G-protein Coupled Receptors (GPCRs) Lecture 3 2023 PDF

G-Protein Coupled receptors Rhodopsi n Receptor 🞂​ Introducethe basic aspects of GPCRs, their classification, and structure 🞂​ IntroduceG proteins and their characteristics 🞂​ Introducethe effect of post- translational modifications on the action of different proteins, especially the GPCR...

G-Protein Coupled receptors Rhodopsi n Receptor 🞂​ Introducethe basic aspects of GPCRs, their classification, and structure 🞂​ IntroduceG proteins and their characteristics 🞂​ Introducethe effect of post- translational modifications on the action of different proteins, especially the GPCR 🞂​ Part 1: GPCR general features and classes 🞂​ Part 2: G-proteins 🞂​ Part 3: Post-translational modifications  GPCR general features and classes Seven Transmembrane Receptors (7TMR) Hepta-helical Receptors Serpentine Receptors 🞂​ The Nobel Prize in Chemistry was awarded to Robert J. Lefkowitz and Brian K. Kobilka for their work in characterizing G-protein-coupled receptors (GPCRs) — the proteins that enable cells to sense and respond to their environment Robert J. Brian K. Lefkowitz Kobilka Natur 492, 57, 2012 Roth and Marshall, Nature, β2-adrenergic receptor Phosphorylatio n sites for PKA Phosphorylation sites for GRK2 Top-down view of a hormone receptor with an adrenaline molecule bound between the membrane spanning segments. The receptor is viewed from the extracellular surface. The N-terminal domain is glycosylated. The two catechol hydroxyls of adrenaline interact with the serine residues present in the membrane spanning α- helix E. Ostrowski et al. Annu Rev Pharmacol Toxicol.  7 Transmembrane 🞂​ Extracellular domains ◦ α helices are highly conserved 🞂​ 3 extracellular loops (EL) 🞂​ 3 or 4 intracellular loops (IL) Intracellular  S 🞂​ S bridge between extracellular loops bridge 1 and 2 (EL1 and EL2) 🞂​ Consensus sequences : Aspartate- Arginine- Tyrosine (DRY) in intracellular loop 2 (IL2). Importance: ◦ Coupling to G proteins ◦ Switch to regulate receptor activity: DRY motif is important for DRY isomerization of receptors between inactive and activate conformations ◦ In the absence of DRY, receptors are constitutively active  Inactive GPCR conformation Active GPCR conformation Structure of Rhodopsin viewed from the cytoplasmic side. Activation of GPCRs probably involves a separation of the third transmembrane domain (TM-III) and TM-VI (double arrow in part a) and a twist in TM- VI, which ‘pulls’ the third cytoplasmic loop (IL3) into the cell (part b). Such conformational changes probably result in GPCR–G-protein interactions along IL3. Chalmers and Behan, 2002, Nature, 🞂​ Largest known receptor family – ◦ Constitutes > 1% of the human genome. ◦ ~ 800 – 950 genes 🞂​ Comprises receptors for a diverse array of molecules: ◦ neurotransmitters ◦ odorants ◦ lipids ◦ neuropeptides ◦ large glycoprotein hormones 🞂​ Target of the majority of best-selling drugs (40%-50% of all prescribed pharmaceuticals on the market). 🞂​ GPCRs: nearly 300 different kinds – grouped into 6 main subfamilies/class: ◦ Rhodopsin-like group – includes most of the GPCRs. Class A ◦ Glucagon-like group. Class B ◦ Metabotropic glutamate (mGlu) and GABAB receptor family. Class C 🞂​ Grouped according to > 20 % sequence homology. Conn et al. Nature Reviews,  🞂​ Majority are of class I Chalmers and Behan, 2002, Nature, VOLUME 🞂​ Ligand Receptor  G protein  Effectors Ligand size can vary 🞂​ Binding of low-molecular-mass ligands ◦ Amines ◦ Nucleotides ◦ Eicosanoids 🞂​ bind to sites within the hydrophobic core 🞂​ Binding of low-molecular-mass ligands ◦ Peptide hormones (40 Da (Ca2+) to more than 100 kDa) 🞂​ Peptide ligands are accommodated on the exterior face of the receptor 🞂​ Binding of low-molecular-mass ligands ◦ Glycoprotein hormones 🞄 TSH 🞄 LH 🞄 FSH 🞂​ Protein ligands are accommodated on the exterior face of the receptor 🞂​Calcium sensors and metabotropic receptors ◦ Ca2+ ◦ Glutamate ◦ GABAB 🞂​ They bind to large N-terminal extensions. 