Activation and inhibition of G protein-coupled receptors by cell-penetrating membrane-tethered peptides

Abstract

Classical ligands bind to the extracellular surface of their cognate receptors and activate signaling pathways without crossing the plasma membrane barrier. We selectively targeted the intracellular receptor-G protein interface by using cell-penetrating membrane-tethered peptides. Attachment of a palmitate group to peptides derived from the third intracellular loop of protease-activated receptors-1 and -2 and melanocortin-4 receptors yields agonists and/or antagonists of receptor-G protein signaling. These lipidated peptides--which we have termed pepducins--require the presence of their cognate receptor for activity and are highly selective for receptor type. Mutational analysis of both intact receptor and pepducins demonstrates that the cell-penetrating agonists do not activate G proteins by the same mechanism as the intact receptor third intracellular loop but instead require the C-tail of the receptor. Construction of such peptide-lipid conjugates constitutes a new molecular strategy for the development of therapeutics targeted to the receptor-effector interface.

Figure 1

Membrane-tethered PAR1 i3 loop peptides activate regulated Ca²⁺ signaling and aggregation in platelets. (A) The topological arrangement of the membrane-spanning segments (TM1–7), extracellular loops (e1-e4), and intracellular loops (i1-i4) of PAR1 is based on the x-ray structure of rhodopsin (1) and is illustrated on the left. Thrombin cleaves the extracellular domain (e1) at the R₄₁—S₄₂ bond creating a new N terminus, S₄₂FLLRN, which functions as a tethered PAR1 agonist. The composition of the peptides used in this study is shown on the right, and their corresponding effects on platelet Ca²⁺ are shown immediately below. (B) Platelets from healthy volunteer donors were isolated by gel filtration chromatography, and Ca²⁺ measurements were performed as described (11). Intracellular Ca²⁺ concentration was monitored as the ratio of fluorescence excitation intensity at 340/380 nm. (C) PAR1 i3 loop peptides cause full platelet aggregation. Individual aggregation traces of platelets stimulated with 10 μM of indicated peptides or 200 μM palmitic acid are shown. Platelet aggregation was monitored as percent of light transmittance of stirred platelets at 37°C, as described (19).

Figure 2

Membrane-tethered PAR1 i3 loop peptides require the presence of their cognate receptor to activate signaling. (A and B) PAR1-Rat1 cells (24) were challenged with 1 nM to 50–100 μM i3 peptide. PLC-β activity was determined by measuring total [³H]-InsP formation (17). PLC-β activity was converted to percent of the full response relative to 0.1 nM thrombin (100%) and plotted as a function of peptide concentration by using a two-site equation that fits the biphasic activation and inhibition profile. The full PAR1 thrombin responses for individual experiments were 12.4-fold for P1pal-19 and P1pal-19/untransfected Rat1 cells, 18-fold for P1pal-19Q (Pal-RCLSSSAVANQSQQSQALF), 12.4-fold for P1pal-19E (Pal-RCESSSAEANRSKKERELF), 7.6-fold for P1pal-13, and 9.4-fold for P1pal-12 and P1pal-7.

Figure 3

Lipidated PAR1 i3 loop peptides are cell-penetrating and do not activate receptor at the extracellular ligand-binding site. Human platelet Ca²⁺ responses were determined as in Fig. 1B. (A) Palmitoylated i3 loop peptides do not inhibit Ca²⁺ flux pathways downstream of PAR4. Platelets were stimulated with either 3 μM SFLLRN or 2 μM P1pal-19 alone, followed by 2 mM GYPGKF. (B) Palmitoylated i3 loop peptides penetrate intact cells. Flow cytometry was conducted on platelets or Rat1 fibroblasts stably transfected with PAR1 (24) that were treated with Fluor-labeled peptides, Fluor-Pal-i3 (Fluor-P1pal-19), or Fluor-i3 (Fluor-P1-i3–19), as indicated. Cells were incubated with 10 μM Fluor-Pal-i3 or Fluor-i3 for 2 min in PBS/0.1% FCS and then treated with 2 units of pronase for 15 min at 37°C and washed before flow cytometry. (C) Platelets were stimulated with 3 μM SFLLRN or 3 μM P1pal-19 alone. In the right trace, platelets were pretreated with the anti-PAR1 small molecule, 1 μM BMS-200661, then sequentially challenged with 3 μM SFLLRN and 3 μM P1pal-19.

