title: Role of the PAR1 receptor 8th helix in signaling: the 7-8-1 receptor activation mechanism.
abstract: The protease-activated receptors are tethered ligand G protein-coupled 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 alpha-aneurysm with a downwards-directed 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.
authors.affiliation: Molecular Oncology Research Institute, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
authors.forename: Steven
authors.initials: S
authors.lastname: Swift
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Leger
authors.forename: Joyce
authors.initials: J
authors.lastname: Talavera
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.forename: Andrew
authors.initials: A
authors.lastname: Bohm
authors.forename: Athan
authors.initials: A
authors.lastname: Kuliopulos
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000502:physiology; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D044463:Receptor, PAR-1 / Q000737:chemistry* / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000494:pharmacology
chemical_list: D044463:Receptor, PAR-1; D013917:Thrombin; D043802:GTP-Binding Protein alpha Subunits, Gq-G11