interaction within the ligand-binding pocket. In addition, the every-other-residue periodicity of the effects on granisetron affinity strongly suggests that this region of the ligand-binding site of the 5HT3R (and by inference, other members of the ligand-gated ion channel family) is in a beta-strand conformation.     

Random mutagenesis of the M3 muscarinic acetylcholine receptor expressed in yeast. Identification of point mutations that "silence" a constitutively active mutant M3 receptor and greatly impair receptor/G protein coupling

The M3 muscarinic receptor is a prototypical member of the class I family of G protein-coupled receptors (GPCRs). To facilitate studies on the structural mechanisms governing M3 receptor activation, we generated an M3 receptor-expressing yeast strain (Saccharomyces cerevisiae) that requires agonist-dependent M3 receptor activation for cell growth. By using receptor random mutagenesis followed by a genetic screen in yeast, we initially identified a point mutation at the cytoplasmic end of transmembrane domain (TM) VI (Q490L) that led to robust agonist-independent M3 receptor signaling in both yeast and mammalian cells. To explore further the molecular mechanisms by which point mutations can render GPCRs constitutively active, we subjected a region of the Q490L mutant M3 receptor that included TM V-VII to random mutagenesis. We then applied a yeast genetic screen to identify second-site mutations that could suppress the activating effects of the Q490L mutation and restore wild-type receptor-like function to the Q490L mutant receptor. This analysis led to the identification of 12 point mutations that allowed the Q490L mutant receptor to function in a fashion similar to the wild-type receptor. These amino acid substitutions mapped to two distinct regions of the M3 receptor, the exofacial segments of TM V and VI and the cytoplasmic ends of TM V-VII. Strikingly, in the absence of the activating Q490L mutation, all recovered point mutations severely reduced the efficiency of receptor/G protein coupling, indicating that the targeted residues play important roles in receptor activation and/or receptor/G protein coupling. This strategy should be generally applicable to identify sites in GPCRs that are critically involved in receptor function.     

Physical characterization of native opiate receptors. Additional information from detailed binding analysis of a radiation-inactivated receptor

Target size analyses of the etorphine receptor were performed on frozen rat brain P2 homogenates using the radiation inactivation technique. Multi-point saturation curves at each radiation dose revealed that the apparent dissociation constant for the binding of this ligand to its receptor is a function of the dose. Analysis of the results shows clearly that the ligand-binding macromolecule is functionally coupled to at least one other macromolecule. When the coupling is destroyed the ligand dissociation constant becomes larger by over an order of magnitude. Thus, the variation of the dissociation constant with dose yields interesting new information on the nature of the native receptor which has implications with respect to the conformation of the binding site and to solubilization and cloning methods directed towards sequencing the ligand-binding component of opiate receptors.     

Evidence for transmembrane modulation of the ligand-binding site of the hepatocyte galactose/N-acetylgalactosamine-specific receptor

The ligand-binding activity of the galactose/N-acetylgalactosamine-specific receptor (Gal/GalNAc receptor) present on the surface of hepatocytes can be modulated under a number of conditions in the intact cell. The carboxylic acid ionophores monensin and nigericin inhibit endocytosis by the Gal/GalNAc receptor in a concentration-dependent manner. Monensin at a concentration of 100 microM reduces the number of binding sites for asialo-orosomucoid and a tri-branched glycopeptide (F2) 5-10-fold; however, the number of Gal/GalNAc receptor subunits detected at the cell surface by a competitive radioimmunoassay and by immunoprecipitation of surface labeled receptor is not significantly altered. Replacement of NaCl in the medium with either N-methylglucamine or sorbitol to isotonicity also inhibits binding and endocytosis. The monensin, nigericin, N-methylglucamine, and sorbitol treatments have in common the ability to alkalinize the cytosol of the hepatocyte. None of these agents has any effect on binding by the isolated Gal/GalNAc receptor nor is the intracellular pH shift of such a magnitude that it would alter binding by the isolated Gal/GalNAc receptor. This has led us to conclude that the ligand-binding properties of the Gal/GalNAc receptor at the cell surface can be modulated in a transmembrane fashion by events other than those involving pH or Ca2+ regulation at the ligand-binding site itself. Such transmembrane modulation of ligand binding by the Gal/GalNAc receptor may provide a rapid and efficient mechanism for mediating ligand release and immediate return of the receptor to the cell surface.     

In vitro characterization of ligand-induced oligomerization of the S. cerevisiae G-protein coupled receptor,Ste2p

Background

The S. cerevisiae α-factor receptor, Ste2p, is a G-protein coupled receptor that plays key roles in yeast signaling and mating. Oligomerization of Ste2p has previously been shown to be important for intracellular trafficking, receptor processing and endocytosis. However the role of ligand in receptor oligomerization remains enigmatic.

Methods

Using functional recombinant forms of purified Ste2p, atomic force microscopy, dynamic light scattering and chemical crosslinking are applied to investigate the role of ligand in Ste2p oligomerization.

Results

Atomic force microscopy images indicate a molecular height for recombinant Ste2p in the presence of α-factor nearly double that of Ste2p alone. This observation is supported by complementary dynamic light scattering measurements which indicate a ligand-induced increase in the polydispersity of the Ste2p hydrodynamic radius. Finally, chemical cross-linking of HEK293 plasma membranes presenting recombinant Ste2p indicates α-factor induced stabilization of the dimeric form and higher order oligomeric forms of the receptor upon SDS-PAGE analysis.

Conclusions

α-factor induces oligomerization of Ste2p in vitro and in membrane.

General significance

These results provide additional evidence of a possible role for ligand in mediation of Ste2p oligomerization in vivo.     

Helix 1/8 interactions influence the activity of nuclear receptor ligand-binding domains

The ligand-binding domain (LBD) of apo-nuclear receptors in solution is thought to be a very dynamic structure with many possible conformations. Upon ligand binding, the structure is stabilized to a more rigid conformation. The dynamic stabilization assay is a LBD reassembly assay that takes advantage of the high specificity of the intramolecular interactions that comprise the ligand-bound LBD. Here, we demonstrate dynamic stabilization for the nuclear receptors peroxisome proliferator-activated receptor (PPAR)gamma and nerve growth factor inducible (NGFIB)beta and identify residues important for stabilization of the intramolecular interactions induced by PPARgamma ligands. Site-directed mutagenesis studies identified residues in helices 1 and 8 required for LBD reassembly. Further, disrupting the helix 1/8 interaction in the context of the holo-LBD alters the response of the receptor in a compound-specific manner, suggesting that residues far from the ligand-binding pocket can influence the stability of the ligand-bound receptor. Thus, these results support and extend models of the apo-LBD of PPARgamma as a dynamic structure.