Exam 1 Review Slides 2024 PDF

FIGURE 2 | Crystal structures of representative GPCR-ligand complexes from classes A, B, C, and F presenting diverse ligand-binding sites. Class A GPCRs ar classified into rhodopsin (bRho, PDB ID: 2HPY) and nonrhodopsin GPCRs. The representative structures of class A nonrhodopsin GPCRs which are fur...

FIGURE 2 | Crystal structures of representative GPCR-ligand complexes from classes A, B, C, and F presenting diverse ligand-binding sites. Class A GPCRs ar classified into rhodopsin (bRho, PDB ID: 2HPY) and nonrhodopsin GPCRs. The representative structures of class A nonrhodopsin GPCRs which are further subdivided into aminergic-like (β2 AR, PDB ID: 3P0G), nucleotide-like (A2A AR, PDB ID: 3QAK), peptide-like (µ-OR, PDB ID: 5C1M), and lipid-like receptors (CB1 PDB ID: 5XRA) along with their bound ligands are shown. Similarly, representative structures for class B (CRF1 [PDB ID: 4K5Y], GCGR [PDB ID: 5EE7], full-lengt 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 allosteric modulator) are sho Receptors are shown in cartoon representation and the ligands are shown as stick models with transparent surfaces. Agonists are represented as red sticks, 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 founddifferent near thecrystallization C-terminus.techniques came in 2007 (Cherezov BOOMING AGE GPCR BIOLOGY The ligand binding pocket is positioned in the extracellular The pioneering study of two-dimensional (2D) structure for bovine rhodopsin (bRho) in 1983 residues from helices II, III, V, VI,marked and VII.the beginning of GPCR structural biology (Hargrave et al., 1983). A decade later, 2D projection map was calculated from the solved 2D crystals of bRho using electron cryomicroscopy, which served as the basis for the construction of the receptor molecular model (Baldwin, 1993; Schertler et al., 1993). However, the first threedimensional (3D) structure of bRho in its inactive state was released only in 2000 (Palczewski et al., 2000). Despite relentless efforts, elucidation of GPCR structures remained challenging 2007). Moreover, the first crystal structures for GPCR c B, C, and F have been solved (Hollenstein et al., 2013; half of 2013; the bundle is formed byexperimental structu et al., Wu et and al., 2014). So far, 44 distinct GPCRs and ∼205 ligand-receptor complexes co all the four classes, A–C, and F are available, of which belong to the Class A subfamily (Hauser et al., 2017). I be noted that most of the existing GPCR structures are in ones, bound to an inhibitor. In the last year (2017) more than 40 GPCR crystal structures have been deter which are listed in Table 2. GPCR structural studies revealed the arrangement of the TM domains, location orthosteric, allosteric, bitopic, and biased ligand binding FIGURE 2 | Crystal structures of representative GPCR-ligand complexes from classes A, B, C, and F presenting diverse ligand-binding sites. Class A GPCRs ar classified into rhodopsin (bRho, PDB ID: 2HPY) and nonrhodopsin GPCRs. The representative structures of class A nonrhodopsin GPCRs which are further subdivided into aminergic-like (β2 AR, PDB ID: 3P0G), nucleotide-like (A2A AR, PDB ID: 3QAK), peptide-like (µ-OR, PDB ID: 5C1M), and lipid-like receptors (CB1 PDB ID: 5XRA) along with their bound ligands are shown. Similarly, representative structures for class B (CRF1 [PDB ID: 4K5Y], GCGR [PDB ID: 5EE7], full-lengt 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 allosteric modulator) are sho Receptors are shown in cartoon representation and the ligands are shown as stick models with transparent surfaces. Agonists are represented as red sticks, 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 AGE of the class A receptors except that class B receptors have an N-terminal BOOMING OF GPCR STRUCTURAL different crystallization techniques came in 2007 (Cherezov 2007). Moreover, the first crystal structures for GPCR c BIOLOGY extracellular