Brightness Analysis by Z-Scan Fluorescence Fluctuation Spectroscopy for the Study of Protein Interactions within Living Cells

Fluorescence fluctuation spectroscopy (FFS) quantifies interactions of fluorescently labeled proteins inside living cells by brightness analysis. Conventional FFS implicitly requires that the sample thickness exceeds the size of the observation volume. This condition is not always fulfilled when measuring cells. Cytoplasmic sections, especially, can be thinner than the axial size of the observation volume. The finite sample thickness introduces a brightness bias which, if not recognized, leads to an erroneous interpretation of the data. To avoid this artifact, we introduce z-scan FFS which consists of a fluorescence intensity z scan through the sample followed by an FFS measurement. To model the experimental z-scan data, a new PSF model had to be introduced. We use the intensity z scan together with the PSF model to determine the geometry of the sample and then extract the brightness from the FFS data. Cells expressing EGFP serve as a model system for testing the experimental approach. We demonstrate that z-scan FFS abolishes the brightness artifact and use the method to determine the oligomerization of cytoplasmic nuclear transport factor 2.     

Probing intermolecular protein-protein interactions in the calcium-sensing receptor homodimer using bioluminescence resonance energy transfer (BRET).

The calcium-sensing receptor (CaR) belongs to family C of the G-protein coupled receptor superfamily. The receptor is believed to exist as a homodimer due to covalent and non-covalent interactions between the two amino terminal domains (ATDs). It is well established that agonist binding to family C receptors takes place at the ATD and that this causes the ATD dimer to twist. However, very little is known about the translation of the ATD dimer twist into G-protein coupling to the 7 transmembrane moieties (7TMs) of these receptor dimers. In this study we have attempted to delineate the agonist-induced intermolecular movements in the CaR homodimer using the new bioluminescence resonance energy transfer technique, BRET2, which is based on the transference of energy from Renilla luciferase (Rluc) to the green fluorescent protein mutant GFP2. We tagged CaR with Rluc and GFP2 at different intracellular locations. Stable and highly receptor-specific BRET signals were obtained in tsA cells transfected with Rluc- and GFP2-tagged CaRs under basal conditions, indicating that CaR is constitutively dimerized. However, the signals were not enhanced by the presence of agonist. These results could indicate that at least parts of the two 7TMs of the CaR homodimer are in close proximity in the inactivated state of the receptor and do not move much relative to one another upon agonist activation. However, we cannot exclude the possibility that the BRET technology is unable to register putative conformational changes in the CaR homodimer induced by agonist binding because of the bulk sizes of the Rluc and GFP2 molecules.     

Agonist-induced formation of FGFR1 homodimers and signaling differ among members of the FGF family

Fibroblast growth factor receptor 1 (FGFR1) is known to be activated by homodimerization in the presence of both the FGF agonist ligand and heparan sulfate glycosaminoglycan. FGFR1 homodimers in turn trigger a variety of downstream signaling cascades via autophosphorylation of tyrosine residues in the cytoplasmic domain of FGFR1. By means of Bioluminescence Energy Resonance Transfer (BRET) as a sign of FGFR1 homodimerization, we evaluated in HEK293T cells the effects of all known FGF agonist ligands on homodimer formation. A significant correlation between BRET² signaling and ERK1/2 phosphorylation was observed, leading to a further characterization of the binding and signaling properties of the FGF subfamilies. FGF agonist ligand-FGFR1 binding interactions appear as the main mechanism for the control of FGFR1 homodimerization and MAPK signaling which demonstrated a high correlation. The bioinformatic analysis demonstrates the interface of the two pro-triplets SSS (Ser–Ser–Ser) and YGS (Tyr–Gly–Ser) located in the extracellular and intracellular domain of the FGFR1. These pro-triplets are postulated participate in the FGFR1 homodimerization interface interaction. The findings also reveal that FGF agonist ligands within the same subfamily of the FGF gene family produced similar increases in FGFR1 homodimer formation and MAPK signaling. Thus, the evolutionary relationship within this gene family appears to have a distinct functional relevance.     

