Bioc 325 Lecture 8 PDF

Orphan receptors Protease-Activated Receptors (PARs) 1 Introduce a special subset of GPCR family, Protease Activated Receptors (PARs) Review the mechanisms by which cell-surface proteolysis activates PARs to initiate signal transduction....

Orphan receptors Protease-Activated Receptors (PARs) 1 Introduce a special subset of GPCR family, Protease Activated Receptors (PARs) Review the mechanisms by which cell-surface proteolysis activates PARs to initiate signal transduction. 2 3 An orphan receptor is an apparent receptor that has a similar structure to other identified receptors but whose endogenous ligand has not yet been identified. Human genome contains ~800 gene encoding for GPCR half encodes for sensory receptors the remaining, ~360 receptors ◦ ~210 receptors are of identified ligand ◦ ~160 are Orphan receptors with no known ligand or function If a ligand for an orphan receptor is later discovered, the receptor is referred to as an "adopted orphan". GPCR orphan receptors are usually given the name "GPR" followed by a number, for example GPR1. 4  39%: Class I, knowns I 37%: Class I, orphans Known I 6% : Class II, orphans II 5%: Class II, knowns Orphans I 5 Chalmers and Behan, 2002, Nature, VOLUME 1 Orphan and liganded GPCRs (non-sensory receptors) Wise et al. Annu. Rev. Pharmacol. Toxicol. 2004. 44:43–666 Screening 7 Wise et al. Annu. Rev. Pharmacol. Toxicol. 2004. 44:43–66 Screening “Reverse pharmacology" approach 8 Wise et al. Annu. Rev. Pharmacol. Toxicol. 2004. 44:43–66 Calcium measurement cAMP measurement Internalization of tagged receptor Arachidonic acid release (PLA2) Electrophysiology 9 Examples of adopted orphan receptors in the GPCR family include 1 ◦ Histamine H4 receptor ◦ Orexin receptor ◦ Prolactin Releasing Peptide receptor Examples of adopted orphan receptors in the nuclear receptor group include 2 ◦ farnesoid X receptor (FXR), ◦ liver X receptor (LXR), ◦ and peroxisome proliferator-activated receptor (PPAR) 1 Szekeres PG, Receptors Channels. 2002;8(5-6):297-308. 2 Fagg GE, Neurosci. Lett. (May 1987). 76 (2): 221–7 10 11 Family of GPCR activated by proteolytic cleavage by Serine proteases Cleavage of part of the extracellular amino terminus exposes a tethered ligand domain, which binds to and activates the receptors to initiate multiple signaling cascades 12 contribute to tissue responses to injury by ◦ repair ◦ homeostasis ◦ cell survival ◦ inflammation ◦ pain transmission PAR family acts through the actions of Gi (cAMP inhibitory), 12/13 (Rho and Ras activation) and q (calcium signaling) to cause cellular actions. 13 Serine proteases ◦ Thrombin acts on PARs 1, 3 and 4 ◦ Trypsin acts on PAR 2 many are produced during tissue damage many cofactors aid in the binding between receptor and proteases Serine proteases rely on serine residue in catalytic domain in the enzyme to induce proteolysis Lysine (K) and Arginine (R) are the cleavage sites 14 comprise ~2% of human genome diverse biological functions membrane bound or soluble can cleave ligand or receptor can activate or terminate signal transduction 15 both TACE and ACE cleave the precursor of the active soluble hormones TACE: TNFα converting enzyme ACE: Angiotensin converting enzyme 16 Ossovskaya and Bunnett, Physiol Rev, 2004 Neutral endopeptidase degrades and inactivate neuropeptide substance P (SP) 17 Ossovskaya and Bunnett, Physiol Rev, 2004 Cleave special GPCR (PARs) on cell membrane ◦ Activate receptor (thrombin cleaving PAR1) ◦ Inactivate receptor (Cathepsin G cleaving PAR1on different site) 18 Ossovskaya and Bunnett, Physiol Rev, 2004 PAR1 activation and signalling α-Th: Thrombin 19 Arora P et al. J Cell Sci 2007;120:921-928 Tethered ligand Important for Thrombin binding to PAR 1 and 3 20 VALERIA S. OSSOVSKAYA AND NIGEL W. BUNNETT Thrombin binds to PAR 1/3 due to the presence of Hirudin like site cleaves peptide sequence of N terminal allows the binding of tethered ligand to receptor 21 Ossovskaya and Bunnett, Physiol Rev, 2004 binding to one receptor facilitates cleavage of another receptor on the same cell PAR 3 contains Hirudin like site, binds thrombin PAR 4 lacks this site 22 Ossovskaya and Bunnett, Physiol Rev, 2004 Model of PAR 3 co-factoring of PAR4 Lin eta al., Pharmaco Reviews 2013. 23 cleaved receptor can activate uncleaved receptor 24 Ossovskaya and Bunnett, Physiol Rev, 2004 Lin eta al., Pharmaco Reviews 2013. 25 2 types ◦ constitutive PARs internalization ◦ Activated PARs internalization after activation, receptors’ activity is regulated by ◦ Ubiquitin mediated internalization ◦ Clathrin coated pits mediated internalization 26  27 Soh et al. British Journal of Pharmacology (2010)  28 Ossovskaya and Bunnett, Physiol Rev, 2004 29 30  Plasma PK cleavage site PAR1 NATLDPR SFLLRN PAR2 GTNRSSKGR SLIGKV PAR3 NDTNNLAKPTLPIK TFRGAP HB-EGF PAR LPAPR GYPGQV N-Terminus HB-EGF Tethered ligand domain EGFR ADAM MAPK p42/p44, JNK 31  PK PAR-1 PAR-2 PAR-4 HEK-293 GFP Internalizaton? 32  Activation of protease activated receptors (PARS) by plasma PK A B C D PAR1 Panels A-D: HEK 293 cells transfected with GFP-PAR1. A: Control; B: Thrombin 15 min; C: Kallikrein 5 min; D: Kallikrein 30 min E F G H PAR 2 Panels E-H: HEK 293 cells transfected with GFP-PAR 2. E: Control; F: Trypsin15 min; G: Kallikrein 5 min; H: Kallikrein 30 min I J K L PAR 4 Panels I-L: HEK 293 cells transfected with GFP-PAR 4. I: Control; J: Trypsin15 min; K: Kallikrein 5 min; L: Kallikrein 30 min 33 34 35