Chrivia GPCR Lectures 1 2024 PDF

Cell Signaling & G Protein-coupled Receptors Topics Signal Trans.: From Extracellular Signal to Cellular Response Cell-Surface Receptors & Signal Transduction Proteins G Protein-coupled Receptors (GPCRs): Structure and Mechanism GPCR signaling from endosomes and organelles GPCR scaffolds Goals Learn...

Cell Signaling & G Protein-coupled Receptors Topics Signal Trans.: From Extracellular Signal to Cellular Response Cell-Surface Receptors & Signal Transduction Proteins G Protein-coupled Receptors (GPCRs): Structure and Mechanism GPCR signaling from endosomes and organelles GPCR scaffolds Goals Learn the properties of signaling molecules (ligands), cell-surface receptors, & intracellular signal transduction components. Learn the G protein cycle of reactions involved in GPCR signaling. Learn about the effect of GPCR and transducer/effector localization Learn about Biased Signally Cell Signaling How cells receive, process and respond to information from the environment and from other cells. This mediated by “extracellular” signaling molecules called first messengers (ligands) e.g. hormones, neurotransmitters, local regulators and drugs that bind to receptors PM Organelles Endosomes Cytoplasm Nucleus Membrane bound receptors Cytoplasm/nucleus located receptors Why should we care about cell signaling? A. Activation of signaling pathways controls most physiological responses. Norepinephrine- increases contraction of cardiac muscle cells- this regulates cardiac output and blood pressure. Insulin-increases glucose uptake by muscle and liver cells and adipocytesregulates energy metabolism Aldosterone increases Na+ reabsorption by kidney- regulates Electrolytes in plasma and blood pressure B. Signaling pathways are target of drugs that: 1. Treat diseases- e.g. Aldosterone receptor inhibitors- treatment hypertension 2. Cause side effects- e.g. Aspirin used to treat inflammation and fever but also impairs coagulation of platelets resulting in abnormal bleeding General Principles of Signaling In animals, signaling systems are classified based on the distance over which they act. Endocrine signaling acts over long distances within the organism (e.g., insulin). Paracrine signaling acts over very short distances, for example between neighboring cells. Neurotransmitters and developmental signals typically act in this manner. In autocrine signaling, cells release ligands that bind to their own surface receptors, modulating activity. Many growth factors act in this manner. Finally, signaling systems involving plasma membrane-attached proteins act via direct cell-to-cell contact. General Principles of Signal Transduction Signal transduction refers to the overall process of converting extracellular signals into intracellular responses. Key players in signal transduction are 1. signaling molecules also called first messengers Signaling molecules 2. receptors 3. signal transducer recognise the active recepter and activate the effector 4. effectors. 4. second messengers, Cells respond to signals by changing the activity of existing enzymes (fast) and/or the levels of expression of enzymes and cell components (slower) by gene regulation There are a larger number of different types signal molecules and receptors in cells. In contrast, there are relatively few types of intracellular signal transduction systems. How does this provide specific signals?? Transducers Effectors Second Messengers Transducer activate Effector generate First Messengers/hormones/drugs/ligands First Messengers can bind to specific receptors expressed on cells membrane located through out cells. For example: PM, endosomes and subcellular organelles Usually are hydrophilic in nature but there are exceptions can change the conformation of the receptor Neurotransmitters Acetylcholine Serotonin Norepinephrine Growth hormones and Peptide hormones First Messengers/hormones/drugs/ligands First Messengers also can bind to specific intracellular receptors Usually are nonpolar (hydrophobic) in nature Gases NO Steroid Hormones Testosterone Estradiol