Role of the Vam3p transmembrane segment in homodimerization and SNARE complex formation

Intracellular membrane fusion in eukaryotic cells is mediated by SNARE (soluble N-ethylmaleimide sensitive factor (NSF) attachment protein receptor) proteins and is known to involve assembly of cognate subunits to heterooligomeric complexes. For synaptic SNAREs, it has previously been shown that the transmembrane segments drive homotypic and support heterotypic interactions. Here, we demonstrate that a significant fraction of the yeast vacuolar SNARE Vam3p is a homodimer in detergent extracts of vacuolar membranes. This homodimer exists in parallel to the heterooligomeric SNARE complex. A Vam3p homodimer also formed from the isolated recombinant protein. Interestingly, homodimerization depended on the transmembrane segment. In contrast, formation of the quaternary SNARE complex from recombinant Vam3p, Nyv1p, Vti1p, and Vam7p subunits did not depend on the transmembrane segment of Vam3p nor on the transmembrane segments of its partner proteins. We conclude that Vam3p homodimerization, but not quaternary SNARE complex formation, is promoted by TMS-TMS interaction. As the transmembrane segments of Vam3p and other SNARE homologues were previously shown to be critical for membrane fusion downstream of membrane apposition, our results may shed light on the functional significance of SNARE TMS-TMS interactions.     

Characterization of the B2 receptor and activity of bradykinin analogs in SHP-77 cell line by Cytosensor microphysiometer

The Cytosensor microphysiometer device (Molecular Devices, Sunnyvale, CA) is capable of measuring the rate at which cells acidify their environment in response to ligand–receptor binding. By measuring the extracellular acidification response (ECAR) we characterized some aspects of ligand–B₂ receptor interaction in SHP-77 cell line. SHP-77 cells maximally acidified their environment within 30 s after the exposure to bradykinin (BK) or the BK agonist, B9972, with the maximum effect seen at a ligands concentration of 1 μM. Fetal bovine serum (FBS) modulated the binding of BK or B9972, showing that B9972 is a partial agonist. In addition, the binding of BK agonist or antagonist to the B₂ receptor showed different ECAR and different interaction with other intracellular and plasma membrane proteins. Our microphysiometrical results showed that two parameters, antagonist binding affinity (pD₂) and antagonist potency (pIC₅₀) are required to characterize BK antagonist activity for the B₂ receptor in the SHP-77 cell line. The previously used parameter of B₂ antagonist activity, pA₂, had high variation and poor correlation with the inhibition of SHP-77 cell growth in vitro and suppression of tumor growth when SHP-77 cells were injected to mice. Our results permit us to conclude that BK agonists and antagonists differ in their interactions with the B₂ receptor and consequently elicit different cell responses. Based on our results, we have developed a new microphysiometrical assay for analyzing the activity of BK agonists and antagonist in SHP-77 cells, which may facilitate the discovery of new potent anticancer drugs.     

Constitutive Dimerization of the G-Protein Coupled Receptor, Neurotensin Receptor 1, Reconstituted into Phospholipid Bilayers

Neurotensin receptor 1 (NTS1), a Family A G-protein coupled receptor (GPCR), was expressed in Escherichia coli as a fusion with the fluorescent proteins eCFP or eYFP. A fluorophore-tagged receptor was used to study the multimerization of NTS1 in detergent solution and in brain polar lipid bilayers, using fluorescence resonance energy transfer (FRET). A detergent-solubilized receptor was unable to form FRET-competent complexes at concentrations of up to 200 nM, suggesting that the receptor is monomeric in this environment. When reconstituted into a model membrane system at low receptor density, the observed FRET was independent of agonist binding, suggesting constitutive multimer formation. In competition studies, decreased FRET in the presence of untagged NTS1 excludes the possibility of fluorescent protein-induced interactions. A simulation of the experimental data indicates that NTS1 exists predominantly as a homodimer, rather than as higher-order multimers. These observations suggest that, in common with several other Family A GPCRs, NTS1 forms a constitutive dimer in lipid bilayers, stabilized through receptor-receptor interactions in the absence of other cellular signaling components. Therefore, this work demonstrates that well-characterized model membrane systems are useful tools for the study of GPCR multimerization, allowing fine control over system composition and complexity, provided that rigorous control experiments are performed.     

Probing ligand protein binding equilibria with fluorescence fluctuation spectroscopy

