Targeting biased signaling by PAR1: Function and molecular mechanism of parmodulins

Figures

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  Figure 1.. Overview of PAR1 signaling. The proteolytic cleavage of the amino-terminus of PAR1 exposes a specific tethered ligand that can bind to an extracellular binding pocket and activate the receptor, leading to signaling via intracellular G proteins and β-arrestin. The amino acid sequence of the protease-sensitive region of PAR1 and the cleavage sites of representative proteases are shown. The different intracellular signaling pathways and the effects on cells triggered by PAR1 via coupling to different G proteins and β-arrestin are indicated. Direct coupling of PAR1 via Gαi is not fully established and may depend on the interaction of PAR1 with coreceptors, for example, EPCR and sphingosine 1-phosphate receptor 1 (S1P1).69,70 EC, endothelial cells; MMP1 and MMP13, matrix metalloproteinase-1 and -13; PR3, proteinase 3.

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  Figure 2.. Overview of PAR1 modulators. (A) Thrombin activation of PAR1 triggers coupling to Gαq, Gα12/13, and β-arrestin 2 leading to cytodisruptive effects. (B) aPC activation of PAR1 selectively triggers coupling to Gα12/13 and β-arrestin 2 but not Gαq and elicits cytoprotective effects. (C) Orthosteric inhibitors (eg, vorapaxar, atopaxar) block the ligand binding pocket and completely block intracellular signaling. (D) Parmodulins target the intracellular surface of PAR1 and selectively inhibit some (eg, Gαq) but not all downstream signaling pathways, mimicking the effect of aPC.

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  Figure 3.. Chemical structures of parmodulins and comparison with those of other intracellularly bound GPCR ligands. (A) Chemical structures of JF5 (parmodulin 1), ML159 (parmodulin 3), ML160 (parmodulin 4), ML161 (parmodulin 2), and NRD-21. Other intracellular GPCR ligands are shown below the parmodulins. (B) Largest common substructure TC between parmodulins and other intracellular GPCR ligands. The receptor or receptor family targeted by the substances is indicated on the left-hand side of the matrix.

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  Figure 4.. Summary of the data on the site of action of parmodulins and comparison with the location of intracellular ligand binding sites in other GPCRs. (A) Schematic diagram of PAR1 showing putative sites of action of parmodulins (orange). Replacement of ICL3 and the sequence after Cys365 of PAR1, comprising the cytosolic half of TM7, H8, and the C-terminus, by the corresponding sequence from PAR4 led to a partial or complete loss of the parmodulin effect.35 (B) Sequence alignment of PAR1 with PAR2 to PAR4 and with other GPCRs, which were crystallized bound to an intracellular ligand, β2AR (protein data bank [PDB]: 5X7D),57 CCR2 (PDB: 5T1A),58 CCR7 (PDB: 6QZH),60 CCR9 (PDB: 5LWE),59 and CXCR2 (PDB: 6LFL).61 The alignment is shown over the positions that form the intracellular ligand binding pocket in those GPCRs. The PAR1 sequence on the top is colored by the sequence conservation grade calculated with ConSurf.71 The elements of the multiple sequence alignment are colored by the amino acids’ chemical properties. (C) Refined 3-dimensional structure of PAR1 (PDB: 3VW7)22 downloaded from GPCR database (GPCRdb)62 showing voids on the cytosolic side of the receptor structure (gray closed…