Progesterone Glucocorticoids Text The effect of a first messenger depends on: 1. The receptor it binds (location important) 2. The tranducer and the effector used and intracellular signaling cascades activated (location important) 3. The target proteins present in cells (location important) General Concepts of membrane Receptors Localized to the membrane Receptors are proteins Three domains A) ligand binding domain that nonconvalently but specifically binds the first messenger B) transmembrane domain that connects the two components C) Intracellular domain responds to the first messenger by changing conformation to allow transducer function Signaling Components: Receptors Binding of a natural occurring hormone or neurotransmitter or some drugs act as agonist and causes a conformational change in the receptor that leads to the transduction of an intracellular signal. Binding of many drugs/ligands/hormones act as antagonist which do not lead to an intracellular signal. These inhibit function of agonist. Binding specificity and affinity are determined by the extent of molecular complementarity between the ligand and the receptor. Signaling Components: Receptors A given receptor may exhibit specificity for a ligand or a group of closely related (structurally) ligands. A given ligand may bind to different types of receptors, that activate different transducers/effectors (different cell responses). Further, two receptors that bind different ligands, may signal via the same transducer/effector, even within a single cell. Membrane Receptors Receptor types are defined by how signal is transduced G-protein coupled Receptors Enzyme-linked receptors when they change their shape the series of reactions occur so thet the binding occusr Transducer Effector Signaling Components: Effectors enzymes General Properties A. In many cases produces a signal that can move from membrane to activate intracellular events B. In most cases –Amplification of signal C. The signal can be distributed in cytoplasm to influence several processes at the same time or localized to activate a specific response. receptors are at the plazma membrane, golgi, endosomes, where it will give a very specific response Two Methods for effectors to signal A) Generation of Second Messengers-small molecules B) Activation of series of enzymes usually kinases Signal Components: 2nd Messengers While there are a large number of extracellular receptor ligands ("first messengers"), there are relatively few small molecules used in intracellular signal transduction ("second messengers"). These are cAMP, cGMP, 1,2-diacylglycerol (DAG), and inositol 1,4,5trisphosphate (IP3), and calcium and phosphoinositides. Second messengers are small molecules that diffuse rapidly through the cytoplasm to their protein targets. Another advantage of second messengers is that they facilitate amplification of an extracellular signal. Text stays in the membrane and have effects neaar membrane , hydrophobic Signal Amplification in Signaling Pathways At each step of many signal transduction pathways, the number of activated participants in the pathway increases. This is referred to as signal amplification, and hormone signaling pathways are often referred to as amplification cascades. For example, one epinephrineactivated GPCR activates many adenylyl cyclases, that each produce hundreds of cAMP molecules, and so on. The overall amplification associated with epinephrine signaling is estimated to be ~108fold. Regulation of Signaling ON/OFF : GTPase Switches Heterotrimeric (large) G proteins, Monomeric (small) G proteins GTPase switch proteins also play important roles in intracellular signal transduction. GTPases are active when bound to GTP and inactive when bound to GDP. The timeframe of activation depends on the GTPase activity (the timer function) of these proteins. Proteins known as guanine nucleotideexchange factors (GEFs) promote exchange of GTP for GDP and activate GTPases. Proteins known as GTPaseactivating proteins (GAPs), stimulate the rate of GTP hydrolysis to GDP and inactivate GTPases. Activate