domain (ECD) of ~120 amino acids in addition to the 7TM domain. B, C, and F have been solved (Hollenstein et al., 2013; et al., 2013; Wu et al., 2014). So far, experimental structu The pioneering study of two-dimensional (2D) structure for 44 distinct GPCRs and ∼205are ligand-receptor complexes co bovine (bRho) to in these 1983 receptors marked the beginning of Peptiderhodopsin agonist binding follows a two-domain model. The peptides ~ 30– GPCR structural biology (Hargrave et al., 1983). A decade later, all the four classes, A–C, and F are available, of which 40 projection amino acids in length and their C-terminal region binds to belong the ECDtoand to affinity 2D map was calculated from the solved 2D crystals the contribute Class A subfamily (Hauser et al., 2017). I of bRho using electron cryomicroscopy, which served as the be noted that most of the existing GPCR structures are in of binding ones, bound to an inhibitor. In the last year (2017) basis for the construction of the receptor molecular model more than 40 GPCR crystal structures have been deter (Baldwin, 1993; Schertler et al., 1993). However, the first threewhich are listed in Table 2. GPCR structural studies dimensional (3D) structure of bRho in its inactive state was The N-terminal region of the peptide binds to the 7TM helicalrevealed bundlethe to activate the receptor arrangement of the TM domains, location released only in 2000 (Palczewski et al., 2000). Despite relentless orthosteric, allosteric, bitopic, and biased ligand binding efforts, elucidation of GPCR structures remained challenging Fig. 4 | Activ indicating th intermediate Activation, in 90º VFT (blue arrows state (green change withi GABA B TMDs demonstrate GB2 TMD. Du TM5 (inactiv decreases fro 90º from 13.1 Å to TMD c, Distributio active agonis Inactive int-1 simulations. Int-1 int-2 GB2 upon ag www.nature.com/aps Int-2 active GB2 GB1 npg Chun L et al G protein. e, b c 1 TM5–TM5 inactive 6 314 apo state, th 7 TM5–TM5 active PAM-bound a Inactive TM6–TM6 inactive 2 6 GB2 GB1 TM6–TM6 active conformatio 4 5 4 3 4 VFT of GB1, b 5 3 1 VFTs propag 6 7 2 FIGURE 2 | Crystal structures of representative GPCR-ligand complexes from classes A,2 B, C, and F presenting diverse ligand-binding sites. Classreorientatio A GPCRs ar 2 Active 7 further stabi classified into rhodopsin (bRho, PDB ID: 2HPY) and nonrhodopsin GPCRs. The representative structures of class A nonrhodopsin GPCRs which are further 6 GB2 0 1 3 4 intracellular subdivided into aminergic-like (β2 AR, PDB ID: 3P0G), nucleotide-like (A ID: 5C1M), and lipid-like receptors (CB1 2A AR,5PDB ID: 3QAK), peptide-like0 (µ-OR, 10 PDB20 30 40 G proteins or PDB ID: 5XRA) along with their bound ligands are shown. Similarly, representative structures for class B (CRF1 [PDB ID: 4K5Y], GCGR [PDB ID: 5EE7], full-lengt Fraction observed (%) a The class C/Glutamate family GPCRs comprise fifteen receptors in humans that are activated by small molecules such as amino acids and ions. This family includes receptors for the neurotransmitter glutamate, the GABA receptors, and calcium sensing receptor. The ECD called “venus flytrap domain” d GLP-1Rcontains [PDB ID: 5NX2], and CTR [5UZ7]), class C (mGlu R [PDB ID: 4OR2]), class F (SMO [PDB ID: with 4QIN] the bound to negative allosteric modulator) are sho 1The (VFT) the entire ligand binding site. VFT forms and a bi-lobed structure ligand Receptors are shown in cartoon representation and the ligands are shown as stick models with transparent surfaces. Agonists are represented as red sticks, antagonists are shown as purple and negative modulator is shownexist as blue model.and binding of agonist binding pocket situated insticks, a central cleft. allosteric The class C receptors asstick dimers (Fig. 3a) and enhances interaction of ECDs and changes orientation of transmembrane domains the TMD of Intracellular 4 3 90º 7 BOOMING AGE OF GPCR STRUCTURAL e Inactive BIOLOGY 4 1 6 2 5 