The lateral organization of a prototypical G protein-coupled receptor, the neurokinin-1 receptor (NK1R), was investigated in living cells by fluorescence resonance energy transfer (FRET) microscopy, taking advantage of the recently developed acyl carrier protein (ACP) labeling technique. The NK1R was expressed as fusion protein with ACP to which small fluorophores were then covalently bound. Our approach allowed the recording of FRET images of receptors on living cells with unprecedented high signal-to-noise ratios and a subsequent unequivocal quantification of the FRET data owing to (i) the free choice of optimal fluorophores, (ii) the labeling of NK1Rs exclusively on the cell surface, and (iii) the precise control of the donor-acceptor molar ratio. Our single-cell FRET measurements exclude the presence of constitutive or ligandinduced homodimers or oligomers of NK1Rs. The strong dependence of FRET on the receptor concentration further reveals that NK1Rs tend to concentrate in microdomains, which are found to constitute Ϸ1% of the cell membrane and to be sensitive to cholesterol depletion.ACP labeling ͉ G protein-coupled receptor (GPCR) oligomerization G protein-coupled receptors (GPCRs) were for a long time presumed to be distributed in the plasma membrane exclusively in a monomeric form (1, 2), but recent reports have unveiled a more complex behavior; in particular, dimeric structures have been found for several GPCRs using biochemical and biophysical methods (3-9). Dimerization can occur between receptors of the same subtype (homodimerization) or of different subtypes (heterodimerization). Some GPCRs remain dimeric all of the time, whereas others cycle between monomeric and dimeric states in a ligand-regulated process (7). Although GPCR homodimerization seems to be important for receptor ontology and trafficking, heterodimerization might result in altered ligand selectivity and distinctive coupling to signal transduction pathways, providing an additional possibility for the fine tuning of cellular signaling.In addition to dimerization, the lateral distribution of GPCRs in cell membranes has been extensively debated recently. Several reports based on biochemical (10), plasmon-resonance spectroscopy (11), single-molecule microscopy (12), and fluorescence recovery after photobleaching experiments (13) propose that GPCRs are localized in microdomains, but a clear demonstration of the existence and nature of such microdomains in living cells remains elusive, in particular because the interpretation of biochemical data can be rather equivocal (14-17). Compartmentalization in form of microdomains was proposed to explain the efficiency of signal transduction at the low physiological surface concentrations of the signaling partners by their enrichment inside specialized signaling platforms (10, 18).Bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET) experiments have gained increasing interest to investigate these two central questions on GPCR signaling. (i) They can be p...

CD8؉ cytotoxic T lymphocyte (CTL) can recognize and kill target cells that express only a few cognate major histocompatibility complex class I-peptide (pMHC) complexes. To better understand the molecular basis of this sensitive recognition process, we studied dimeric pMHC complexes containing linkers of different lengths. Although dimers containing short (10 -30-Å) linkers efficiently bound to and triggered intracellular calcium mobilization and phosphorylation in cloned CTL, dimers containing long linkers (>80 Å) did not. Based on this and on fluorescence resonance energy transfer experiments, we describe a dimeric binding mode in which two T cell receptors engage in an antiparallel fashion two pMHC complexes facing each other with their constant domains. This binding mode allows integration of diverse low affinity interactions, which increases the overall binding and, hence, the sensitivity of antigen recognition. In proof of this, we demonstrated that pMHC dimers containing one agonist and one null ligand efficiently activate CTL, corroborating the importance of endogenous pMHC complexes in antigen recognition.A hallmark of CD8ϩ CTLs 1 is their extraordinary sensitivity in recognizing and killing target cells that express only very few cognate pMHC complexes (1, 2). Although soluble TCR and CD8 have been shown to bind pMHC complexes typically with low affinities, fast dissociation kinetics, and in an independent manner (3, 4), the molecular basis of this highly sensitive recognition process is not clear.For several hormone, cytokine, and chemokine receptors, it has been shown that receptor dimerization is a means to strengthen receptor ligand binding and to promote receptor signaling (5, 6). Dimerization and aggregation, pMHC driven or not, have also been proposed for TCR (7-10); however, the evidences provided never gained general acceptance, leaving this issue open (11-13). In addition, none of these studies provided a plausible structural explanation for TCR dimerization. In view of the wealth of structural information on pMHC, TCR, CD8, pMHC-TCR, and pMHC-CD8 complexes, there should be a structural explanation for TCR dimerization, if it indeed exists. The crystal structure of TCR has uncovered three features that are unique to TCR, i.e. are not found in other immunoglobulin (Ig) super-family members. 1) In the V␣ domain, the CЈ strand forms hydrogen bonds with the D strand and not with the CЉ strand (14). 2) C␣ has only 12-18% sequence homology with other Ig-constant domains, and 12-15 residues are missing, resulting in a less ordered structure and a flatter outer surface as compared with C␤ (15). 3) C␤ has a prominent, surface-exposed FG loop (15). Because of these structural features, TCR␣ chains are more likely to dimerize than TCR␤ chains (14).Because TCR are naturally membrane-integrated and associated with CD3 units, studies on TCR dimerization/aggregation should be performed on cells (11). The CD3⑀␥␦ chains each contain an extracellular Ig domain and a cytoplasmic tail harboring one immunotyrosine...