🞂​ This induces a conformational change in the extension, which then interacts with the receptor.  receptors (PARs) are 🞂​ Proteinase-activated cleaved and the newly exposed N- terminus acts as an auto-ligand. 🞂​ Thefreed peptide may also interact separately with another receptor  GPCRs might GPCRs might Agonist (yellow) binding 2 be silent 3 display basal 4 activates G protein- (blue) activity (pink) dependent and β arrestin- dependent effects. GPCRs are synthesized in the RER. An interacting protein 1 (green) is needed to route some GPCRs, to the plasma membrane Desensitization of the receptor by internalization 5 maintains activation of β arrestin-dependent effects. G protein-dependent effects might also continue. Vassart and Costagliola 2011, NATURE, Vol  Loss–of-Function (LOF) Mutations of GPCRs B: Specific LOF mutations affect only basal activity C: Some LOF mutations D: Classic LOF mutations affect gross cause constitutive protein structure, trapping the desensitization receptor in the RER Vassart and Costagliola 2011, NATURE, Vol Loss –of-Function (LOF) Mutations of GPCRs Cnt’d E: Some LOF mutations F: Other LOF G: Other LOF mutations affect the interacting mutations interfere interfere with the protein function with agonist binding intramolecular required to route some conformational change GPCRs to the plasma involved in activation membrane. Vassart and Costagliola 2011, NATURE, Vol Loss–of-Function (LOF) Mutations of GPCRs Cnt’d H: Other LOF mutations I: Mutations affecting interaction of GPCRs interfere with the ability with one G protein, when the receptor is to bind G proteins coupled to multiple G proteins (1), βarr (2) or interacting proteins (3) cause biased activation. Vassart and Costagliola 2011, NATURE, Vol Blue: Wild- type, silent Yellow: Wild- type, with Red :mutated basal with increased activity basal activity Wild-type (blue and yellow) or mutated (red with a yellow dot) GPCRs might display very different levels of constitutive activity and response to their normal agonist. The curves to the right illustrate the basal activity and responses of wild-type GPCRs (totally silent, or with basal activity) and two examples of mutants with increasing constitutive activity, red and orange curves). Vassart and Costagliola 2011, NATURE, Vol Some GOF mutations might cause increased sensitivity to the normal agonist with minimal change in basal activity Other GOF mutations might render a GPCR sensitive to a normally inert positive allosteric modulator, also resulting in an increase in sensitivity to the normal agonist. Vassart and Costagliola 2011, NATURE, Vol Heng et al. Biotechnol Adv, G-protein-coupled receptor signaling Rockman et al. 2002 NATURE, VOL G-proteins (Hetero-trimeric G-Proteins) 🞂​ Guanine nucleotide binding proteins ◦ Participate in reversible, GTP-mediated interactions 🞂​ Bind GDP and GTP with high affinity ◦ Adopt different structure depending on the bound nucleotide 🞂​ GTPbound complex has high affinity for other proteins (acceptors), affecting their enzymatic activity 🞂​ Possess intrinsic GTPase activity ◦ Usually activated by interaction with regulatory proteins (e.g. GAPs) 🞂​ Covalentattachment of various lipids (Myristoylation, palmitoylation..) is responsible for membrane targeting ◦ Heterotrimeric G-proteins: directly couple to receptors (GPCR) and enzymes 🞄 Coupled to 7TM spanning receptors 🞄 They serve as a bridge between GPCR and effectors 🞄 All members are heterotrimeric consisting of α, β, and γ subunits ◦ For simplicity, it will be further referred to as: G-protein  and γ are attached to the plasma 🞂​ α membrane by lipid anchors 🞂​ α depicts the function of G-protein and subsequently the GPCR bound to it 🞂​ αcan bind either GDP or GTP depending on the activity status: GDP bound: 2010 Nature Education, Modified. 1 4 2 3 Vassart and Costagliola 2011, NATURE, Vol 🞂​ GPCR/Gαs 🞂​ Activates Adenylyl Cyclase AC 🞂​ Produces PKA cAMP 🞂​ Activates PKA 🞂​ GPCR/Gαq 🞂​ Activates Phospholypase C (PLC) Produces IP3 and 🞂​ DAG ◦ IP3 induces Ca2+ release ◦ DAG activates PKC 🞂​ RGSs:Regulators of G-protein Signaling; promote the hydrolysis of GTP into GDP, thus stopping the activation of G-protein ◦ decrease life span of GTP-bound alpha subunits 🞂​ GEFs: Guanine-nucleotide Exchange Factors; promote dissociation of GDP and subsequently the binding of GTP  G-protein Gαs Gαi Cholera Pertussi toxin s toxin sensitiv sensitiv e e 🞂​ Cholera-toxin (CTX): 1. CTX causes ADP- ribosylation of Gαs 2. Gαs loses GTPase activity 3. Gαs trapped in active state: active AC, high cAMP 4. cAMP regulates secretion of fluid into intestine 5. enormous loss of liquid and electrolytes  Pertussis 🞂​Pertussis-toxin toxin (PTX): 1. PTX causes ADP- ribosylation of Gαi 2. Gαi becomes blocked from interacting with GPCR 3. Gαi is now trapped in an inactive state: can’t inhibit AC, High cAMP Post-Translational Modifications (PTMs) 🞂​ It is the chemical modification of protein after its translation. 🞂​ Key role in functional Proteomics. 🞂​ They regulate activity, localization and interaction with other cellular molecules such as proteins, nucleic acids, lipids and cofactors. PTMs could modulate the following: ◦ Stability of protein ◦ Biochemical activity (activity regulation) ◦ Protein targeting (protein localization) ◦ Protein signaling (protein-protein interaction, cascade amplification) 🞂​ Phosphorylatio PTMs that n control GPCR or G-proteins 🞂​ Glycosylation 🞂​ Lipidation 🞂​ Ubiquitination 🞂​ S-Nitrosylation 🞂​ Methylation 🞂​ N-Acetylation 🞂​ Proteolysis 🞂​ Addition of phosphate group to a protein. 🞂​ Principally on serine, threonine or tyrosine residues. 🞂​ Also known as Phosphoregulation. 🞂​ Critical role in cell cycle, growth, apoptosis and signal transduction pathways. 🞂​ Addition of glycosyl group or carbohydrate group to a protein. 🞂​ Principally on Asparagine, hydroxylysine, serine or threonine. 🞂​ Significant effect on protein folding, conformation, distribution, stability and activity.  Lipidation/Membrane Lipidation Anchoring is a method to target proteins to membranes in (endoplasmic reticulum [ER], Golgi apparatus, mitochondria), organelles vesicles (endosomes, lysosomes) and the plasma membrane. Addition of phosphatidylinositol, anchors/transfers cell surface proteins to the plasma membrane. Lipidation/Membrane Anchoring Extracellular Intracellular  🞂​ Ubiquitin is a small regulatory protein that can be attached to the proteins and label them for destruction. 🞂​ Effects in cell cycle regulation, control of proliferation and differentiation, programmed cell death (apoptosis), DNA repair, immune and inflammatory processes and organelle biogenesis. Wertz and Dixit, Cell Death and Differentiation, 2010