Extracellular 5 1 180º 2 7 6 3 4 5 The high Figure 1. GPCRs. different crystallization techniques came inC2007 (Cherezov orthosteric (A) Structural organiza tion of class C GPCRs. Class carboxylic 2007). Moreover, the first crystal structures for GPCR a c Int-1 Int-2 Active C GPCRs have a common structure consisting of a interact Agonist Agonist Agonist the B, C, and VFT F have been solved (Hollenstein et al., 2013; with two lobes (lobe 1 and lobe 2) separating binding of et al., 2013;byWu et al.,orthosteric 2014). So far, experimental The pioneering study of two-dimensional (2D) structure for a cleft as site, a 7TM and a CRD structu The basic a The crystal structure for all butand GABA 44 distinct GPCRs ∼205 ligand-receptor complexes coH bovine rhodopsin (bRho) in 1983 marked the beginning of B receptor. tions with of classes, mGlu3 receptor was used for GPCR structural biology (Hargrave et al., 1983). A decade later, all the four A–C, (PDB andIDF2E4W) are available, of which key hydrog the VFT and CRD. The bovine rhodopsin crystal Video 2). T 2D projection map was calculated from the solved 2D crystals belong to the Class A subfamily (Hauser et al., 2017). I EC the 7TM. 1 1 1 6(PDB 6 4 3 for 5 5 2 5 5 2 4 3 6 6 used structure ID 1GZM)1 was 2 side chains 23 2 3 4 2 4 7 of bRho using electron cryomicroscopy, which served the be noted that most of the existing GPCR structures are in 3 5 2 1 1 2 7 6as 6 4 3 5 1 4 7 7 1 representation of two prototypical (B) Schematic confirmed class as heterodimer (GABA B receptor), ones, bound toC GPCRs an inhibitor. In the last year (2017) basis for the construction of the receptor molecular model Most resid or homodimer (mGlu receptor).For GABAB receptor, more than 40 GPCR crystal structures have been deter (Baldwin, 1993; Schertler et al., 1993). However, the first threeIC GB2 GB1 the VFT of PAM the VFT is directly linked to the 7TM. Two subwhich are units, listed inB1 Table 2. B2GPCR structural dimensional (3D) structure of bRho in its inactive state was shift by abo G ABA and G ABA , form an obligator y studies revealed the arrangement the TM for domains, location released only in 2000 (Palczewski et al., 2000). Despite relentless The most heterodimer. GABAB1 of is responsible endogenous ligands binding, while GABA responsible for binding This closure brings the two LB2s in contact, andbitopic, decreases the distance structure a B2 is orthosteric, allosteric, and biased ligand efforts, elucidation of GPCR structures remained challenging 3 GB2 GB1 (inac GB1(active) Schematic structure of class GB2 (inactive) GB2 (active) 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 (nucleus, Golgi) Endosomes Cytoplasm Nucleus Membrane bound receptors chaperon-hichack Cytoplasm/nucleus located receptors The effect of a first messenger depends on: 1. The receptor it binds (location important) Ione ligand can bind Multiple receptors nAch R Na+ channel mAch R GPCR 2. The tranducer and the effector used and intracellular signaling cascades activated (location important) 1. 3. The target proteins present in cells(location important) B1 membrane AC/cAMP/PKA Versus B1 golgi AC/cAMP/ EPAC/PLCe/DAG PKD 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. RGS domain binds Ga Syrovatkina et al. Page 26 Author Manuscript Author Manuscript Author Manuscript Author Manuscript Syrovatkina et al. Figure 3. Figure 1. Phylogenetic relationship of human and mouse G subunits and their expression. Author Manuscript Author Manuscript Author Manuscript Author Manuscript Author Manusc Author Manus 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 Do receptors bind all combinations? Table 1 G subunit interacting proteins Author Manuscript 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 Ga12 G p115RhoGEF, LARG and PDZ-RhoGEF Gap1, rasGap, Btk tyrosine kinase, Radixin, Hax-1, Cadherins, -SNAP, p120caterin, Integrin lllb 3 olf 12, G 13 (+) indicates