enzyme Regulation of Signaling ON/OFF : Kinases/Phosphatases Kinases use ATP to phosphorylate amino acid sidechains in target proteins. Kinases typically are specific for tyrosine or serine/threonine sites. Phosphatases hydrolyze phosphates off of these residues. Kinases and phosphatases act together to switch the function of a target protein on or off. There are about 600 kinases and 100 phosphatases encoded in the human genome. Activation of many cell-surface receptors leads directly or indirectly to changes in kinase or phosphatase activity. Note that some receptors are themselves kinases (e.g., the insulin receptor). Adding a phosphate Can also inactivate proteins Map Kinase Signal Cascade-Ser/Thr kinases 100 1000 10000 RAF MEK membrane bound receptor G-protein coupled receptors form the largest family of membrane receptors approximately 850 members. Over 30% of drugs target these receptors. They are named GPCR because they interact with and activate heterotrimeric Gproteins. All G-protein coupled receptors have consist of a single polypeptide chain that spans the plasma membrane 7 times. GPCR- Diversity of ligands, receptors and Biological functions Text G-protein Couple Receptors and heterotrimeric G protein They bind to heterotrimeric G proteins hence their name G protein coupled receptors. The G protein transduces the signal to a membrane bound enzyme or to an ion channel a subunit: N terminal Myristoylation (myristic acid ) and Palmitoylation (palmitic acid). g subunit prenylation of the γ subunit (prenyl groups -3-methylbut-2-en-1-yl) FIGURE 2 | Crystal structures of representative GPCR-ligand complexes from classes A, B, C, and F presenting diverse ligand-binding s classified into rhodopsin (bRho, PDB ID: 2HPY) and nonrhodopsin GPCRs. The representative structures of class A nonrhodopsin GPCR subdivided into aminergic-like (β2 AR, PDB ID: 3P0G), nucleotide-like (A2A AR, PDB ID: 3QAK), peptide-like (µ-OR, PDB ID: 5C1M), and l PDB ID: 5XRA) along with their bound ligands are shown. Similarly, representative structures for class B (CRF1 [PDB ID: 4K5Y], GCGR [P GLP-1R [PDB ID: 5NX2], and CTR [5UZ7]), class C (mGlu1 R [PDB ID: 4OR2]), and class F (SMO [PDB ID: 4QIN] bound to negative allos Receptors are shown in cartoon representation and the ligands are shown as stick models with transparent surfaces. Agonists are repres antagonists are shown as purple sticks, and negative allosteric modulator is shown as blue stick model. Class A/Rhodopsin family GPCRs Contains hundreds of receptors and consist of a 7-transmembrane (7TM) in the membrane with three extracellular and three intracellular loops connecting the individual helices and an extracellular N-terminus and intracellular C-terminus). The seven TM helices form a bundle; and a short helix VIII runsOF parallel to theSTRUCTURAL membrane is often found different near thecrystallization C-terminus. techniques came BOOMING AGE GPCR 2007). Moreover, the first crystal struc C, and F have been solved (Hollens The ligand binding pocket is positioned in the extracellularB, of theWu bundle is So far, ex ethalf al., 2013; et al., and 2014). The pioneering study of two-dimensional (2D) structure for 44 distinct GPCRs and ∼205 ligand-rece bovine (bRho) 1983 marked beginning of formedrhodopsin by residues frominhelices II, III, the V, VI, and VII. GPCR structural biology (Hargrave et al., 1983). A decade later, all the four classes, A–C, and F are a 2D projection map was calculated from the solved 2D crystals belong to the Class A subfamily (Haus of bRho using electron cryomicroscopy, which served as the be noted that most of the existing GPC ones, bound to an inhibitor. In the basis for the construction of the receptor molecular model more than 40 GPCR crystal structures (Baldwin, 1993; Schertler et al., 1993). However, the first threewhich are listed in Table 2. GPCR dimensional (3D) structure of bRho in its inactive state was revealed the arrangement of the TM d released only in 2000 (Palczewski et al., 2000). Despite relentless orthosteric, allosteric, bitopic, and