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 membranetethered 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. G protein-coupled receptors (GPCRs) play a vital role in the signaling processes that control cellular metabolism, cell growth, and motility, inflammation, neuronal signaling, and blood coagulation. Although remarkably diverse in sequence and function, all GPCRs share a highly conserved topological arrangement of a seven-transmembrane helical core domain joined by three intracellular loops, three extracellular loops, and N-and C-terminal domains (1). A key event for the switch from inactive to active receptor is ligand-induced conformational changes of transmembrane helices 3 (TM3) and 6 (TM6) (2). These helical movements in turn alter the conformation of the intracellular loops of the receptor to promote activation of associated heterotrimeric G proteins.Mutagenesis studies (3-5) demonstrated that the third intracellular loop (i3) mediates a large part of the coupling between receptor and G protein. i3 loops expressed as minigenes have also been shown to directly compete with ␣1B-adrenergic receptors for G q binding (6). Okamoto and colleagues (7) localized a G protein activator region in the C-terminal end of the third cytoplasmic loop of the human ␤2-adrenergic receptor. They showed that a soluble peptide corresponding to this region (R 259 -K 273 ) activates G s protein under cell-free conditions. Moreover, related peptides found in wasp venom, such as mastoparan, stimulate GDP-GTP exchange from purified G proteins (8). These amphiphilic cationic peptides act in the absence of receptors to directly stimulate G i and G o and compete with intact receptor for the G protein ␣ subunit (9). However, there are currently no effective strategies to directly study the mechanism of receptor-G protein coupling in a controlled fashion under in vivo conditions.Here, we present an approach to study receptor-mediated G protein activation by using palmitoylated peptides as receptormodulating agents based on the i3 loops of the protease-activated receptors (PAR), PAR...

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...

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