Long and short distance movements of ß(2)-adrenoceptor in cell membrane assessed by photoconvertible fluorescent protein dendra2-ß(2)-adrenoceptor fusion.

ABSTRACT

Local movements of receptors in the plasma membrane have been extensively studied, as it is generally believed that the dynamics of membrane distribution of receptors regulate their functions. However, the properties of large-scale (>5µm) receptor movements in the membrane are relatively obscure. In the present study, we addressed the question as to whether the large-scale movement of receptor in the plasma membrane at the whole cell level can be explained quantitatively by its local diffusive properties. We used HEK 293 cells transfected with human ß2-adrenoceptor fused to photoconvertible fluorescent protein dendra2 as a model system; and found that 1) functional integrity of the dendra2-tagged receptor remains apparently intact; 2) in a mesoscopic scale (~4µm), ~90% of the receptors are mobile on average, and receptor influx to, and out-flux from a membrane area can be symmetrically explained by a diffusion-like process with an effective diffusion coefficient of ~0.1µm(2)/s; 3) these mobility parameters are not affected by the activity state of the receptor (assessed by using constitutively active receptor mutants); 4) in the macroscopic scale (4-40µm), although a slowly diffusing fraction of receptors (with D<0.01µm(2)/s) is identifiable in some cases, the movement of the predominant fraction is perfectly explained by the same effective diffusion process observed in the mesoscopic scale, suggesting that the large scale structure of the cell membrane as felt by the receptor is apparently homogeneous in terms of its mesoscopic properties. We also showed that intracellular compartments and plasma membrane are kinetically connected even at steady-state.