stimulation. (-) represents inhibition. Syrovatkina et al. Page 33 Author Manuscript 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). Gas and Gas bind at distinct sites on Adenylate cyclase Table 1. Regulatory properties of transmembrane adenylyl cyclase (AC) isoforms a AC isoform G protein regulators stimulatory inhibitory Group I AC1 AC8 AC3 Gs! Gs! Gs! G! i, z, o, G"# G"# G"# Group II AC2 AC4 AC7 Gs!, G"# Gs!, G"# Gs!, G"# Group III AC5 AC6 Gs!, G"# Gs!, G"# Group IV AC9 Gs! Protein kinases 10 stimulatory 9 12 12 12 inhibitory PKC! (weak) PKC! (weak) CaMK II RGS2 Other C1 3 4 N d CaM d CaM d CaM* PAM PP2A Gi! f 1b CC1b PKC! PKC (!, $) C1 Calcium 6 5 7 1 1111 2 CaMK IV PKC! G! i, z G! i, z 8 b PKC! PAM C2 PKA PKA, PKC (%, &) f free Ca2+ f free Ca2+ f f PKC f via calcineurin C2 PAM, Ric8a PAM, Snapin Gs! Making cAMP Binding of C1 to C2 forms nearly identical tertiary structures, as predicted from Classification of Isoforms catalytic site. These have basal if we stop the interactin, we come back to the basic activity, their sequence similarities, despite the fact that these activity structures were solved with a C1 domain from type 5 AC Membrane-bound ACs are often classified into four Fig. 1. Structure of adenylyl cyclase. a Crystal structure of cyto- tor (dark blue). Membrane spans are modeled from the 12-memand a C2 domain from type 2 AC. The pseudosymmetry different categories based on regulatory properties. Gas binds C2 increasing its affinity plasmic domains of AC in complex with GTP"S-Gs!2+ , forskolin brane spanning transporters. b Alternate view from cytocreates two related sites along the domain interface, a inhibitor, Group I-dideoxy-3 consists of Ca. Shown -stimulated 1, 3 and 8; group side, showing forskolin and catalytic site more clearly. (FSK) and P-site 2 ! 5 ! ! ATP are plasmic AC for C1 10x substrate-binding site and a related forskolin C1 site. Both II consists of G " # -stimulated AC 2, 4 and 7; group III isis indicated by an arrow. (yellow), C2 (rust), Gs! (green), FSK (cyan), and P-site inhibi- Interaction site of Gi! with C1 domain pockets are well affinity defined and Gai binds C1 and decrease forare C2structurally related. comprised of Gi!/Ca2+-inhibited AC5 and 6, while There are notable differences between the C1a and C2 group IV contains forskolin-insensitive AC9 (table 1). structures, particularly comparing the regions that play Note that although significant sequence homology eximportant affinity role in the binding of Gs! (C2 domain) or ists within members of groups II and III, members of Gbg bind to C2 an decrease in the formation of a P-loop structure that binds pyrogroup I are more distantlyThe related. This is Topology reflected protein for detailed biochemical characterization. Adenylyl Cyclases: and Structure and to C1/C2 tophosphate increase affinity in the active site (C1 domain). It is ofexpression note that overall regulatory patterns for the various groups of thein twothe catalytic domains of AC in Esche- Forskolin also increases C1/C2 interaction 10x Gas and forskolin together -100x Gi binding inhibits despite forskolin activation Activation of Gene Transcription by GPCR Signaling GPCRs regulate gene transcription by cAMP and PKA signaling. cAMP-released PKA catalytic domains enter the nucleus and phosphorylate the CREB (CREbinding) protein, which binds to CRE (cAMP-response element) sequences upstream of cAMPregulated genes. p-CREB interacts with CBP/p300 to help assemble the RNA Pol II transcription machinery at these promoters. CREB, CREM, ATF1 And splice variants bind different promoters Phosphodiesterase Breaks down cAMP IP3/DAG Signaling Elevates Cytosolic Ca2+ The steps downstream of PLC that make up the IP3/DAG signaling pathway. IP3 diffuses from the cytoplasmic membrane to the smooth ER where it binds to and triggers the opening of IP3-gated Ca2+ channels. Another kinase, protein kinase C (PKC) binds to DAG and calcium in the