G protein-coupled receptors (GPCRs) are important targets for medicinal agents. Four different G protein families, G(s), G(i), G(q), and G(12), engage in their linkage to activation of receptor-specific signal transduction pathways. G(12) proteins were more recently studied, and upon activation by GPCRs they mediate activation of RhoGTPase guanine nucleotide exchange factors (RhoGEFs), which in turn activate the small GTPase RhoA. RhoA is involved in many cellular and physiological aspects, and a dysfunction of the G(12/13)-Rho pathway can lead to hypertension, cardiovascular diseases, stroke, impaired wound healing and immune cell functions, cancer progression and metastasis, or asthma. In this study, regulator of G protein signaling (RGS) domain-containing RhoGEFs were tagged with enhanced green fluorescent protein (EGFP) to detect their subcellular localization and translocation upon receptor activation. Constitutively active Galpha(12) and Galpha(13) mutants induced redistribution of these RhoGEFs from the cytosol to the plasma membrane. Furthermore, a pronounced and rapid translocation of p115-RhoGEF from the cytosol to the plasma membrane was observed upon activation of several G(12/13)-coupled GPCRs in a cell type-independent fashion. Plasma membrane translocation of p115-RhoGEF stimulated by a GPCR agonist could be completely and rapidly reversed by subsequent application of an antagonist for the respective GPCR, that is, p115-RhoGEF relocated back to the cytosol. The translocation of RhoGEF by G(12/13)-linked GPCRs can be quantified and therefore used for pharmacological studies of the pathway, and to discover active compounds in a G(12/13)-related disease context.

G protein-coupled receptors (GPCRs) constitute a large class of seven transmembrane proteins, which bind selectively agonists or antagonists with important consequences for cellular signaling and function. Comprehension of the molecular details of ligand binding is important for the understanding of receptor function and in turn for the design and development of novel therapeutic compounds. Here we show how ligand−receptor interaction can be investigated in situ with high sensitivity on sensor surfaces by total internal reflection fluorescence (TIRF) measurements. A generally applicable method for the surface immobilization of membrane proteins was developed using the prototypic seven transmembrane neurokinin-1 receptor. The receptor was expressed as a biotinylated protein in mammalian cells. Membranes from cell homogenates were selectively immobilized on glass surfaces covered with streptavidin. TIRF measurements showed that a fluorescent agonist binds to the receptor on the sensor surface with similar affinity as to the receptor in live cells. This approach offers the possibility to investigate minute amounts of membrane protein in an active form and in its native environment without purification.

We report on an in vivo single-molecule study of the signaling kinetics of G protein-coupled receptors (GPCR) performed using the neurokinin 1 receptor (NK1R) as a representative member. The NK1R signaling cascade is triggered by the specific binding of a fluorescently labeled agonist, substance P (SP). The diffusion of single receptor-ligand complexes in plasma membrane of living HEK 293 cells is imaged using fast single-molecule wide-field fluorescence microscopy at 100 ms time resolution. Diffusion trajectories are obtained which show intra- and intertrace heterogeneity in the diffusion mode. To investigate universal patterns in the diffusion trajectories we take the ligand-binding event as the common starting point. This synchronization allows us to observe changes in the character of the ligand-receptor-complex diffusion. Specifically, we find that the diffusion of ligand-receptor complexes is slowed down significantly and becomes more constrained as a function of time during the first 1000 ms. The decelerated and more constrained diffusion is attributed to an increasing interaction of the GPCR with cellular structures after the ligand-receptor complex is formed.