bias efforts, elucidation of GPCR structures remained challenging BIOLOGY Class A; ligand binding sites ECL2 (A) ECL1 ECL3 N-term TM4 TM2 TM3 TM5 TM1 H8 TM7 TM6 ICL3 ICL1 (B) 2x60 23x50 7x42 1 2x63 7x4 3x28 7x38 3x29 7x35 3x32 7x34 0.4 0.3 0.2 3x33 7x31 3x36 3x37 x52 0.1 6x58 6x55 6x52 2x60 23x50 7x42 1 2x63 7x4 3x28 7x38 3x29 7x35 3x32 7x34 0.4 0.3 0.2 3x33 7x31 3x36 3x37 x52 C-term ICL2 0.1 6x58 6x55 6x5 2x60 23x50 7x42 1 2x63 7x4 3x28 7x38 3x29 7x35 3x32 7x34 0.4 0.3 0.2 3x33 7x31 3x36 3x37 x52 6x58 6x55 6x52 Class A receptors: When ligand binds TM6 rotates and swings away from center of the helical GPCR, the β adrenergic receptor (β AR; PDB ID: 2RH1); (B) A bundle, accompanied by (PDB inward movements of TM 7 Reviews Review ID: 3SN6); and (C) an active GPCR binds a G protein Chemical ID: 2 2 the G protein α-subunit. membrane helices (gray), three extracellular loops (ECLs) and an l terminus (purple). The transmembrane domain consists of the Figure 6. Structural rearrangements during GPCR activation. Inactive (light pink) and active (dark purple) conformations of the β2AR show Cartoon representation of the β2AR highlighting transmembrane differences in helix position and side-chain orientation in three distinct regions of the GPCR: the binding pocket (top, left); the connector region, or conserved core triad (bottom, left); and the intracellular coupling site (top and bottom, right). ential for the transduction of a signal across the cell mbrane, is composed of a bundle of seven α-helices GPCR Dynamics: Structures in bedded in the cell membrane connected by three motion. Chemical review Latorraca et al 2017 FIGURE 2 | Crystal structures of representative GPCR-ligand complexes from classes A, B, C, and F presenting diverse ligand-binding s classified into rhodopsin (bRho, PDB ID: 2HPY) and nonrhodopsin GPCRs. The representative structures of class A nonrhodopsin GPCR subdivided into aminergic-like (β2 AR, PDB ID: 3P0G), nucleotide-like (A2A AR, PDB ID: 3QAK), peptide-like (µ-OR, PDB ID: 5C1M), and l PDB ID: 5XRA) along with their bound ligands are shown. Similarly, representative structures for class B (CRF1 [PDB ID: 4K5Y], GCGR [P GLP-1R [PDB ID: 5NX2], and CTR [5UZ7]), class C (mGlu1 R [PDB ID: 4OR2]), and class F (SMO [PDB ID: 4QIN] bound to negative allos Receptors are shown in cartoon representation and the ligands are shown as stick models with transparent surfaces. Agonists are repres antagonists are shown as purple sticks, and negative allosteric modulator is shown as blue stick model. The class B/Secretin family- GPCRs comprise fifteen receptors in humans that are activated by peptide endocrine hormones, peptide paracrine factors, and neuropeptides. The topology of class B receptors is similar to that of the class A receptors except that class B receptors have an N-terminal BOOMING AGE OF GPCR STRUCTURAL different crystallization techniques came 2007). Moreover, the first crystal struc BIOLOGY extracellular domain (ECD) of ~120 amino acids in addition to the 7TM domain. B, C, and F have been solved (Hollens et al., 2013; Wu et al., 2014). So far, ex The pioneering study of two-dimensional (2D) structure for 44 distinct GPCRs and ligand-rece bovine rhodopsin 1983 marked thea beginning of model. Peptides binding(bRho) to theseinreceptors follows two-domain The peptides are ∼205 ~ 30–40 GPCR structural biology (Hargrave et al., 1983). A decade later, all the four classes, A–C, and F are a amino acids inmap length their C-terminal region to the ECDtoand to affinity 2D projection was and calculated from the solved 2D binds crystals belong thecontribute Class A subfamily (Haus of bRho using electron cryomicroscopy, which served as the be noted that most of the existing GPC of binding ones, bound to an inhibitor. In the basis for the construction of the receptor molecular model The N-terminal regionetofal., the peptide bindsthe to first the 7TM bundle toGPCR activate the structures more than 40 crystal (Baldwin, 1993; Schertler 1993). However, three- helical which are listed in Table 2. GPCR dimensional (3D) structure