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.
Highlights
To a receptor have been clearly described
We previously used the high resolution fluorescence recovery after photobleaching (FRAP) approach at variable radius to study T7-EGFP-human mu opioid (hMOP) receptor diffusion in the plasma membrane of SH-SY5Y neuronal cells: we showed that diffusion of receptors in the basal state is confined to joined permeable domains [21]
To study the origins and links to functional processes of receptor organization and dynamics, we examined the effects after agonist binding of the inhibition of two phenomena: clathrin recruitment and G-protein activation
Summary
To a receptor have been clearly described. Signal transduction following activation of the heterotrimeric G-protein and effectors drives the cell response. Numerous experiments involving fluorescence recovery after photobleaching (FRAP) or single molecule tracking and concerning GPCR have been reported. These studies all indicate that GPCR diffusion is restricted to domains. To study the origins and links to functional processes of receptor organization and dynamics, we examined the effects after agonist binding of the inhibition of two phenomena: clathrin recruitment and G-protein activation. We found two distinct patterns of compartmentalization, each associated with one of the major events occurring at the plasma membrane after receptor activation.
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