Turning Receptors On and Off with Intracellular Pepducins: New Insights into G-protein-coupled Receptor Drug Development

G-protein-coupled receptors (GPCRs) are a large family of remarkably versatile membrane proteins that are attractive therapeutic targets because of their involvement in a vast range of normal physiological processes and pathological diseases. Upon activation, intracellular domains of GPCRs mediate signaling to G-proteins, but these domains have yet to be effectively exploited as drug targets. Cell-penetrating lipidated peptides called pepducins target specific intracellular loops of GPCRs and have recently emerged as effective allosteric modulators of GPCR activity. The lipid moiety facilitates translocation across the plasma membrane, where pepducins then specifically modulate signaling of their cognate receptor. To date, pepducins and related lipopeptides have been shown to specifically modulate the activity of diverse GPCRs and other membrane proteins, including protease-activated receptors (PAR1, PAR2, and PAR4), chemokine receptors (CXCR1, CXCR2, and CXCR4), sphingosine 1-phosphate receptor-3 (S1P3), the melanocortin-4 receptor, the Smoothened receptor, formyl peptide receptor-2 (FPR2), the relaxin receptor (LGR7), G-proteins (Gαq/11/o/13), muscarinic acetylcholine receptor and vanilloid (TRPV1) channels, and the GPIIb integrin. This minireview describes recent advances made using pepducin technology in targeting diverse GPCRs and the use of pepducins in identifying potential novel drug targets. G-protein-coupled receptors (GPCRs) are a large family of remarkably versatile membrane proteins that are attractive therapeutic targets because of their involvement in a vast range of normal physiological processes and pathological diseases. Upon activation, intracellular domains of GPCRs mediate signaling to G-proteins, but these domains have yet to be effectively exploited as drug targets. Cell-penetrating lipidated peptides called pepducins target specific intracellular loops of GPCRs and have recently emerged as effective allosteric modulators of GPCR activity. The lipid moiety facilitates translocation across the plasma membrane, where pepducins then specifically modulate signaling of their cognate receptor. To date, pepducins and related lipopeptides have been shown to specifically modulate the activity of diverse GPCRs and other membrane proteins, including protease-activated receptors (PAR1, PAR2, and PAR4), chemokine receptors (CXCR1, CXCR2, and CXCR4), sphingosine 1-phosphate receptor-3 (S1P3), the melanocortin-4 receptor, the Smoothened receptor, formyl peptide receptor-2 (FPR2), the relaxin receptor (LGR7), G-proteins (Gαq/11/o/13), muscarinic acetylcholine receptor and vanilloid (TRPV1) channels, and the GPIIb integrin. This minireview describes recent advances made using pepducin technology in targeting diverse GPCRs and the use of pepducins in identifying potential novel drug targets. G-protein-coupled receptors (GPCRs) 2The abbreviations used are: GPCRG-protein-coupled receptorPARprotease-activated receptorTMtransmembrane domainH8eighth helixNBD12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl))PSphosphatidylserineMC4melanocortin-4DICdisseminated intravascular coagulation. represent a large family of cell-surface receptors that are activated by an array of ligands, including odors, hormones, peptides, and large proteins (Fig. 1). Despite significant sequence and functional diversity between members of the GPCR family, all GPCRs have a conserved molecular architecture of seven transmembrane domains. The GPCR superfamily, with an estimated number of >1000 members (1Takeda S. Kadowaki S. Haga T. Takaesu H. Mitaku S. Identification of G-protein-coupled receptor genes from the human genome sequence.FEBS Lett. 2002; 520: 97-101Crossref PubMed Scopus (324) Google Scholar, 2Fredriksson R. Lagerström M.C. Lundin L.G. Schiöth H.B. The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints.Mol. Pharmacol. 2003; 63: 1256-1272Crossref PubMed Scopus (2257) Google Scholar), is divided into six main subfamilies (Classes A–F) based on signature conserved residues and ligand interactions (Fig. 1A) (3Kolakowski Jr., L.F. GCRDb: a G-protein-coupled receptor database.Receptors Channels. 1994; 2: 1-7PubMed Google Scholar, 4Bockaert J. Pin J.P. Molecular tinkering of G-protein-coupled receptors: an evolutionary success.EMBO J. 1999; 18: 1723-1729Crossref PubMed Scopus (1242) Google Scholar). The Class A subfamily represents the largest group, and many Class A receptors are major drug targets (5Lagerström M.C. Schiöth H.B. Structural diversity of G-protein-coupled receptors and significance for drug discovery.Nat. Rev. Drug. Discov. 