Agonist-selective dynamic compartmentalization of human Mu opioid receptor as revealed by resolutive FRAP analysis.
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.
Saulière-Nzeh Ndong
Aude
A
IPBS (Institute of Pharmacology and Structural Biology), CNRS, 205 route de Narbonne, F-31077 Toulouse, France.
Saulière-Nzeh
Aude Ndong
AN
Millot
Claire
C
Corbani
Maithé
M
Mazères
Serge
S
Lopez
André
A
Salomé
Laurence
L
D000701:Analgesics, Opioid / Q000494:pharmacology; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / Q000494:pharmacology; D036681:Fluorescence Recovery After Photobleaching; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009020:Morphine / Q000494:pharmacology; D009447:Neuroblastoma / Q000378:metabolism*; D037342:Pertussis Toxin / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D017450:Receptors, Opioid, mu / Q000819:agonists* / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000701:Analgesics, Opioid; D017450:Receptors, Opioid, mu; C485184:enhanced green fluorescent protein; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D049452:Green Fluorescent Proteins; D009020:Morphine; D037342:Pertussis Toxin