Reversible translocation of p115-RhoGEF by G(12/13)-coupled receptors.

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.

Covalent labeling of cell-surface proteins for in-vivo FRET studies.

Fluorescence resonance energy transfer (FRET) is a powerful technique to reveal interactions between membrane proteins in live cells. Fluorescence labeling for FRET is typically performed by fusion with fluorescent proteins (FP) with the drawbacks of a limited choice of fluorophores, an arduous control of donor-acceptor ratio and high background fluorescence arising from intracellular FPs. Here we show that these shortcomings can be overcome by using the acyl carrier protein labeling technique. FRET revealed interactions between cell-surface neurokinin-1 receptors simultaneously labeled with a controlled ratio of donors and acceptors. Moreover, using FRET the specific binding of fluorescent agonists could be monitored.

FRET imaging reveals that functional neurokinin-1 receptors are monomeric and reside in membrane microdomains of live cells.

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 ligand-induced 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 approximately 1% of the cell membrane and to be sensitive to cholesterol depletion.

Kinetics of the initial steps of G protein-coupled receptor-mediated cellular signaling revealed by single-molecule imaging.

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.

CD8+ cytotoxic T lymphocyte activation by soluble major histocompatibility complex-peptide dimers.

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-A) linkers efficiently bound to and triggered intracellular calcium mobilization and phosphorylation in cloned CTL, dimers containing long linkers (> or = 80 A) 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 anti-parallel 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.