Single particle tracking is a powerful tool for probing the organization and dynamics of the plasma membrane constituents. We used this technique to study the micro -opioid receptor belonging to the large family of the G-protein-coupled receptors involved with other partners in a signal transduction pathway. The specific labeling of the receptor coupled to a T7-tag at its N-terminus, stably expressed in fibroblastic cells, was achieved by colloidal gold coupled to a monoclonal anti T7-tag antibody. The lateral movements of the particles were followed by nanovideomicroscopy at 40 ms time resolution during 2 min with a spatial precision of 15 nm. The receptors were found to have either a slow or directed diffusion mode (10%) or a walking confined diffusion mode (90%) composed of a long-term random diffusion and a short-term confined diffusion, and corresponding to a diffusion confined within a domain that itself diffuses. The results indicate that the confinement is due to an effective harmonic potential generated by long-range attraction between the membrane proteins. A simple model for interacting membrane proteins diffusion is proposed that explains the variations with the domain size of the short-term and long-term diffusion coefficients.

A functional fluorescent neurokinin NK2 receptor, EGFP-NK2, was previously used to follow, by fluorescence resonance energy transfer measurements in living cells, the binding of its fluorescently labeled agonist, bodipy-neurokinin A (NKA). Local agonist application suggested that the activation and desensitization of the NK2 receptors were compartmentalized at the level of the plasma membrane. In this study, fluorescence recovery after photobleaching experiments are carried out at variable observation radius (vrFRAP) to probe EGFP-NK2 receptor mobility and confinement. Experiments are carried out at 20°C to maintain the number of receptors constant at the cell surface during recordings. In the absence of agonist, 35% EGFP-NK2 receptors diffuse within domains of 420 ؎ 80 nm in radius with the remaining 65% of receptors able to diffuse with a long range lateral diffusion coefficient between the domains. When cells are incubated with a saturating concentration of NKA, 30% EGFP-NK2 receptors become immobilized in small domains characterized by a radius equal to 170 ؎ 50 nm. Biochemical experiments show that the confinement of EGFP-NK2 receptor is not due to its association with rafts at any given time. Colocalization of the receptor with ␤-arrestin and transferrin supports that the small domains, containing 30% of activated EGFP-NK2, correspond to clathrin-coated pre-pits. The similar amount of confined EGFP-NK2 receptors found before and after activation (30 -35%) is discussed in term of putative transient interactions of the receptors with preexisting scaffolds of signaling molecules.The general mechanisms underlying the activation of Gprotein-coupled receptors (GPCRs) 1 and the following attenuation of the cellular response seem well established at the molecular level. The prevailing view is that the intracellular signal results from a cascade of transient and sequential molecular interactions starting with the activation of the heterotrimeric G-protein upon ligand/receptor interaction. The biological effect then can be attenuated by three main mechanisms: (i) agonists removal; (ii) agonist-mediated desensitization of the receptor molecules by uncoupling of the activated receptors from heterotrimeric G-proteins; and (iii) agonist-stimulated receptor endocytosis followed by either recycling or degradation. The remaining key questions are to understand the architecture governing the molecular interactions of the signaling cascade and how these multiple interactions take place in space and time, notably starting with the lateral organization of the partners at the level of the plasma membrane (1-4). Here we investigate these different aspects using the neurokinin NK2 receptor expressed in HEK293 cells as a model system.Neurokinins form a family of neuropeptides acting at three distinct GPCRs, namely NK1, NK2 and NK3. The NK2 receptor shows preferential binding for the endogenous neurokinin A (NKA) (5) and is mostly found in peripheral nervous tissues where it participates mainly to smooth muscle contraction. In pr...

We synthesized 3β-hydroxy-pregn-5-ene-21-(1-methylpyrenyl)-20-methylidene (Py-met-chol), consisting of cholesterol steroid rings connected to a pyrene group via a linker without polar atoms. This compound has interesting spectroscopic properties when probing membranes: 1), The pyrene has hypochromic properties resulting from probe self-association processes in membranes. Using liposomes of various lipid compositions, we determined the association constants of the probe (K): KDOPC ≫ KPOPC ≫ KDMPC > KDMPC/15 mol % Chol > KDMPC/30 mol % Chol. This indicates a better probe solvation in saturated than in unsaturated lipids, and this effect is enhanced as the cholesterol concentration increases. 2), The pyrene fluorophore is characterized by monomer (I1–I5) and excimer (IE) emission bands. In model membranes, I1/I3 and IE/I3 ratios revealed a correlation between the polarity of the lipid core of the membrane and the amount of cholesterol. 3), Using this probe, we monitored the first steps of the signaling pathway of the mouse δ-opioid receptor, a G-protein-coupled receptor. The thickness of the membrane around this receptor is known to change after agonist binding. Fluorescence spectra of living Chinese hamster ovary cells overexpressing mouse δ-opioid receptor specifically revealed the agonist binding. These results indicate that Py-met-chol may be useful for screening ligands of this family of receptors.