Agonist-selective Dynamic Compartmentalization of Human Mu Opioid Receptor as Revealed by Resolutive FRAP Analysis

Aude Ndong Saulière-Nzeh 1,2, Claire Millot, Maithé Corbani3, Serge Mazères, André Lopez and Laurence Salomé 4

 

1.From the IPBS (Institute of Pharmacology and Structural Biology), CNRS, 205 route de Narbonne, F-31077 Toulouse, France and the University of Toulouse, UPS, IPBS, F-31077 Toulouse, France

4 To whom correspondence should be addressed: IPBS 205, route de Narbonne 31077 Toulouse cedex, France. Fax: 33-0-5-61-17-59-94; E-mail: laurence.salome@ipbs.fr.

 

1 Recipient of postgraduate fellowships from MENESR. Current address: Inserm; U858; F-31432 Toulouse, France.
2 Current address: Université de Toulouse; UPS; Institut de Médecine Moléculaire de Rangueil; F-31432 Toulouse, France.
3 Current address: Institut de Génomique Fonctionnelle, CNRS UMR 5203-INSERM U.661, Université Montpellier I et II, 141 rue de la Cardonille, 34094 Montpellier Cedex 05, France.

 

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.