Sphingosine-1-phosphate receptor 1 reporter mice reveal receptor activation sites in vivo
Mari Kono, … , Ewa M. Turner, Richard L. Proia
Mari Kono, … , Ewa M. Turner, Richard L. Proia
Published March 25, 2014
Citation Information: J Clin Invest. 2014;124(5):2076-2086. https://doi.org/10.1172/JCI71194.
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Technical Advance Inflammation

Sphingosine-1-phosphate receptor 1 reporter mice reveal receptor activation sites in vivo

Abstract

Activation of the GPCR sphingosine-1-phosphate receptor 1 (S1P1) by sphingosine-1-phosphate (S1P) regulates key physiological processes. S1P1 activation also has been implicated in pathologic processes, including autoimmunity and inflammation; however, the in vivo sites of S1P1 activation under normal and disease conditions are unclear. Here, we describe the development of a mouse model that allows in vivo evaluation of S1P1 activation. These mice, known as S1P1 GFP signaling mice, produce a S1P1 fusion protein containing a transcription factor linked by a protease cleavage site at the C terminus as well as a β-arrestin/protease fusion protein. Activated S1P1 recruits the β-arrestin/protease, resulting in the release of the transcription factor, which stimulates the expression of a GFP reporter gene. Under normal conditions, S1P1 was activated in endothelial cells of lymphoid tissues and in cells in the marginal zone of the spleen, while administration of an S1P1 agonist promoted S1P1 activation in endothelial cells and hepatocytes. In S1P1 GFP signaling mice, LPS-mediated systemic inflammation activated S1P1 in endothelial cells and hepatocytes via hematopoietically derived S1P. These data demonstrate that S1P1 GFP signaling mice can be used to evaluate S1P1 activation and S1P1-active compounds in vivo. Furthermore, this strategy could be potentially applied to any GPCR to identify sites of receptor activation during normal physiology and disease.

Authors

Mari Kono, Ana E. Tucker, Jennifer Tran, Jennifer B. Bergner, Ewa M. Turner, Richard L. Proia

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Figure 1

Generation of S1P1 GFP signaling reporter mice.

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Generation of S1P1 GFP signaling reporter mice. (A) Schematic of the “Ta...
(A) Schematic of the “Tango” design to monitor S1P1-β-arrestin interactions. Ligand activation of GPCRs leads to their phosphorylation and subsequent recognition by arrestins. The target GPCR, in this case S1P1, is modified by linking the tetracycline-controlled transactivator (tTA) to its C terminus through a TEV protease recognition sequence (tevs). Ligand binding to the receptor stimulates the recruitment of a β-arrestin-TEV protease fusion protein, triggering the release of tTA from the C terminus of modified S1P1. Free tTA enters the nucleus and stimulates histone H2B-GFP reporter gene activity. (B) Design of the S1pr1 knockin vector. Coding sequences for the 2 fusion proteins, S1P1-tTA and mouse β-arrestin-2-TEV (mArrb2-TEV) protease, connected by an IRES, were included along with the neomycin resistance gene (NeoR) flanked by loxP sites. This knockin segment was flanked by 2.4 kb of 5′ and 3.8 kb of homologous 3′ genomic sequences adjacent to the second exon of S1pr1. The herpes simplex virus thymidine kinase (TK) gene was added outside of the homologous sequence to minimize random integration. Schematics of the targeting vector, WT S1pr1 allele, and S1pr1 knockin allele are shown. PA, polyadenylation sequence. (C) Mouse mating scheme to obtain S1P1 GFP signaling (S1P1GS) mice. S1pr1 knockin mice were crossed with histone-EGFP reporter (H2B-GFP) mice, in which human histone 1 protein H2bj and EGFP fusion protein are expressed under the control of a tetracycline-responsive promoter element and cytomegalovirus minimal promoter.