cytoplasmic membrane and is activated. phosphatidic acid IP2 IP4 FIGURE 2. The cell-surface receptors responsible for InsP3 formation belong to two main classes, the G protein-coupled receptors (GPCRs) and the protein tyrosine kinase-linked receptors (PTKRs) that are coupled to different phospholipase C (PLC) isoforms. The the GPCRs use the PLC! isoforms, whereas the receptor tyrosine kinases (RTKs) are coupled to the PLC-" isoforms. During the transduction process, the precursor lipid PtdIns4,5P2 is hydrolyzed by PLC to produce both InsP3 and diacylglycerol (DAG). The InsP3 released from the membrane diffuses into the cytosol where it engages the InsP3 receptors (InsP3Rs) to release Ca2! from the endoplasmic reticulum. The Ca2!-mobilizing function of InsP3 is terminated through its metabolism by either InsP3 3-kinase or InsP3 5-phosphatase. The resulting InsP2 and InsP4 enter an inositol phosphate metabolic pathway and are recycled back to free inositol. The DAG is recycled back to the precursor CDP-DAG, which then combines with inositol to reform the phosphatidylinositol (PtdIns) that is returned to the plasma membrane to be phosphorylated to the PtdIns4,5P2 precursor to maintain the InsP3 signaling pathway. A key component of this metabolic pathway is the inositol monophosphatase (IMPase) that hydrolyzes InsP1 to free inositol. This IMPase is inhibited by lithium (Li!), which thus acts to reduce the supply of inositol resulting in a decline in the activity of the InsP3/Ca2! signaling pathway. The action of InsP3 is terminated by either by conversion to InsP3 3-kinase or InsP3 5phosphatase. The resulting InsP2 and InsP4 are recycled back to free inositol. The DAG is recycled back to the precursor CDP-DAG, which then combines with inositol to reform the phosphatidylinositol (PtdIns) that is returned to the plasma membrane to be phosphorylated to the PtdIns 4,5P2 precursor to maintain the InsP3 signaling pathway. Some Galpha subunits activate Rho GEFS which activate RhoA RhoA is a member of the Rho GTPase family and serves as an intracellular molecular switch, cycling between a GTP-bound active form and a GDP-bound inactive form. G12/13 Gq p115 RhoGEF LARG RhoGEF PDZ RhoGEF p63 RhoGEF RhoA RhoA A number of events contribute to the termination of signaling by a GPCR. Example: Galpha s signaling 1. Decrease in cAMP levels a. Decrease AC activity b. Hydrolysis of cAMP via cAMP phosphodiesterase c. Reassembly of PKA holoenzyme- R2C2 d. Hydrolysis of GTP by Gas enhanced by GAPS/GDIs 2. Decrease transcription a. Dephosphorylation of CREB by protein phosphatase 1 b. Dissociation of CBP from CREB and decreased transcription 3. Receptor desensitization of receptor and receptor down regulation a. Phosphorylation of receptor by kinases such as PKA and G-proteincoupled receptor kinase GRKs and binding of b-arrestin and GPCRs can be `removed from the membrane by endocytosis Following receptor activation/G-proteins and dissociate/-receptor is phosphorylated and arrestin binds. This prevents G-proteins from rebinding. Receptor is “desensitized” Receptor internalized into endosome vesicle and either degraded (Down regulated) or recycled to surface G-protein-coupled receptor kinase Recycling (class A) Or degradation (class B) Endosome GPCR kinases β-adrenergic receptor kinases (GRKs 2 and 3); ubiquitously expressed. Located in the cytoplasm and have a C- terminal pleckstrin homology domain that binds Gβγ subunits. Translocate to membrane when receptor is activated and Gβγ released. Kinase activity “turned on “ by binding activated receptor Author Manuscript GRK phosphorylation and b-arrestin binding sites C-terminal domain ICL3 Class B- Tight binder Author Manuscript at least 2 full complete phosphorylation codes at C-terminal end Binds barrestin 1/2 Class A- lose binder A Within C-terminal none, a partial or at most 1 complete phosphorylation codes Partial sites in ICL3 Binds preferential