FTY720 (Gilenya) Phosphate Selectivity of Sphingosine 1-Phosphate Receptor Subtype 1 (S1P1) G Protein-coupled Receptor Requires Motifs in Intracellular Loop 1 and Transmembrane Domain 2

Abstract

FTY720 phosphate (FTY720P) is a high potency agonist for all the endothelial differentiation gene family sphingosine 1-phosphate (S1P) receptors except S1P receptor subtype 2 (S1P 2 ). To map the distinguishing features of S1P 2 ligand recognition, we applied a computational modeling-guided mutagenesis strategy that was based on the high degree of sequence homology between S1P 1 and S1P 2 . S1P 2 point mutants of the ligand-binding pocket were characterized. The head group-interacting residues Arg3.28, Glu3.29, and Lys7.34 were essential for activation. Mutation of residues Ala3.32, Leu3.36, Val5.41, Phe6.44, Trp6.48, Ser7.42, and Ser7.46, predicted to interact with the S1P hydrophobic tail, impaired activation by S1P. Replacing individual or multiple residues in the ligand-binding pocket of S1P 2 with S1P 1 sequence did not impart activation by FTY720P. Chimeric S1P 1 /S1P 2 receptors were generated and characterized for activation by S1P or FTY720P. The S1P 2 chimera with S1P 1 sequence from the N terminus to transmembrane domain 2 (TM2) was activated by FTY720P, and the S1P 2 (IC1-TM2) S1P1 domain insertion chimera showed S1P 1 -like activation. Twelve residues in this domain, distributed in four motifs a-d, differ between S1P 1 and S1P 2 . Insertion of 78 RPMYY in motif b alone or simultaneous swapping of five other residues in motifs c and d from S1P 1 into S1P 2 introduced FTY720P responsiveness. Molecular dynamics calculations indicate that FTY720P binding selectivity is a function of the entropic contribution to the binding free energy rather than enthalpic contributions and that preferred agonists retain substantial flexibility when bound. After exposure to FTY720P, the S1P 2 (IC1-TM2) S1P1 receptor recycled to the plasma membrane, indicating that additional structural elements are required for the selective degradative trafficking of S1P 1 .The lipid signaling molecule sphingosine 1-phosphate (S1P) 3 is an important regulator of the cardiovascular and immune systems and functions in numerous physiological and pathophysiological conditions (for reviews, see Refs. 1-3). Although S1P has several intracellular targets and can function as a second messenger (4), many of its biological effects are mediated by activation of five members of the endothelial differentiation gene family of G protein-coupled receptors, S1P 1-5 . The five S1P receptors share high amino acid identity, ranging from 33 to 51% (5), and the individual receptor subtypes couple to distinct as well as overlapping cellular signaling pathways, allowing S1P to mediate its diverse cellular effects (6, 7).The S1P receptor modulator FTY720 (Gilenya) has been approved as frontline treatment for relapsing/remitting multiple sclerosis (8, 9). FTY720 is phosphorylated in vivo by sphingosine kinase 2 to form the active metabolite FTY720 phosphate (FTY720P), which is a high affinity agonist of all the endothelial differentiation gene family S1P receptors except S1P 2 (10 -12). This type of receptor selectivity of FTY720P appears to be important to its...

Integrating the puzzle pieces: The current atomistic picture of phospholipid–G protein coupled receptor interactions

“…Consideration of docked complexes or even crystallographic complex structures and specific interactions within those complexes can provide insights into the enthalpic differences between different complexes, but do not aid in identification of entropic differences that can dramatically influence specificity. We recently undertook an extensive study to define the source of the FTY720-phosphate selectivity against the S1P 2 receptor [43]. An extensive set of first site mutations and then S1P 1 /S1P 2 receptor chimeras surprisingly identified intracellular loop 1 as providing this selectivity.…”

“…Aromatic ring position has a tremendous impact on receptor activation profiles, with S1P 2 activity evident for analogs in which the aromatic ring is placed at or near the end of the tail region, S1P 3 /S1P 4 potency showing an optimum when the aromatic ring appears near the middle of the hydrophobic tail, and S1P 1 /S1P 5 showing about a ten-fold preference for the aromatic ring to be adjacent to the linker [8]. An atomistic explanation for the anti-S1P 2 selectivity of the analogs with aromatic rings adjacent to the linker was difficult to find, as it stems not from any single amino acid substitution, but from differences in the amino acid sequence in the first intracellular loop, located quite distant from the ligand binding pocket [43]. Extensive molecular dynamics simulations indicate that the differences in the intracellular loop sequence impact the mobility of the hydrophobic tail in a manner dependent on the presence or absence of an aromatic ring.…”

An update on sphingosine-1-phosphate receptor 1 modulators

“…47 However, FTY720 displays various side effects in humans that correlate with its receptor promiscuity; the monophosphate acts as a substrate for all S1P receptors except S1P 2 . 43,48 Agonism of S1P 1 was reported to cause bradycardia, and activation of S1P 3 has been linked to the development of hypertension. 49,50 The original hypothesis was that bradycardia observed in humans with FTY720 was soley due to agonism of S1P 3 , based on rat studies.…”

Molecular Targets of FTY720 (Fingolimod)

“…Once internalized, FTY720-P, but not S1P, induces S1P 1 polyubiquitination at multiple sites in its C-terminus by the WWP2 ubiquitin E3 ligase, and subsequent degradation via the proteasome [125]. Residues in the intracellular loop and transmembrane domain 2 of S1P 1 confer FTY720-P binding and receptor degradation [126]. The mechanisms of this selective ubiquitination and degradation of S1P 1 by FTY720-P, and not S1P, however, remain unknown.…”