Agonist-selective dynamic compartmentalization of human Mu opioid receptor as revealed by resolutive FRAP analysis

Abstract

Techniques for analyzing the membrane diffusion of molecules are the most promising methods for investigating the compartmentalization of G-protein-coupled receptors, particularly as relevant to receptor signaling processes. Here, we report fluorescence recovery after photobleaching (FRAP) measurements performed at variable spot radius for human mu opioid (hMOP) receptors on SH-SY5Y neuroblastoma cells in the presence of ligands. Although an antagonist did not affect the behavior of the receptors compared with the basal state, two different agonists, DAMGO and morphine, caused markedly different changes to receptor diffusion. Like receptors in the absence of ligand, receptors bound to morphine exhibited diffusion confined to joined semipermeable domains, but with smaller domain size and diffusion coefficient. This effect was inhibited by pertussis toxin, strongly suggesting that this dynamic behavior is associated with early steps of signaling. In the presence of DAMGO, half of the receptors displayed free long-range diffusion and the other half were confined to smaller isolated domains. Hypertonic sucrose buffer suppressed this effect, which we attribute to receptor entry into clathrin-coated pits. It is likely that the observation of distinct receptor dynamics in the presence of DAMGO and morphine involves the agonist-selective phosphorylation of the receptor.

FIGURE 1.

Recovery curves of FRAP measurements on T7-EGFP-hMOP receptors with an observation radius r = 3. 45 μm: raw, averaged data, and fit. Fluorescence intensities, plotted as a function of time after bleaching, are normalized with respect to the extent of the decrease of the intensity just after the bleaching pulse. a, raw data for a single measurement (one cell: gray squares), average of a set of about 30 measurements on distinct cells (1 day: empty squares) and average of 4 sets of measurements (4 days: black full squares). b, fit of this recovery curve, average over 4 sets of measurements (4 days), by a diffusion equation (see text) assuming two-diffusing populations (2D fit) minimizes the sum of the quadratic deviations compared with that assuming a single population (1D fit).

FIGURE 2.

FRAP analysis of the dynamic organization of T7-EGFP-hMOP receptors in the presence of DAMGO and morphine. Plots of the mobile fraction M against the reciprocal of the observation radius R. Each data set corresponds to a single run of measurements. For each ligand, the reference measurements in the basal state were performed the same day at the same temperature. T7-EGFP-hMOP receptor diffusion was analyzed in the presence of agonists at a concentration of 1 μm: DAMGO (■) and basal state (□) at 14 °C, and morphine (■) and basal state (□) at room temperature.

FIGURE 3.

Distribution of T7-EGFP-hMOP receptors in fractions of SH-SY5Y membranes separated on sucrose gradients. Membranes without treatment (open bars) and after exposure to morphine (hatched bars) or DAMGO (filled bars) were solubilized in 0.35% of Triton X-100 at 4 °C and separated on a sucrose density gradient. EGFP fluorescence was measured in DRM (fractions 3–5), pellet (fraction 9), and solubilized fractions (fractions 1–2 and 6–8). The data are expressed as relative percentages of the total fluorescence obtained from two independent experiments.

FIGURE 4.

FRAP analysis of the dynamic organization of T7-EGFP-hMOP receptors in the presence of DAMGO and hypertonic sucrose. Plots of the mobile fraction M against the reciprocal of the observation radius R. Each data set corresponds to a single run of measurements, all performed on the same day. At room temperature, T7-EGFP-hMOP receptors bound to DAMGO in the presence of hypertonic sucrose (▴) have a behavior similar to that in the basal state in the presence of hypertonic sucrose (□). The presence of DAMGO in the absence of sucrose is accompanied by the expected dynamic change (■) at 14 °C (see Fig. 2).

FIGURE 5.

FRAP analysis of the dynamic organization of T7-EGFP-hMOP receptor in the presence of morphine and PTX. Plots of the mobile fraction M against the reciprocal of the observation radius R. At room temperature, T7-EGFP-hMOP receptors bound to morphine preincubated with PTX (▴) have a behavior similar to that in the basal state (□). The presence of morphine in the absence of pretreatment by PTX is accompanied by the expected dynamic change (■) (see Fig. 2).

FIGURE 6.

Schematic model for the dynamic organization of T7-EGFP-hMOP receptors in the SH-SY5Y membrane in the basal state (A), and in the presence of morphine (B) or DAMGO (C). A, before activation by the agonist, receptors diffuse in joined semi-permeable domains allowing long-range diffusion. B, after morphine binding the receptors diffuse in smaller joined semi-permeable domains. This modification is also observed in the presence of hypertonic sucrose but inhibited by a pretreatment with PTX. C, DAMGO binding induces the slow diffusion confined to small isolated domains for half of T7-EGFP-hMOP receptors; the other half has a more rapid free diffusion. This change is also observed in the case of a pretreatment with PTX but inhibited in the presence of hypertonic sucrose.