__________2 type of ligands___________ 1 G-Protein Coupled Receptors GPCRs • One of the largest protein families: > 1000 type of GPCRs in mammals >3% of the human genes • Major drug targets: ~ 60 % of approved drugs interact with specific GPCR G-Protein Coupled Receptors GPCRs 2 G-Protein Coupled Receptors GPCRs Extracellular N-terminus seven 1 2 3 4 5 6 7 membrane-spanning helices Intracellular C-terminus G-Protein Coupled Receptors GPCRs modulate activity of a wide variety of biological processes including: • Neurotransmission • chemoattraction • cardiac function • olfaction • vision, etc. 3 Physiological roles of GPCRs Classes of GPCR * Class A Rhodopsin like • Class C Metabotropic glutamate / pheromone •(Rhod)opsin •Metabotropic glutamate •Olfactory •Calcium-sensing like •Prostanoid •Putative pheromone receptors •Nucleotide-like •GABA-B •Gonadotropin-releasing hormone •Thyrotropin-releasing hormone Class A: Rhodopsin like •Melatonin Class B: Secretin like Class C: Metabotropic glutamate / pheromone • Class B Secretin like Class D: Fungal pheromone •Calcitonin Class E: cAMP receptors •Corticotropin releasing factor •Gastric inhibitory peptide Putative (proposed) families: •Glucagon * Ocular albinism proteins •Growth hormone-releasing hormone * Insect odorant receptors •Parathyroid hormone •PACAP * Plant Mlo receptors •Secretin * Nematode chemoreceptors •Vasoactive intestinal polypeptide * Vomeronasal receptors (V1R & V3R) •Diuretic hormone * Taste receptors T2R 4 G-protein coupled receptors (GPCR) Extracellular N-terminus Intracellular C-terminus seven membrane-spanning helices Binding domain for Ligand Signaling peptides (hormones) Binding domain for G-Proteins 5 about "G" proteins • Are compossed by 3 subunits: α, β, γ, •bind Guanosine Triphosphate (GTP) •the α subint associate to GPCRs only when in “GDP” mode • The α subunit has GTPase activity • α and γ subunits are linked to membrane by covalently attached lipids • G-proteins become activated by GPCRs: Active state: bound GTP Inactive state: bound GDP about "G" proteins 6 the type of G-Protein determine intracellular effects Summary of GPCR signaling 7 the G-protein coupled receptor Kinase family (GRK) • Family of six mammalian serine/threonine kinases • They Phosphorylate the GPCRs after ligand-activation • upon binding to GPCRs, they also bind to cytoplasmic inhibitory protein arrestins o GRK1 (originally called rhodopsin kinase) o GRK2 (originally called β-adrenergic receptor kinase-1, βARK1) o GRK3 (originally called β-adrenergic receptor kinase-2, βARK2) o GRK4 (originally called IT-11) o GRK5 o GRK6 Structure of G-protein coupled receptor Kinase (GRK) Receptor recognition Membrane targeting 8 GPCRs Desensitization Importance of GPCR desensitization …..decreased responsiveness of receptor over time (despite presence of agonist) ‘feedback’ mechanism preventing acute and chronic overstimulation of GPCR signal transduction pathways filtering of several inputs into one cell and integration into relevant biological response Limits therapeutic applicability of drugs acting on GPCR Leads to unwanted side effects (due to compensatory receptor upregulation) such as hypertension, heart failure… 9 Desensitization Concept 1: HOMEOSTHASIS : When cells are perturbed by any signal (a stimulant, an agonist), -very quickly after the signaling occurs- the cell engages multiple mechanisms aimed to return the cell to its basal state. ! even though the stimulation or perturbation continues ! Data from HEK-293 cells Transfected with the angiotensin receptor Rapid increase of diacylglycerol angiotensin still present ! angiotensin types of GPCR desensitization 1.Homologous desensitization • agonist-dependent • Rapid (sec~min) 2.Heterologous desensitization (down-regulation) • agonist-independent • Slow (min~hrs) 10 types of GPCR desensitization Homologous desensitization: • through phosphorylation by: •G protein-coupled receptor kinases ( GRK ) or •Second messenger-dependent protein kinases ( PKA/ PKC ) • receptor stays in the plasma membrane • generally reduced responsiveness Heterologous desensitization: • through phosphorylation by GRK • receptor is internalized • reduced responsiveness only to specific agonist 11 mechanisms of GPCRs desensitization Loop -like feedback mechanisms induce desensitization independently mechanisms of GPCRs desensitization of arrestins Kinase recognize the activated receptor C-terminus of Receptor is Phosphorylated Phosphorylation favors binding of specific interacting protein This block the binding of arrestins enhanceThe G the desenzitization mediated by GRK proteins -protein !! 