of bRho in its inactive state was receptor revealed the arrangement of the TM d released only in 2000 (Palczewski et al., 2000). Despite relentless orthosteric, allosteric, bitopic, and bias efforts, elucidation of GPCR structures remained challenging a 90º VFT 90º TMD GB2 1 Inactive GB2 c 7 2 6 GB1 5 Fraction observed (%) b Inactive Int-1 Int-2 GB1 4 int-1 int-2 active TM5–TM5 TM5–TM5 TM6–TM6 TM6–TM6 6 4 inactive active inactive active The class C/Glutamate family GPCRs comprise fifteen receptors in humans that are activated by FIGURE 2 | Crystal structures of representative GPCR-ligand complexes from classes A, B, C, and F presenting diverse ligand-binding s small molecules such as amino ions. This GPCRs. family The includes receptors for of theclass neurotransmitter classified into rhodopsin (bRho, PDB ID: acids 2HPY) and and nonrhodopsin representative structures A nonrhodopsin GPCR subdivided into aminergic-like (β2 AR, PDB ID: 3P0G), nucleotide-like (A2A AR, PDB ID: 3QAK), peptide-like (µ-OR, PDB ID: 5C1M), and l glutamate, the GABA receptors, and calcium sensing receptor. The ECD is called “venus flytrap PDB ID: 5XRA) along with their bound ligands are shown. Similarly, representative structures for class B (CRF1 [PDB ID: 4K5Y], GCGR [P d [5UZ7]), class C (mGlu R [PDB ID: 4OR2]), and class F (SMO [PDB ID: 4QIN] bound to negative allos GLP-1R [PDB ID: 5NX2], and CTR 1 domain” (VFT) contains the entire ligand binding site. The VFT forms a bi-lobed structure with the Receptors are shown in cartoon representation and the ligands are shown as stick models with transparent surfaces. Agonists are repres antagonists are shown as purple sticks,in and modulator shown as blue stick ligand binding pocket situated a negative centralallosteric cleft. The classis C receptors existmodel. as dimers and binding of agonist enhances interaction of ECDs and changes orientation of transmembrane domains 3 4 5 1 3 6 7 2 2 Active GB2 4 7 6 3 2 0 1 0 5 10 20 30 Intracellular 4 3 6 2 7 5 BOOMING AGE OF GPCR STRUCTURAL BIOLOGY e Inactive Int-1 4 3 1 GB2 Agonist The pioneering study of two-dimensional (2D) structure for bovine rhodopsin (bRho) in 1983 marked the beginning of GPCR structural biology (Hargrave et al., 1983). A decade later, 2D projection map was calculated from the solved 2D crystals 1 1 5 5 2 5 5 of bRho using electron cryomicroscopy, which served as 2the 2 3 4 2 3 4 6 7 1 6 7 1 basis for the construction of the receptor molecular model (Baldwin, 1993; Schertler et al., 1993). However, the first threeGB1 dimensional (3D) structure GB2 of bRho in its inactive state was released only in 2000 (Palczewski et al., 2000). Despite relentless efforts, elucidation of GPCR structures remained challenging Extracellular 1 5 90º 40 180º 2 7 6 3 4 5 different crystallization techniques came GB1 (inac GB1(active) GB2 (inactive) GB2 (active) 2007). Moreover, the first crystal struc Int-2 Active B, C, and F Agonist have been solved Agonist (Hollens et al., 2013; Wu et al., 2014). So far, ex 44 distinct GPCRs and ∼205 ligand-rece all the four classes, A–C, and F are a belong to the Class A subfamily (Haus EC 1 1 6 6 43 4 3 6 6 be noted that most2of the existing GPC 2 7 1 2 3 4 5 3 4 5 7 1 2 ones, bound to an inhibitor. In the more than 40 GPCR crystal structures IC PAM which are listed in Table 2. GPCR revealed the arrangement of the TM d orthosteric, allosteric, bitopic, and bias Binding of ligand enhances G protein binding and binding of G proteins Agonist affinity Chemical Reviews Review Figure 1. GPCR signaling: (A) an orthosteric ligand (orange) binds an inactive GPCR, the β2 adrenergic receptor (β2AR; PDB ID: 2RH1); (B) A ligand-bound GPCR undergoes a conformational change to its active state (PDB ID: 3SN6); and (C) an active GPCR binds a G protein (PDB ID: 3SN6), which subsequently promotes nucleotide release from, and activation of, the G protein α-subunit. Syrovatkina et al. Figure 6. G-protein Receptor Activation Ligand binding changes the conformation of the receptor which allows binding of G protein. The receptor acts as GEF