2008; 7: 339-357Crossref PubMed Scopus (1160) Google Scholar). Class A receptors are further divided into subgroups on the basis of ligand specificity, such as the protease-activated receptors (PARs). To date, four PARs have been identified: PAR1, PAR2, PAR3, and PAR4. PARs are activated when cleaved by proteases at the N-terminal extracellular domain (6Vu T.K. Hung D.T. Wheaton V.I. Coughlin S.R. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation.Cell. 1991; 64: 1057-1068Abstract Full Text PDF PubMed Scopus (2746) Google Scholar). Proteolytic cleavage exposes a new N terminus that binds to the body of the receptor in an intramolecular mode that activates G-protein signaling. G-protein-coupled receptor protease-activated receptor transmembrane domain eighth helix 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)) phosphatidylserine melanocortin-4 disseminated intravascular coagulation. Aside from a number of conserved motifs, there is modest sequence homology across family classes, especially in the extracellular and intracellular loops. Functionally important conserved residues in transmembrane regions within each receptor class have been identified (7Probst W.C. Snyder L.A. Schuster D.I. Brosius J. Sealfon S.C. Sequence alignment of the G-protein-coupled receptor superfamily.DNA Cell Biol. 1992; 11: 1-20Crossref PubMed Scopus (704) Google Scholar, 8Wess J. Molecular basis of receptor/G-protein coupling selectivity.Pharmacol. Ther. 1998; 80: 231-264Crossref PubMed Scopus (374) Google Scholar). In Class A receptors, the conserved (D/E)RY motif, located at the interface between TM3 and intracellular loop 2, is involved in formation of salt bridges that maintain the receptor in the inactive conformation. In rhodopsin, this salt bridge formation, also referred to as an “ionic lock,” occurs between Arg-135 in TM3 and Glu-247 in TM6. Another essential motif is the NPXXY sequence in TM7, which connects ligand binding with the intracellular eighth helix and G-protein activation. A specific cholesterol-binding site was observed in proximity to a conserved tryptophan residue from TM4 present in a large number of Class A members, suggesting a potential functional role in receptor binding and thermal stability (9Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le Trong I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Crystal structure of rhodopsin: a G-protein-coupled receptor.Science. 2000; 289: 739-745Crossref PubMed Scopus (5095) Google Scholar). Another important conserved motif in many Class A receptors is an intramolecular disulfide bond between extracellular cysteines in the first and second extracellular loops. The cytoplasmic surface of rhodopsin is shown in Fig. 1B and identifies three cytoplasmic loops (C1, C2, and C3) and a C-terminal tail (C4). Within the C-terminal domain is the highly conserved eighth helix (H8) previously identified in rhodopsin (9Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le Trong I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Crystal structure of rhodopsin: a G-protein-coupled receptor.Science. 2000; 289: 739-745Crossref PubMed Scopus (5095) Google Scholar) and other Class A receptors, including PAR1 and PAR2 (10Swift S. Leger A.J. Talavera J. Zhang L. Bohm A. Kuliopulos A. Role of the PAR1 receptor 8th helix in signaling: the 7-8-1 receptor activation mechanism.J. Biol. Chem. 2006; 281: 4109-4116Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). H8 is anchored to the membrane by palmitoylation of C-terminal cysteine residues. Swift et al. (10Swift S. Leger A.J. Talavera J. Zhang L. Bohm A. Kuliopulos A. Role of the PAR1 receptor 8th helix in signaling: the 7-8-1 receptor activation mechanism.J. Biol. 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This that targeting loops of the GPCRs receptor both within the have identified pepducins that of signaling within a which for drug et al. J. K. J. Kuliopulos A. M. S. A. T. L. L. protease-activated with pepducins in J. Full Text Full Text PDF PubMed Scopus Google Scholar) signaling on the loops of PAR1 in targeting the PAR1 loop and activation. In the PAR1 loop pepducin but on activation and This minireview the of the new class of pepducin and of GPCRs and other cell-surface that these lipopeptides be and to specifically target their cognate which have to of the structure of GPCRs and interactions with pepducins and G-proteins the of pepducin allosteric of the lipid and of the peptide sequence may but also the and of In pepducins represent a new class of for the of GPCRs and their and may be as potential in a of diseases.