barrestin2 This form blocks G protein binding regarding !arr fun important to dete GPCR!!arr confo barcode or other tant to obtain stru microscopy, of GP ciate how specific macologic perspe nists, both as tool c and as potential novel insights into the cell. FIGURE 4. Structural mechanisms for !arr activation and signaling. A, !arr activation occurs through disruption of the polar core (“phosphate sensor”) by the phosphorylated C terminus of the receptor, thereby allowing specific motifs in electron !arr (“activation sensor,” including the finger and lariat loops) to Single particle microscopy Identified Core conformation with bind to ligand-activated structure, Protein Data Bank Acknowledgments— identified tailthe conformation with receptor (inactive Interactions between the receptor (PDB) 1G4M; active alternative models for the finger interactions between the structure, C-terminalPDB tail of4JQI). B, transmembrane CRobert Lefkowitz fo loop interaction from the rhodopsin!finger loop peptide domains structureand (yellow, the receptor with barr (phosphate sensor terminal(cyan, end with (activation PDB 4PXF) and the rhodopsin!arrestin-1 structure PDBbarrs 4ZWJ) with the only) sensor and phosphate sensor). disactive receptor (green). C, single particle electron microscopy identifies tinct conformations of !2AR!!arr, with a tail conformation with interactions References between the C-terminal tail of the receptor with !arr (phosphate sensor only) Phosphate sensor =polar core of + charges 1. Lohse, M. J., Ben and a core conformation with interactions between the transmembrane (1990) !-Arresti domains and !arrs (activation sensor and phosphate sensor). EM images Cell signaling (A) Arres n response (D) Dimeriza!on ac!va!on Cell signaling (E) Transac!va!on XXXXXXXXXXX Clathrin la ce A pERK PM Cell signaling Figure 2. Schematic Co Current View of the (F) Biphasic ac!va!on Impact of Localized Sign Nuclear duced by b-Arrestins a targets pa thw ay Sig na lin g Downstream effector pERK Cell signaling ay hw pat ing nal Sig protein-coupled recept mediated activation of b-a src Time P P plasma membrane (PM) is t duce a rapid ERK respo access nuclear targets. endosomes produces dela vation and sequesters ER Cytoplasmic Endosome plasm, thereby preventin targets G-protein β-arres!n nuclear targets. (B) GPCRvation of Gs in the PM pro Time A and transient response cAMP-dependent targets cytoplasm. Activation in en (B) G protein response duces Time a delayed response tially accesses nuclear targ PM of the cAMP-responsive g Cytoplasmic G shown as an example). Fo targets Figure 1. Classical and Novel Modes of G-Protein-Coupled Receptor (GPCR) Activation. (A) Classical mode, (B) biased activation, (C) intracellular activation, are capable of efficient recy endosome-initiated activat (D) dimerization activation, (E) transactivation, and (F) biphasic activation. Time occur repeatedly in the p sence of agonist through m 368 Trends in Pharmacological Sciences, April 2018, Vol. 39, No. 4 of endocytosis and recyclin curved arrows). Dotted line Endosome Nuclear the b-arrestin function at th targets not require the continued e.g., PCK1 GPCR. cAMP Cell signaling cAMP Barrestin mediated internalization Is required for delayed signaling in endosomes G Time CRE How is GPCR localization to sites in PM or Subcellular sites? Receptor compartmentalization occurs via Scaffold proteins containing PDZ domain and non-PDZ scaffolds 1. Localized to specific site in the plasma membrane 2. Localized to specific Intracellular site Signaling molecules localized by non-PDZ scaffolds (b-arrestin and AKAPs) hydrophopic hydrophilic are antigonist so they block the receptor were fixed with 4% formaldehyde and imaged on a LEICA DMi8 microscope in confocal mode with a 20 x air lens. NE β1AR Oct3 Plasma membrane cAMP PDE3 Oct3 Golgi Gs cAMP mAKAP ! PI4P IP2+DAG PLCε EPAC β1AR Golgi PKD Cytosol Nucleus Figure 9. Signal transduction by cell surface and Golgi b1ARs. b1ARs are located on both the plasma membrane and the Golgi apparatus in cardiac myocytes. Stimulation of cell surface b1ARs leads to production of cytosolic cAMP but this cAMP cannot access the Epac/PLCe/mAKAPb due to PDE3 dependent hydrolysis of cAMP. To PKD=protein kinase D Activate transcription factors b-arrestins have multiple functions in addition to Desensitization and Internalization MINIREVIEW: The !