Ligand- and cell-dependent determinants of internalization and cAMP modulation by delta opioid receptor (DOR) agonists

Signaling bias refers to G protein-coupled receptor ligand ability to preferentially activate one type of signal over another. Bias to evoke signaling as opposed to sequestration has been proposed as a predictor of opioid ligand potential for generating tolerance. Here we measured whether delta opioid receptor agonists preferentially inhibited cyclase activity over internalization in HEK cells. Efficacy (τ) and affinity (KA) values were estimated from functional data and bias was calculated from efficiency coefficients (log τ/KA). This approach better represented the data as compared to alternative methods that estimate bias exclusively from τ values. Log (τ/KA) coefficients indicated that SNC-80 and UFP-512 promoted cyclase inhibition more efficiently than DOR internalization as compared to DPDPE (bias factor for SNC-80: 50 and for UFP-512: 132). Molecular determinants of internalization were different in HEK293 cells and neurons with βarrs contributing to internalization in both cell types, while PKC and GRK2 activities were only involved in neurons. Rank orders of ligand ability to engage different internalization mechanisms in neurons were compared to rank order of E _max values for cyclase assays in HEK cells. Comparison revealed a significant reversal in rank order for cyclase E _max values and βarr-dependent internalization in neurons, indicating that these responses were ligand-specific. Despite this evidence, and because kinases involved in internalization were not the same across cellular backgrounds, it is not possible to assert if the magnitude and nature of bias revealed by rank orders of maximal responses is the same as the one measured in HEK cells.     

Temporal variation and structure of macro-epifauna associated with macrophytes in the Bizerte lagoon (Tunisia,SW Mediterranean Sea)

The temporal variation in composition and abundance of the macro-epifauna associated with seaweeds (Ulva lactuca, Cladophora sp., Gracilariopsis longissima and Gracilaria bursa-pastoris) and seagrass (Cymodocea nodosa) was investigated at Menzel Jemil station in the Bizerte lagoon. Twelve replicate samples (25 x 25 cm frame) were collected monthly from October 2009 to September 2010. Sampling data yielded 21,575 invertebrate specimens from floating seaweeds and the seagrass, belonging to 40 taxa, of which 18 species were arthropods, 13 molluscs, three echinoderms, three fishes, two polychaetes and one cnidarian. Crustaceans were the most dominant and the most species-rich group and Idotea balthica basteri the most abundant species, accounting for 28.94% of the total abundance. Minimum (9.25 ± 5.56 species) and maximum (19.75 ± 3.2 species) values of mean species richness were observed in December and June, respectively. Species richness and density of macro-epifauna showed similar trends to plant biomass and the highest values of both indices coincided with periods of maximal plant biomass. Mean values of Shannon’s index H′ and evenness J′ were low, ranging from 0.34 ± 0.02 bit to 1.08 ± 0.06 bit, and from 0.23 ± 0.03 to 0.52 ± 0.07, respectively. The high dominance of herbivores and detritivores highlighted the substantial role played by macrophytes as food source for associated macro-epifauna species. Multivariate analyses also indicated significant seasonal differences for the macro-epifauna assemblage and revealed significant relationships with environmental variables. According to the canonical correspondence analysis, concentrations of phosphates and nitrites, and temperature were the variables that explained the variation of the macro-epifauna abundance in the Bizerte lagoon.     

Delta opioid receptors recycle to the membrane after sorting to the degradation path

Soon after internalization delta opioid receptors (DOPrs) are committed to the degradation path by G protein-coupled receptor (GPCR)-associated binding protein. Here we provide evidence that this classical post-endocytic itinerary may be rectified by downstream sorting decisions which allow DOPrs to regain to the membrane after having reached late endosomes (LE). The LE sorting mechanism involved ESCRT accessory protein Alix and the TIP47/Rab9 retrieval complex which supported translocation of the receptor to the TGN, from where it subsequently regained the cell membrane. Preventing DOPrs from completing this itinerary precipitated acute analgesic tolerance to the agonist DPDPE, supporting the relevance of this recycling path in maintaining the analgesic response by this receptor. Taken together, these findings reveal a post-endocytic itinerary where GPCRs that have been sorted for degradation can still recycle to the membrane.