Allosteric Activation of a G Protein-coupled Receptor with Cell-penetrating Receptor Mimetics

Abstract

G protein-coupled receptors (GPCRs) are remarkably versatile signaling systems that are activated by a large number of different agonists on the outside of the cell. However, the inside surface of the receptors that couple to G proteins has not yet been effectively modulated for activity or treatment of diseases. Pepducins are cell-penetrating lipopeptides that have enabled chemical and physical access to the intracellular face of GPCRs. The structure of a third intracellular (i3) loop agonist, pepducin, based on protease-activated receptor-1 (PAR1) was solved by NMR and found to closely resemble the i3 loop structure predicted for the intact receptor in the on-state. Mechanistic studies revealed that the pepducin directly interacts with the intracellular H8 helix region of PAR1 and allosterically activates the receptor through the adjacent (D/N)PXXYYY motif through a dimer-like mechanism. The i3 pepducin enhances PAR1/Gα subunit interactions and induces a conformational change in fluorescently labeled PAR1 in a very similar manner to that induced by thrombin. As pepducins can potentially be made to target any GPCR, these data provide insight into the identification of allosteric modulators to this major drug target class.

Keywords: G protein-coupled receptor (GPCR); biotechnology; cell surface receptor; cell-penetrating peptide (CPP); chemical biology; drug delivery system; nuclear magnetic resonance (NMR); receptor structure-function; signal transduction; thrombin.

FIGURE 1.

Structure of an i3 loop pepducin agonist. A, the NMR structure of the P1pal-19 pepducin was determined by simulated annealing methods using 551 NOE-based distance restraints. The α carbons of the pepducin residues were numbered corresponding to the intact receptor. B, the structure of P1pal-19 (red) corresponded closely (backbone root mean square deviation, 2.1 Å) with the analogous i3 loop region of the on-state of the β₂-adrenergic receptor·G_s complex (35) (yellow) comprising the cytoplasmic α-helical extensions of TM5 and TM6 and an overall root mean square deviation of 3.6 Å with the entire i3 loop region of the PAR1 model. The PAR1-vorapaxar (VPX) structure of the off-state is shown in white (33). After cleavage by thrombin or MMP-1 (23), the new N-terminal tethered peptide ligand SFLLRN--- or PRSFLLRN---, respectively, binds to the outside surface of PAR1 in an intramolecular mode (42) to activate receptor-G protein signaling. C, a superimposed ensemble of 30 individual energy-minimized structures of P1pal-19.

FIGURE 2.

Interrogation of intracellular receptor residues required for activation of PAR1-G protein signaling by pepducin versus extracellular agonists. An array of 29 single, double, and triple mutants covering the i1, i2, i3, and H8 intracellular loop regions of PAR1 were transfected into COS-7 cells. Cells were then challenged with various concentrations of the extracellular agonists thrombin, SFLLRN peptide, and P1pal-19 pepducin. Maximal PLC-β activity was determined by measuring total [³H]InsP formation for 30 min (5) as detailed in Table 2. The effects of each mutation for the respective agonists are depicted according to the color activity scale at the bottom. SW I, Switch I.

FIGURE 3.

The i3 loop peptide directly interacts with the PAR1 H8 helix region. A, deletion of the PAR1 H8 helix causes complete loss of the receptor-PLC-β (InsP) response to P1pal-19 in COS7 cells. B, binding of WT PAR1 or ΔH8 PAR1 and Gα_i to avidin-N-biotin-i3 loop peptides. HEK293 cells were transfected with WT PAR1 or ΔH8, or PAR1-null Rat 1 cells were used. Membrane lysates were incubated with avidin beads that were precoated with the biotinylated WT i3 loop peptide (Bio19) or negative control i3 loop (Bio19E) in the presence of 10 μm GDP or GTPγS. Eluted proteins were resolved by 10% SDS-PAGE, and Western blotting was performed to detect PAR1 (SFLLR-Ab; 1:500) or Gα_i (Gα_i1/2-Ab; 1:1000). C and D, Gα_i inhibitor pertussis toxin (PTX) suppresses PAR1-PLC-β activity of PAR1 Rat 1 cells stimulated by either thrombin or P1pal-19. E, top, P1pal-19 and thrombin enhance binding of Gα_i to PAR1. PAR1 Rat 1 cells were stimulated for 10 min with P1pal-19 or thrombin (Thr). Cell lysates were then incubated with PAR1-Ab-coated protein A beads, and bound Gα_i protein was detected by Western blotting. Bottom, schematic representation of the PAR1·G protein complex bound to P1pal-19. Error bars represent S.D. IP, immunoprecipitation.

FIGURE 4.

