Abstract
Although sphingosine-1-phosphate receptor 1 (S1P1) has been shown to trigger several S1P targeted functions such as immune cell trafficking, cell proliferation, migration, or angiogenesis, tools that allow the accurate detection of endogenous S1P1 localization and trafficking remain to be obtained and validated. In this study, we developed and characterized a novel monoclonal S1P1 antibody. Mice were immunized with S1P1 produced in the yeast Pichia pastoris and nine hybridoma clones producing monoclonal antibodies were created. Using different technical approaches including Western blot, immunoprecipitation and immunocytochemistry, we show that a selected clone, hereinafter referred to as 2B9, recognizes human and mouse S1P1 in various cell lineages. The interaction between 2B9 and S1P1 is specific over receptor subtypes, as the antibody does not binds to S1P2 or S1P5 receptors. Using cell-imaging methods, we demonstrate that 2B9 binds to an epitope located at the intracellular domain of S1P1; reveals cytosolic and membrane localization of the endogenous S1P1; and receptor internalization upon S1P or FTY720-P stimulation. Finally, loss of 2B9 signal upon knockdown of endogenous S1P1 by specific small interference RNAs further confirms its specificity. 2B9 was also able to detect S1P1 in human kidney and spinal cord tissue by immunohistochemistry. Altogether, our results suggest that 2B9 could be a useful tool to detect, quantify or localize low amounts of endogenous S1P1 in various physiological and pathological processes.
Highlights
Sphingosine 1-phosphate receptor 1 (S1P1) is part of the sphingosine 1-phosphate (S1P) receptor family, which comprises five G-protein coupled receptors (GPCR, S1P1, S1P2, S1P3, S1P4, and S1P5, S1P1-5)
To select animals optimally responding to the recombinant proteins, reactivity against S1P1 was assessed by dot blot and Western blot with sera obtained from the animals (Fig 2)
We have previously shown that S1P1 mRNA is present in primary murine embryonic fibroblasts (MEF) suggesting that protein may be expressed [17]
Summary
Sphingosine 1-phosphate receptor 1 (S1P1) is part of the sphingosine 1-phosphate (S1P) receptor family, which comprises five G-protein coupled receptors (GPCR, S1P1, S1P2, S1P3, S1P4, and S1P5, S1P1-5). The drug FTY720 (Fingolimod, Gilenya) which activates S1P1 leading to impaired lymphocyte migration is currently used for the treatment of relapsing remitting multiple sclerosis [9] This drug is phosphorylated, in vivo, and the resulting FTY720-P binds to S1P1 to activate receptors as a true agonist. This process leads to the internalization of S1P1 that are not recycled at the membrane blocking the egress of lymphocytes. The murine anti-S1P1 monoclonal IgG, called E49 [11] was produced using an Escherichia coli-derived human S1P1 full-length antigen Another interesting antibody was the rabbit anti-S1P1 polyclonal antibody H60 raised against amino acids 322–381 of S1P1 of human origin [9, 12, 13]. 2B9 binds to the intracellular part of the receptor, reveals cytoplasmic and membrane bound S1P1 as well as receptor internalization upon S1P and FTY720-P stimulation
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