12 mechanisms of GPCRs desensitization Regulatory domains GPCRs binding here ! G-βγβγβγ subunits binding here ! mechanisms of GPCRs desensitization 13 Arrestins are adaptor proteins for GPCR desensitization • GRK-mediated phosphorylation is not sufficient for full receptor inactivation • cofactor (‘arresting protein’) first identified as binding of a 48-kDa soluble protein to phosphorylated rhodopsin “visual arrestin” •Arrestins bind their GPCR targets in a GRK -dependent manner following agonist stimulation Arrestins are adaptor proteins for GPCR desensitization name tissue localisation Vis. Arrstin retina Cytosol ß-Arrestin 1/ 2 ubiquitous Cytosol cone-Arrestin retina, lungs Cytosol D- E-Arrestin ubiquitous Cytosol Activated-receptor recognition 48 kD phosphorylation recognition 14 The β-arrestin mechanism is bi-functional: DESENSITIZATION & INTERNALIZATION The β-arrestin mechanism is bi-functional Concept 1: They desensitize GPCRs ! Concept 2: at the very same time they work as SINGLE TRANSDUCER UNITS (in its own ability) leading to activation of several biochemical pathways Concept 3: This system can also lead to endocytosis of the GPCRs via clathrin 15 The βββ-arrestin and desensitization of GPCRs arrestins block G-protein signaling thus reducing the production of ... 2nd messengers The βββ-arrestin and desensitization of GPCRs arrestins also function as ADAPTORS βββ-Receptor to recrut precisely the enzymes that DEGRADATE 2nd messengers Adenylate cyclase = cAMP if the GPCR is a βββ-Receptor (which is a Stimulative regulative G-protein (Gs)-coupled receptor), then 2nd messenger will be cAMP, and the recruted protein would be the cAMP-phosphodiesterase PDE4D 16 The βββ-arrestin and desensitization of GPCRs mACh-Receptor phospholipase C (PLC) = DAG if the GCPR is a muscarinic ACh-Receptor, (which is a Gq-αααsubunit -coupled receptor) then the 2nd messenger is DAG and the recruted protein would be the DAG-Kinase (which induces DAG degradation) The β-arrestin bi-functional mechanisms ( 1st Mechanism ) They slow the rate of 2nd messenger generation promote the rate of 2nd messenger degradation 17 The β-arrestin mechanism is bi-functional: DESENSITIZATION & INTERNALIZATION βββ-arrestins work as adaptor to facilitate the Clathrin-mediate endocytosis of GCPRs in response to their agonists ubiquitination of βββ-arrestins promotes de interaction with other proteis involved in endocytosis βββ-arrestins require Ubiquitination 18 internalization of “agonist-bound” phosphorylated receptor is β-arrestin-dependent • β-arrestin binds clathrin and adaptor protein-2 (AP-2) • receptors cluster in the pit • pit then pinched off the cell surface by dynamin (large GTPase) GPCRs in diseases 19 GPCRs in diseases β-arrestin-biased D(2)R agonist antipsychotic effects drugs that target GPCR • Zyprex D4-receptors, bipolar disorder & schizophrenia (Eli Lilly co) • Clarinex antihistamine against seasonal & year-round allergies • Zantac treat and prevent ulcers in the stomach and intestines; histamine receptor antagonists, GlaxoSmithKline • Zolmig 5HT1-receptors, migraine (Astra Zeneca) 20 The role of G protein-coupled receptors in the pathology of Alzheimer's disease • GPCRs exert their multiple functions through a complex network of intracellular signalling pathways. • Molecular Mechanism: 1. Ligand-binding 2. GPCRs activate heterotrimeric G protein 3. Exchange of GDP–GTP is induced 4. GTP-binds to the alpha subunit 5. the βγ dimer is releasease. 6. Alpha subunit activates specific secondary effector molecules: • adenylyl cyclase (AC) • phospholipase C (PLC) • phospholipase A2 (PLA2) This leads to the generation of 2nd messengers that activate: • Extracellular signal-regulated kinase 1/2 (ERK1/2) • Janus kinase (JAK) • phophoinositide 3-kinase (PI3K) and the modulation of the α-secretase pathway. 21.