-Arrestins mologous desensitiz the cell (heterologou zation is often medi Gprotein signaling phos desensitization, idues is predominate GRK isoforms: GRK tem, GRK2, GRK3 expressed, and GRK4 tract (30). Importan be absolutely requ intracellular phosph deficient receptor m negative GRK, abo and internalization ( !arr binding requi change in the GPCR GRK-mediated phos FIGURE 1. The spectrum of !arr-mediated signaling. !arrs regulate a wide limiting aspect of rec array of pathways downstream of GPCRs (see text). 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Òš¾ší$æÒ ìçŒ$‡æç ‡åæ$ô‡æ$Tí TŸ æ«ç aö¶ Ì$í‡Òç Ò${í‡66$í{ 7Sp)* –[8JS7p )bh )b n–[*Sb7 hS*fih rb DqT a!EwTFx:*2M gfi––h mfi fi h*[ 5fi3 b5fioligonucleotide Gi Gs heterologous second messenger analy G proteins by protein Gα Gα (endogenous or kinase A )‡æ«Õ‡S $í æ«ç Ò‡ìç Õ‡S ‡Ò Í$tåTš)6çŒ Ñçåç)æTÑÒ Xfirb fi [hh[nS7p rb Shb fi7[–S7fiwS73:8fi3 yTDq )bh )b n–[*Sb7M k[–:fih and h)bm7 CTX 1 , L M Luttrell, R6Twx[ heterologous) Gq/11 Gi/o, G12/13 heterologous messenger analy J Y Daaka J Lefkowitz ‹ç)çíŒ$í{ Tí åç66 æS)çs ‡í $íåÑç‡Òç $í $íæчåç66š6‡Ñ åöa¶ ì‡S fi fihfi7* xfi[7h # h fi br )bh )b n–[*fi3 ]] [73 [ fi fiV fihhfi3 second [h rb–3 treatment, antagonistic ß]s÷− ÷ßs÷÷ constructs Gi/o Gs, sGq co-precipitation, sec Œ$ŸŸçÑçíæ$‡æ$Tí TÑ {ÑTÕæ«endogenous/heterologous ÑçÒš6æ $í åç66š6‡Ñ )ÑT6$Ÿçчæ$Tí S78 fi[hfi b5fi :7h*Sx:–[*fi3 8fi––hM +)fi fiV fihhSb7 br fi8fi *b m[h [h rb––bmh4 Affiliations Gq/11 endogenous/heterologous second messenger analy analysis $í«$¾$æ$Tí TŸ /‡Ÿß ‡íæ‡{Tí$ÆçÒ aö¶ ‡ÑÑçÒæ÷ù åöa¶tŒç)çíŒçíæG13 v l99R messenger fi73bpfi7b:h !EwTFv l9 rxb– xp ( b*fiS7i b5fi fiV fihhfi3 !EwTF b !EwTFx:*GTPγS Gq/11 Gi , Go, G13 endogenous/heterologous labeling, Gα anti PMID: 9363896 or antisense Ì$í‡Òç ‡åæ$ô‡æ$Tí $í m¾ÑT¾6‡ÒæÒ÷Xs÷Y ‡æ ‡ Òæç) $í æ«ç Ò${í‡66$í{ å‡Ò凌ç ]99 rxb– xp ( b*fiS7M k[–:fih rb *)fi Shb fi7[–S7fiwS73:8fi3 S78 fi[hfi br S7* capture [8fi––:w oligonucleotide, second Gq/11 Gi/o a ‡ŸæçÑ /‡Ò ‡åæ$ô‡æ$Tí (í åTíæчÒæs ‡í $íåÑç‡Òç $í $íæчåç66š6‡Ñ åöa¶ $íheterologous –[ 8TyD mfi fi [h rb––bmh4 fi73bpfi7b:h !EwTFv a(A H # 9 L2wrb–3i b5fi fiV messenger fihhfi3 second messenger analysis, PTX Gs Gi, Gq/11 heterologous co-precipitation, sec Gs heterologous GTPγS labeling, co-prec éTÒt² åç66Ò ¾S ‡ ÑTšæç íTæ G13 $íôT6ô$í{ ‡ Ñçåç)æTÑs $íå6šŒ$í{ çþ)ÑçÒÒ$Tí !EwTFv aEY A # ( E2wrb–3i b5fi fiV fihhfi3 !EwTFx:*v aEC L # ( l2wrb–3 fi–[*S5fi *b :7h*Sw transcriptional activity o messenger analysis, TŸ ‡ åTíÒæ$æšæ$ôç6S ‡åæ$ôç ìšæ‡íæ ÍÒ s TÑ çþ)TÒšÑç æT \t¾ÑTìTt x:–[*fi3 8fi––hM p115RhoGEF-RGS Gs Gq/11 endogenous second messenger am \s÷~ Gs Gi/o heterologous GTPγS labeling, second åöa¶ TÑ ŸTÑÒÌT6$ís ÑçÒš6æÒ $í aö¶ Ì$í‡Òç ‡åæ$ô‡æ$Tí (í f%+÷]ù analysis, PTX Gα siRNA Gs Gi,TÑGq/11, GTPγS labeling, second m åç66Òs æÑç‡æìçíæ Õ$æ« ŸTÑÒÌT6$í $ÒT)Ñçí‡6$íç $íåÑç‡ÒçÒ åöa¶heterologous ¾S Gi Gq, G13 heterologous GTPγStranscriptional labeling analysis, XY # ÷ ÷ ‡íŒ ß² # ß Y G12 æ$ìçÒs ÑçÒ)çåæ$ôç6S žTÑÒÌT6$í æÑç‡æìçíæ SRE and CREB Gi Gi, Gs, Gq heterologous GTPγS labeling, second m $팚åçÒ ‡í Î\ ÷ # − ~JtŸT6Œ $íåÑç‡Òç $í aö¶ Ì$í‡Òç )«TÒ)«TÑS6‡t endogenous/heterologousanalysis, GTPγSPTX or 32P-GTP la Gq/11 G13, Gi2 æ$Tís Òš{{çÒæ$í{ 櫇æ åöa¶ ì‡S ìçŒ$‡æç aö¶ Ì$í‡Òç ‡åæ$ô‡æ$Tí heterologous ¾S ‡ Gi Gq/11 co-precipitation, GTP-AA second messenger a Gi Gs heterologous second messenger analy Ñçåç)æTÑt$íŒç)çíŒçíæ )‡æ«Õ‡S fTÕçôçÑs ŸTÑÒÌT6$ítÒæ$ìš6‡æçŒ Gs Gi/o, Gq/11, endogenous/heterologousco-precipitation GTP-AA labeling, PTX sec and