We examine the binding of fluorescent ligands to proteins by analyzing the fluctuation amplitude g(0) of fluorescence fluctuation experiments. The normalized variance g(0) depends on the molecular brightness and the concentration of each species in the sample. Thus a single g(0) measurement is not sufficient to resolve individual species. Titration of the ligand with protein establishes the link between molecular brightness and concentration by fitting g(0) to a binding model and allows the separation of species. We first apply g(0) analysis to binary dye mixtures with brightness ratios of 2 and 4 to demonstrate the feasibility of this technique. Next we consider the influence of binding on the fluctuation amplitude g(0). The dissociation coefficient, the molecular brightness ratio, and the stochiometry of binding strongly influence the fluctuation amplitude. We show that proteins with a single binding site can be clearly differentiated from proteins with two independent binding sites. The binding of fluorescein-labeled digoxigenin to a high-affinity anti-digoxin antibody was studied experimentally. A global analysis of the fluctuation amplitude and the fluorescence intensity not only recovered the dissociation coefficient and the number of binding sites, but also revealed the molecular heterogeneity of the hapten-antibody complex. Two species were used to model the molecular heterogeneity. We confirmed the molecular heterogeneity independently by fluorescence lifetime experiments, which gave fractional populations and molecular brightness values that were virtually identical to those of the g(0) analysis. The identification and characterization of molecular heterogeneity have far-reaching consequences for many biomolecular systems. We point out the important role fluctuation experiments may have in this area of research.     

The CXC chemokine receptor 4 ligands ubiquitin and stromal cell-derived factor-1α function through distinct receptor interactions

Recently, we identified extracellular ubiquitin as an endogenous CXC chemokine receptor (CXCR) 4 agonist. However, the receptor selectivity and molecular basis of the CXCR4 agonist activity of ubiquitin are unknown, and functional consequences of CXCR4 activation with ubiquitin are poorly defined. Here, we provide evidence that ubiquitin and the cognate CXCR4 ligand stromal cell-derived factor (SDF)-1α do not share CXCR7 as a receptor. We further demonstrate that ubiquitin does not utilize the typical two-site binding mechanism of chemokine-receptor interactions, in which the receptor N terminus is important for ligand binding. CXCR4 activation with ubiquitin and SDF-1α lead to similar Gα(i)-responses and to a comparable magnitude of phosphorylation of ERK-1/2, p90 ribosomal S6 kinase-l and Akt, although phosphorylations occur more transiently after activation with ubiquitin. Despite the similarity of signal transduction events after activation of CXCR4 with both ligands, ubiquitin possesses weaker chemotactic activity than SDF-lα in cell migration assays and does not interfere with productive entry of HIV-1 into P4.R5 multinuclear activation of galactosidase indicator cells. Unlike SDF-1α, ubiquitin lacks interactions with an N-terminal CXCR4 peptide in NMR spectroscopy experiments. Binding and signaling studies in the presence of antibodies against the N terminus and extracellular loops 2/3 of CXCR4 confirm that the ubiquitin CXCR4 interaction is independent of the N-terminal receptor domain, whereas blockade of extracellular loops 2/3 prevents receptor binding and activation. Our findings define ubiquitin as a CXCR4 agonist, which does not interfere with productive cellular entry of HIV-1, and provide new mechanistic insights into interactions between CXCR4 and its natural ligands.     

Kinetic and biophysical analysis of the m2 muscarinic receptor

The recombinant Pm2 muscarinic receptor expressed in Chinese hamster ovary (CHO) cells was used as a model system to examine receptor-effector coupling and ligand binding. In CHO cells, equilibrium binding studies and the dependence on receptor number per cell of the maximum response and EC50 values for agonist stimulation of phosphatidylinositol metabolism and inhibition of cAMP formation were consistent with a modified ternary complex model of signal transduction that included a physiologically noncompetent receptor state. Detailed kinetic studies of oxotremorine M (Oxo-M) binding to CHO cell membranes suggested that agonist interactions at the high affinity class of binding sites are complicated and depend on receptor expression levels. At low levels of expression, kinetic data were consistent with a special case of a mechanism in which Oxo-M shifts the equilibrium between two receptor conformations while at high levels of expression, it was necessary to evoke receptor-receptor interactions to explain the kinetic data. Far ultraviolet circular dichroism studies of the purified recombinant receptor showed a high content of alpha-helical secondary structure and small changes in secondary structure upon antagonist, but not agonist, binding.     

Structural adaptation of the glycophorin A transmembrane homodimer to D-amino acid modifications

Protein-protein recognition is an essential process in life. The chemistry of these kind of interactions is predominantly stereospecific (i.e. receptor-ligand, antibody-hapten binding). Here, we investigated whether the hydrophobic nature of the membrane affects this stereospecificity. To this end, we synthesized a diastereomer analogue (2D-GPA) of the glycophorin A transmembrane helix, with two l-valine residues replaced by their d-enantiomer. This ensures a disruption of the secondary structure. We investigated the ability of the diastereomer peptide to recognize the GPA chimera in the ToxR homodimer reporting system, in vivo. The peptide demonstrated a dose-dependent dominant negative effect on the GPA transmembrane in the bacterial ToxR system, suggesting a wild-type like interaction. This result was corroborated in vitro by fluorescence energy transfer between 2D-GPA and all-l GPA. Peptide binding to the bacteria was confirmed through confocal imaging, and Western blot confirmed that ToxR GPA receptor levels are not affected by the presence of the exogenous peptide. In order to understand the structural basis for heterodimer formation, homodimer and heterodimer structures, based on the NMR 3D structure of GPA, were subjected to a molecular dynamics simulation. The resulting heterodimer structure maintained most of the original inter-helical interactions, and its structure is similar to that of the homodimer. We postulate that the need to satisfy all H-bonds can compensate for the structural strain induced by the presence of the d-amino acid residues.     

