S1P1 receptor phosphorylation, internalization, and interaction with Rab proteins: effects of sphingosine 1-phosphate, FTY720-P, phorbol esters, and paroxetine.

Sphingosine 1-phosphate (S1P) and FTY720-phosphate (FTYp) increased intracellular calcium in cells expressing S1P1 mCherry-tagged receptors; the synthetic agonist was considerably less potent. Activation of protein kinase C by phorbol myristate acetate (PMA) blocked these effects. The three agents induced receptor phosphorylation and internalization, with the action of FTYp being more intense. S1P1 receptor-Rab protein (GFP-tagged) interaction was studied using FRET. The three agents were able to induce S1P1 receptor-Rab5 interaction, although with different time courses. S1P1 receptor-Rab9 interaction was mainly increased by the phorbol ester, whereas S1P1 receptor-Rab7 interaction was only increased by FTYp and after a 30-min incubation. These actions were not observed using dominant negative (GDP-bound) Rab protein mutants. The data suggested that the three agents induce interaction with early endosomes, but that the natural agonist induced rapid receptor recycling, whereas activation of protein kinase C favored interaction with late endosome and slow recycling and FTYp triggered receptor interaction with vesicles associated with proteasomal/lysosomal degradation. The ability of bisindolylmaleimide I and paroxetine to block some of these actions suggested the activation of protein kinase C was associated mainly with the action of PMA, whereas G protein-coupled receptor kinase (GRK) 2 (GRK2) was involved in the action of the three agents.