Subject(s)
Antibodies, Monoclonal/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Colonic Neoplasms/drug therapy , Research Design/standards , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal, Humanized , Bevacizumab , Clinical Trials, Phase II as Topic , Clinical Trials, Phase III as Topic , Fluorouracil/administration & dosage , Humans , Leucovorin/administration & dosage , Meta-Analysis as Topic , Randomized Controlled Trials as Topic , Treatment OutcomeABSTRACT
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.
Subject(s)
Receptor, PAR-1/chemistry , Receptor, PAR-1/physiology , Signal Transduction/physiology , Amino Acid Sequence , Animals , COS Cells , Chlorocebus aethiops , GTP-Binding Protein alpha Subunits, Gq-G11/physiology , Humans , Models, Molecular , Molecular Sequence Data , Structure-Activity Relationship , Thrombin/pharmacologyABSTRACT
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.