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.     

D1 Dopamine Receptors Can Interact with Both Stimulatory and Inhibitory Guanine Nucleotide Binding Proteins

Pretreatment of striatal membranes with N-ethylmaleimide in the presence of a D1-specific agonist inactivated endogenous guanine nucleotide binding proteins (G proteins), but not D1 dopamine receptors, resulting in a loss of high-affinity agonist binding sites. Such D1 receptors were solubilized, mixed with exogenous G proteins from cells not containing D1 receptors, and reconstituted into phospholipid vesicles. These reconstituted receptors were able to couple to the exogenous G proteins, and the proportion of agonist high-affinity sites of the receptor (40-57%) was similar to levels obtained with naive receptors coupling to endogenous G proteins (40%) upon solubilization and reconstitution. These hybrid high-affinity sites were fully modulated by guanine nucleotides. Pretreatment of cells with pertussis toxin prior to extraction of G proteins resulted in a 50% decrease in the proportion of high-affinity sites; these sites remained sensitive to guanine nucleotides. When D1 receptors were reconstituted with extracts of cyc- cells, which lack stimulatory G proteins, the proportion of high-affinity sites was reduced to 31% of the total. Pertussis toxin treatment of the cyc- cells completely abolished the formation of high-affinity sites. These results demonstrate that D1-dopaminergic receptors are able to couple to not only stimulatory G proteins (Gs), but also to inhibitory G proteins (Gi).     

Intersubunit cooperativity in the NMDA receptor.

Opening of the NMDA receptor channel requires simultaneous binding of glutamate and glycine. Although the binding sites for each agonist are in different subunits, the presence of one agonist influences the binding of the other. We have localized regions in the S1 binding domain of both subunits required for the transmission of allosteric signals from the glutamate binding NR2A subunit to the glycine binding NR1 subunit. Three-dimensional modeling indicates that these segments are not directly involved in ligand binding, but likely form solvent-accessible loops protruding out of the binding pocket, making them suitable to relay interactions between adjacent subunits. Thus, these segments mediate negative allosteric coupling between the two subunit types that form the NMDA receptor.     

G(i1) and G(oA) differentially determine kinetic efficacies of agonists for kappa-opioid receptor

We examined the diversity of single receptor function by measuring receptor-G protein coupling in the baculovirus-Sf21 expression system. In comparative studies using Sf21 cell membranes expressing kappa-opioid receptor (KOR) plus Galpha(i1)beta(1)gamma(2) or KOR plus Galpha(oA)beta(1)gamma(2), there was no significant difference between both preparations in the K(i) values of various kappa-opioid ligands for the displacement of [(3)H]U69593 binding. However, a marked difference in the rank order of agonists to stimulate [(35)S]GTPgammaS binding was observed between both preparations. These findings suggest that agonist efficacy is dependent on the population of different G proteins expressed in various tissues.     

Identification of the Axin and Frat binding region of glycogen synthase kinase-3.

Glycogen synthase kinase-3 (GSK-3) is a key component of several signaling pathways including those regulated by Wnt and insulin ligands. Specificity in GSK-3 signaling is thought to involve interactions with scaffold proteins that localize GSK-3 regulators and substrates. This report shows that GSK-3 forms a low affinity homodimer that is disrupted by binding to Axin and Frat. Based on the crystal structure of GSK-3, we have used surface-scanning mutagenesis to identify residues that differentially affect GSK-3 interactions. Mutations that disrupt Frat and Axin cluster at the dimer interface explaining their effect on homodimer formation. Loss of the Axin binding site blocks the ability of dominant negative GSK-3 to cause axis duplication in Xenopus embryos. The Axin binding site is conserved within all GSK-3 proteins, and its loss affects both cell motility and gene expression in the nonmetazoan, Dictyostelium. Surprisingly, we find no genetic interaction between a non-Axin-binding GSK-3 mutant and T-cell factor activity, arguing that Axin interactions alone cannot explain the regulation of T-cell factor-mediated gene expression.