The i3 loop pepducin activates the receptor via the TM7 (D/N)PXXY motif and tyrosine propeller. A, identification of essential residues in (D/N)PXXYYY and H8 helix. PAR1 mutants were generated as listed in Table 1, and maximal PLC-β activities ±S.D. for each agonist (P1pal-19, SFLLRN, and thrombin) are depicted by activity score. Bottom, side view of the (D/N)PXXY motif, YYY propeller, and H8 helix. Residues are colored according to the activity score for P1pal-19 agonist. B, PLC-β activity for each PAR1 mutant was converted to a percentage of the full response relative to 1 μm P1pal-19 for WT PAR1 (100%) and plotted as a function of P1pal-19 concentration using a two-site equation that fit the biphasic activation and inhibition profile (5). C, proposed model of activation PAR1 by P1pal-19 by a TM7 tyrosine propeller mechanism. The on-state (silver) and off-state (blue) PAR1 structural models from Fig. 1A were superimposed and docked to the NMR-derived structure of P1pal-19 using the mutagenesis data from A and B. Binding of P1pal-19 to the H8 helix first triggers an inward motion of the N terminus of the H8 helix and C-terminal end of TM7. This inward motion of H8/TM7 leads to a ∼90° rotation of TM7, causing the three tyrosine residues Tyr³⁷¹/Tyr³⁷²/Tyr³⁷³ to also rotate in a propeller motion. Residue Tyr³⁷³ of the (D/P)XXY motif swings into the back of TM6, thereby facilitating a large outward movement of the TM6 and stabilizing the on-state of the receptor. Error bars represent S.D.

FIGURE 5.

Critical pharmacophores in the P1pal-19 agonist. A, cationic side chain residues of P1pal-19 predicted to potentially interact with the PAR1 H8 helix region were sequentially replaced with serine and tested for PLC-β activity for WT PAR1 expressed in COS7 cells (percentage of InsP relative to full response of 0.1 nm thrombin). B, P1pal-19 pepducins (0.6 μm) were tested for the ability to activate PAR1-dependent human platelet aggregation using gel-purified human platelets and light transmission platelet aggregometry. *, p < 0.05; **, p < 0.01; ***, p < 0.001. C, interactions between P1pal-19 i3 loop residues and the TM7/H8 helix region of PAR1. Error bars represent S.D. Pal, palmitate.

FIGURE 6.

The i3 loop pepducin and thrombin induce conformational changes in the cytoplasmic end of TM5 of PAR1. Monobromobimane is a thiol-reactive fluorescent probe and was used to specifically label Cys²⁹⁶ at the TM5/i3 loop junction of PAR1. T7-Ab-agarose beads were used to affinity-purify Cys²⁹⁶ bimane-labeled T7-PAR1C378S/Δ396 (C296Bim) from transfected versus untransfected (HEK) and PAR1 lacking exposed cysteines T7-PAR1C296S/C378S/Δ396 (C296S) cell lysates. A and B, PAR1 agonist 20 nm thrombin (Thr) or 1 or 3 μm P1pal-19 (P1) or buffer was added to 300-μl volumes of the affinity-purified bimane-labeled PAR1 constructs. 30 s after addition of agonists, fluorescence emission intensity was measured at 430–490 nm with excitation at 370 nm. Maximum fluorescence intensity was normalized to the fluorescence of eluates from the T7-Ab beads of the untransfected HEK cells. C, bottom view of PAR1 in the on-state conformation in complex with the agonist pepducin P1pal-19 and the Gα subunit C-terminal α-helix (GαC). D, bottom view of PAR1 in the off-state conformation.

FIGURE 7.

PAR1 forms homodimers/oligomers. A, a dimer model of PAR1 was constructed by first calculating the electrostatic potential of the receptor using Advanced Poisson-Boltzmann Solver assuming 150 mm salt (62). The most intense blue and red coloring represents a potential in excess of +1 kT/e and −1 kT/e, respectively. The dimer interface was selected by minimizing electrostatic repulsions and maximizing favorable interactions. B, bottom view of proposed dimer models of PAR1 depicting the interaction of the i3 loop region of one PAR1 monomer with the H8 helix region of an adjacent PAR1 monomer using the P1pal-19/H8 helix interaction as a pseudodimer template for monomer/monomer interactions. C, co-immunoprecipitations of T7-PAR1 and PAR1-Myc were conducted in protein lysates from transfected HEK293 cells. T7-Ab-agarose beads were used to immunoprecipitate (IP) T7-PAR1 from cell lysates, and bound PAR1-Myc was detected by anti-Myc immunoblot (top). Immunoblotting (IB) with the T7-Ab (bottom) confirmed the presence of T7-PAR1. Pretreatment of HEK cells with 20 nm thrombin for 10 min prior to the collection of cell lysates resulted in complete cleavage and loss of the N-terminal T7 epitope from T7-PAR1 (and binding to beads), confirming that PAR1-Myc did not nonspecifically bind to the T7-Ab-agarose beads. D, PAR1 forms dimers in live COS7 cells. FRET between PAR1-CFP (donor) and PAR1-YFP (acceptor) was quantified in COS7 cells. Fluorescence measurements used 0.5 × 10⁶ cells/ml with excitation at 425 nm and 10-nm slit widths. Top, yellow dashes and blue dots, respectively, represent the signal for PAR1-YFP and PAR1-CFP expressed singly. The FRET signal (gray) was determined by subtracting the background PAR1-CFP (blue) and PAR1-YFP (yellow) signals from the net uncorrected signal from co-expressed receptors (green) as described previously for PAR1-PAR4 heterodimers (22). Bottom, FRET titration between PAR1-CFP and PAR1-YFP co-expressed at different ratios in COS7 cells. The plasmid concentration of donor was kept constant, whereas the acceptor plasmid was varied. Green squares indicate the increase of the FRET amplitude as a function of fluorescence intensity of the acceptor.