Role of the PAR1 Receptor 8th Helix in Signaling

Abstract

The protease-activated receptors are tethered ligand G proteincoupled receptors that are activated by proteolytic cleavage of the extracellular domain of the receptor. The archetypic protease-activated receptor PAR1 strongly activates G q signaling pathways, but very little is known regarding the mechanism of signal transference between receptor and internally located G protein. The recent x-ray structure of rhodopsin revealed the presence of a highly conserved amphipathic 8th helix that is likely to be physically interposed between receptor and G protein. We found that the analogous 8th helix region of PAR1 was critical for activation of G q -dependent signaling. Engineering an 8th helix ␣-aneurysm with a downwardsdirected alanine residue markedly interfered with signal transference to G q . The 8th helix-anchoring cysteine palmitoylation sites were important for the affinity of ligand-dependent G protein coupling but did not affect the maximal signal. A network of H-bond and ionic interactions was found to connect the N-terminal portion of the 8th helix to the nearby NPXXY motif on transmembrane helix 7 and also to the adjacent intracellular loop-1. Disruption of these pairwise interactions caused additive defects in coupling to G protein, indicating that the transmembrane 7-8th helix-i1 loop may move in a coordinated manner to transfer the signal from PAR1 to G protein. This "7-8-1" interaction network was found to be prevalent in G protein-coupled receptors involved in endothelial signaling and angiogenesis.Proteolytic cleavage of the G protein-coupled protease-activated receptors (PARs) 2 activates an extraordinarily diverse array of physiologic responses. These include platelet aggregation and cell-cell adhesion, proliferation/apoptosis, protease homing, cancer invasion, angiogenesis, and the hemostatic and inflammatory responses to vascular injury (1-10). Four protease-activated receptors have been identified: PAR1, PAR2, PAR3, and PAR4. A distinguishing feature of the PARs is that they all contain a preligand sequence located within their extracellular N-terminal domains (1). PAR1 is cleaved by thrombin and other proteases at the Arg 41 -Ser 42 bond to create a fresh 42 SFLLRN 47 N terminus that acts as an intramolecular ligand (11,12). NMR studies indicate that, following cleavage by thrombin, the PAR1 ligand region undergoes a large conformational change and docks to ligand-binding site(s), one of which is located in the N-terminal extracellular domain (13). Synthetic peptides (e.g. SFLLRN) corresponding to the freshly cleaved N terminus can displace the tethered ligand from the ligand binding site(s) and fully activate PAR1 in an intermolecular mode (13).Following intra-or intermolecular liganding, the transmembrane domains of the PARs undergo a conformational change that is transmitted to the intracellular loops (i1-i3) and C-terminal i4 domain. It is these intracellular loops and domains that communicate with the plasma membrane-associated heterotrimeric G proteins. PAR1 can interact with three of t...

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

“…These data are consistent with previous data (43) that showed that the affinity of PAR1 for its extracellular ligand was weakened in the absence of coupled G protein that allosterically stabilizes the receptor in the high affinity state. (44) residues in the H8 helix region (31,45) of the intracellular i4 domain gave 95-100% loss of activation by P1pal-19 with little or no effect on activation by extracellular agonists thrombin and SFLLRN (Fig. 2).…”

“…TM7 contains an essential conserved (D/N)PXXY motif whose tyrosine (Y) residue rotates inside the transmembrane helical bundle to stabilize TM6 in an open on-state (1,49). This lower region of TM7 also interacts with the eighth helix in a tightly coordinated manner during coupling of receptor to G protein (31,49,50). To further delineate the mechanism of activation of PAR1 by the i3 loop pepducin, we tested the activity of a series of point mutants across the entire H8 helix region and adjacent TM7 helix of PAR1 for the ability to be activated by pepducin versus extracellular agonists (Fig.…”

“…The rabbit polyclonal PAR1 antibody (SFLLR-Ab) was generated as described previously (30). PAR1 mutants were made by site-directed mutagenesis in pcDEF3 with QuikChange (Qiagen) or PCR mutagenesis and sequenced to verify the fidelity of the construct as described (31). Cercopithecus aethiops kidney cells (COS7) and human embryonic cells (HEK293) were transfected with T7-PAR1 mutants using Lipofectamine (31) for inositol phosphate (InsP) and fluorescence assays and calcium phosphate for binding studies, and stable PAR1 Rat 1 cells (5) and Rat 1 PAR1-null cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin in 5% CO 2 4 , 0.02% NaN 3 , 1.1 mM deuterated dithiothreitol (DTT), and 0.1 mM 4,4-dimethyl-4-silapentane-1-sulfonic acid at pH 5.12.…”

Targeting Protease-Activated Receptor-1 with Cell-Penetrating Pepducins in Lung Cancer

“…21,22,38 Pepducins based on the i1 and i3 loops have proven to be potent inhibitors of chemokine receptors 32 and PAR4. 21,35 To guide in the design of i1 and i3 pepducins, a model of PAR1 based on the X-ray structure of rhodopsin 39 was generated 40 as shown in Figure 1A. Because different PAR1 intracellular loops are predicted to interact with distinct regions of the heterotrimeric G q , G i , and G 12/13 proteins it was of interest to test whether intracellular blockade of i1 versus i3 loops would preferentially affect different signaling pathways.…”

“…14 Proteolytic cleavage exposes a new N-terminus that binds to the body of the receptor in an unusual intramolecular mode. It recently was shown that matrix metalloprotease-1 also can cleave and activate PAR1 at a distinct site: D 39 -P 40 . 4,15 Synthetic peptides that correspond to the first few amino acids of the freshly cleaved N-terminus of the PARs (eg, SFLLRN PAR1 , TFLLRN PAR1 , PRSFLLRN PAR1 , SLIGRL PAR2 , and AYPGKF PAR4 ), also can function as selective intermolecular agonists to PARs.…”