causing the exchange of GDP for GTP on the α subunit, switching it to the activated state. Relay of the signal to the effector. With bound GTP the α chain dissociates from the b g complex and is able to bind to its effector molecule. bg can in turn also activate downstream effectors. First messenger N a GDP C First messenger b g a b g a GTP C GTP GDP b g 3. Receptor activation of heterotrimeric G-proteins. Basic model RGS (regulators of G-protein signaling) are multi-functional, GTPase-accelerating proteins that promote GTP hydrolysis by the α-subunit of heterotrimeric G proteins 37 RGS domain-containing proteins identified in humans Heterotrimeric G-proteins G proteins are designated by their alpha chains so a G protein with αs is Called Gs. 18 types of alpha Gα subunits Response Gs Adenylate cyclase Gq PLCb Gi Inhibits adenylate cyclase Gb 5 types of beta Gg 12 types of gamma The different alphas (with GTP bound) relay the signal to a different effector to give different responses Page 26 Author Manuscript Author Manuscript al. Syrovatkina et al. Figure 3. Figure 1. Phylogenetic relationship of human and mouse G subunits and their expression. Author Manuscript Author Manuscript Figure 2. Phylogenetic relationship of human G subunits and their expression. Page 25 Phylogenetic relationship of human G subunits and their expression. What combinations of a, b and g can form? Calculated number suggests Several hundred potential combinations Express different combinations of G protein subunits in SF9 cells and determined whether they formed heterotrimeric proteins (tested all alpha, beta and gamma subunits) HA-TAG Page 26 Author Manuscript Author Manuscript al. Syrovatkina et al. Figure 3. Figure 1. Phylogenetic relationship of human and mouse G subunits and their expression. Author Manuscript Author Manuscript Figure 2. Phylogenetic relationship of human G subunits and their expression. Page 25 Phylogenetic relationship of human G subunits and their expression. What combination of a, b and g can form? Calculated number suggests Several hundred potential combinations Example using ai1 and b2 with different g subunits Conclusion: Unrestricted ability of the a, b, g subunits to form heterotrimeric complexes Except no complexes β5 with γ1, γ9, γ11 or γ13 HA-TAG It is known that each GPCR only interact with heterotrimeric proteins with a select subset of Ga proteins. Can all b g proteins be in these complexes? Experiment: Mix Purified receptor in presence of ligand, Ga known to interact with receptor and different combination of b and g proteins. No GTP IP with anti-HA and Western with antibody to receptor and Alpha subunit Neurotensin receptor in presence of 20 uM NT and combinations of Gαi1/β1/ γ IP of HA tagged g subunit of abg complex. look for receptor and Receptor binds better to some Gαi1/β1/ γ complexes Overall Conclusion: Many combinations of a, b and g form but Each GPCRs likely to preferentially bind only a subset Remember both a and bg activate effectors Table 1 G subunit interacting proteins Family G subunit Well-defined G-protein effectors Other G-protein interacting proteins Gas G s, G Adenylate cyclase (+) Tubulin, Calnuc, Src tyrosine kinase, axin Gai G o, G G g, G i1–3, G t1,2, z Adenylate cyclase (-), cGMP phosphodiesterase (+) Rap1Gapll, Calnuc, Src tyrosine kinase, nucleobindin 2 (NUCB2), Tubulin, Pins, Pcp1, LGN, GRIN1, Eya2, Pcp2 Gaq G q, G G 15/16 11, G 14, Phospholipase C- (+), p63RhoGEF GRK2, actin, tubulin, PI3K, TPR1, Btk tyrosine kinase, Phospholipase C- , TRPM8 p115RhoGEF, LARG and PDZ-RhoGEF Gap1, rasGap, Btk tyrosine kinase, Radixin, Hax-1, Cadherins, -SNAP, p120caterin, Integrin lllb 3 Ga12 G olf 12, G 13 (+) indicates stimulation. (-) represents inhibition. al. Page 33 Figure 10. Crystal structures of G in complex with different downstream effectors. (a) Cartoon representation of G s (orange) with adenylyl cyclase (AC) C1A (magenta) and C2A domains (green). (b) Representation of G q (orange) with phospholipase C 3(blue). (c) Representation of G q (orange) with p63RhoGEF (blue) and RhoA (green).