Figure 4

P1pal-19 does not activate C-tail-deleted PAR1 but activates a PAR1 i3-mutant. COS7 cells were transiently transfected with wild-type (WT), S309P, or Δ377 PAR1 receptors. Cells were challenged with (A) thrombin, (B) SFLLRN, or (C) P1pal-19, and PLC-β activity determined by measuring total [³H]-inositol phosphate formation relative to 100% stimulation (9.6-fold) of WT PAR1 with 0.1 nM thrombin.

Figure 5

Receptor selectivity profiles of cell-penetrating PAR1 i3 loop agonists. COS7 cells were transiently transfected with the human receptors PAR1, PAR2, PAR4, CCKA, CCKB, SubP, or rat SSTR2. Transfected cells were challenged with 0.1–10 μM P1pal-19 (A) or P1pal-13 (B), and the highest stimulation of the individual receptors is shown as a black column. The extracellular agonists used to define maximum stimulation for each receptor (open column) were 10 nM thrombin for PAR1, 100 μM SLIGKV for PAR2, 100 nM thrombin for PAR4, 300 nM CCK-8 for CCKA and CCKB, 1 μM AGCKNFFWKTFTSC for SSTR2, and 1.5 μM RPKPQQFFGLM for SubP.

Figure 6

Cell-penetrating PAR2 and MC4 i3 loop peptides activate their cognate receptors. (A) A single point mutation of the C-terminal residue of the PAR2 i3 loop peptide results in full activation of PAR2. COS7 cells were transiently transfected with PAR2 and challenged with 0.1–10 μM P2pal-21 (Pal-MLRSSAMDENSEKKRKRAIK) or P2pal-21F (Pal-MLRSSAMDENSEKKRKRAIF). PLC-β activity was converted to percent of the full response relative to 100 μM SLIGKV (100%) and plotted as in Fig. 2. In these experiments, 100 μM SLIGKV gave 3.9- and 3.1-fold maximal stimulation of PAR2 for the P2pal-21 and P2pal-21F, respectively. (B) Receptor selectivity profile of P2pal-21F. COS7 cells were transiently transfected with the indicated receptors and challenged with 0.1–10 μM of P2pal-21F; the highest stimulation of the individual receptors is shown as a black column. The extracellular agonists used to define maximum stimulation for each receptor (open column) are the same as described in Fig. 5. (C) The lipidated i3 loop peptide of the MC4 obesity receptor stimulates G_s/adenylate cyclase in COS7 cells transiently transfected with human MC4 receptors. Transfected cells were challenged with 3 nM–10 μM MC4pal-14 (Pal-TGAIRQGANMKGAI), and adenylate cyclase activity was converted to percent of the full response relative to 30 nM α-melanocyte-stimulating hormone. Adenylate cyclase activity was measured by using the RIA kit for cAMP (NEN, Lifescience Products).

Figure 7

Inhibitory pepducins derived from the i3 loop sequence of PAR1 and PAR2 are antagonists of their cognate receptors. (A) The cell-penetrating PAR1 i3 loop peptide, P1pal-12, selectively inhibits the Ca²⁺ signal from PAR1. Platelet Ca²⁺ measurements were performed as in Fig. 1. Platelets were pretreated with 3 μM P1pal-12 (open arrowhead) and then stimulated with 3 μM SFLLRN or 200 μM AYPGKF, as indicated. (B) Platelets were preincubated with either vehicle (−) or 3 μM P1pal-12 for 1 min and then sequentially challenged with 3 μM SFLLRN and 200 μM AYPGKF and aggregation monitored. (C) Platelets were pretreated for 1 min with 0.01–5 μM P1pal-12 and challenged with 3 μM SFLLRN. (D) PAR1 and PAR2-expressing COS7 fibroblasts were pretreated with 0.03–100 μM P1pal-12 or P2pal-21 for 5 min, and then challenged with extracellular agonists 0.1 nM thrombin or 100 μM SLIGKV, respectively. Percent InsP inhibition was calculated relative to the full extracellular agonist-stimulated response: 5.2-fold for P1pal-12 and 3.1-fold for P2pal-21. (E) Proposed mechanism of activation and inhibition of receptor–G protein complexes by pepducins. The receptor is shown in its off-state (R) and on-state (R^*) bound to G protein (G). Pepducin agonists are shown as ellipses, pepducin antagonists as rectangles, and the extracellular ligand as a triangle.