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 surfaces). The T4 lysozyme protein, which was inserted in the PAR1 crystal structure to aid crystallization, is removed and helix H8 is added in a possible conformation in the refined PAR1 structure from GPCRdb. The positions that correspond to ligand-binding residues in the β₂AR, CCR2, CCR7, CCR9, and CXCR2 structures are shown as red spheres. The images in the center and on the right-hand side show cytosolic views of the refined PAR1 structure from GPCRdb⁶² (blue) and of AlphaFold predictions of the active state (pink) and Gα_q-bound (yellow) PAR1 structure. The structure models give an impression of how the size of the vestibule accessible from the cytosolic side changes when PAR1 is activated and binds to the Gα_q protein.