aö¶ Ì$í‡Òç )«TÒ)«TÑS6‡æ$Tí $í f%+÷]ù åç66Ò $Ò $íÒçíÒ$æ$ôçheterologous æT Gs Gi PTX treatment, second m G12, G13 messenger analysis, analysis ¾Tæ« ¶IÚ ‡íŒ !tö/+åæ çþ)ÑçÒÒ$Tís $íŒ$å‡æ$í{ æ«‡æ ŸTÑÒÌT6$í Gs/olf Gi/o, Gq, G15 endogenous/heterologous GTPγS or GTP-AA labelin ÕTÑÌÒ ¾S ‡ Œ$ŸŸçÑçíæ ìç嫇í$Òì messenger Gq/11 Gi3ŸÑTì æ«‡æ šÒçŒ ¾S æ«ç !tö/ heterologous GTP-AA analysis, labeling, rece sec fusion, NanoBRET ÉS åTš)6$í{ æT Œ$ŸŸçÑçíæ å6‡ÒÒçÒ TŸ Í )ÑTæç$ís Ñçåç)æTÑÒ heterologous å‡í messenger analysis, Gq Gi second messenger analy Table 1 Non-exhaustive list of GPCRs coupling to non-cognate G proteins. Receptor Class A Table 1 (continued) 5-HT1A Receptor 5-HT1E 5-HT2A Opioid μ 5-HT2B 5-HT2C 3 Oxytocin OT Prostanoid IP 5-HT4A Prostanoid EP2 5-HT4B 5-HT7 Lysophospholipid S1P3 and S1P5 Adenosine A1 Proteinase-activated PAR Adenosine A3 Adrenergic α2 Thyrotropin Adrenergic β1 Adrenergic β2 Vasopressin V1a Angiotensin AT1A Bradykinin B2 Class B Calcitonin Cannabinoid CB1 3 Gq/11, Gi/o, G14 Gs Gi/o Gs Gq/11, Gs, Gq/11, G16 G14/15/16 heterologous endogenous/heterologous Corticotrophin-releasing Cholecystokinin CCK1 hormone Dopamine D1A Glucagon Gs Gq/11 Gs Gs and Gz Gs, Gi, Gq heterologous endogenous Dopamine D3 Glucagon like peptide Galanin GAL2 GLP-1 Gonadotropin releasing Parathyroid hormone Gi Gs Gs, Gz Gi/o, Gq/11 Gi/o, G12 heterologous heterologous endogenous/heterologous Gq/11 Gs Gi, Gs, Gq/11, G14/15 G14/15/16 Gi2 endogenous/heterologous hormone Luteinizing hormone Histamine H2 Vasoactive intestinal Melanin peptide concentrating hormone MCH1 Melanocortin MC4 Class C Calcium sensing Melatonin MT1 CaSR Muscarinic M1 Metabotropic glutamate Muscarinic M2 1a Muscarinic M Metabotropic glutamate 3 5 Muscarinic M4 Gs Gq/11 Gs Gs Gs Gi/o Gq/11, Gi, Go endogenous/heterologous Gi Gi1 Gi, Gq/11, Gi G14/15 Gq, Gs endogenous endogenous heterologous endogenous/heterologous endogenous/heterologous endogenous heterologous s3Cjffi d frrfi8* br *)fi 8TyDw3fi fi73fi7* b*fiS7 JS7[hfi T S7)SXS*b 6wYC b7 yTD GTPase activity, second messenger analysis, pro capture, FRET, NanoBRE second messenger a second messenger analy CRE-luciferase reporter dominant negative Gq/1 GTP-AA labeling co-precipitation second messenger analy GTPγS or α-32P-GTP lab GTP-AA labeling, sec co-precipitation, second messenger analysis messenger analysis second messenger analy GTPγS or GTP-AA lab GTP-AA labeling, second messenger analysis, messenger analysis second messenger analy GTP-AA labeling, sec and CTX messenger analysis, GTP-AA labeling, second messenger analysis GTP-AA labeling, second second messenger a messenger analysis GTPγS labeling, second m antibody capture, βA analysis, PTX JS7[hfi [8*S5[*Sb7M 6fqECl 8fi––h mfi fi S78:X[*fi3 b5fi 7Sp)* S7 hfi :xwr fifi xfi3S[ s3Cjffi A yb3fi– rb !EwTFwxfi3S[*fi3 ;w b*fiS7 hmS*8)S7p *b [8*S5[*fi yTD JS7[hfiM G15 knockout mice, Xfirb fi l9 xS7 fi* fi[*xfi7* mS*) (9 $y 6wYCM uhb fi7[–S7fi a( $y2 b -DT a(9 $y2 D)bh )b n–[*Sb7 br yTD JS7[hfi Xn *)fi !wTF b8fifi3h *) b:p) fi8fi *b 8b: –S7p analysis, Gs GTPγS labeling, second m PTX, CRE repor Gα knockout mice mfi fi [33fi3 *b h[x –fih rb A xS7 S7 *)fi fihfi78fi br 6wYC [73 [8*S5fi yTDq –fi5fi–h *b ;h [73 [8*S5[*Sb7 br DqTM T8*S5[*fi3 DqT )bh )b n–[*fih *)fi !wTF –fi[3S7p *b GTPγS Gi/o, G12/13, Gs endogenous/heterologous or GTP-AA lab Gi2, Gi3 Gq/11 endogenous/heterologous co-precipitation, second 3fi*fi xS7fi3M k[–:fih h)bm7 fi fihfi7* xfi[7h # h fi r bx l fiV fi Sxfi7*h [73 [ fi !wTF messenger fi8fi *b 8b: –S7p *bv [73 [8*S5[*Sb7 brv ;SM ;!" fi–fi[hfi3 r bx *)fi ;Sw8b: –fi3 messenger analysis, PTX Gq/11 analysis, Gq/11 Gi, Gs, endogenous/heterologous GTPγS or GTP-AA labelin fiV fihhfi3 [h rb–3 S78 fi[hfi b5fi :7h*Sx:–[*fi3 8fi––hM [8*S5[*fih yTD JS7[hfi S7 [ t 8w [73 F[hw3fi fi73fi7* [*)m[nM p115RhoGEF-RGS messenger analysis Gq/11 Gi/o g9 Gq/11 Gq/11 Gi/o Gq/11, Gi/o Gi/o, Gs endogenous/heterologous heterologous Gi, Gs, Gq/11 heterologous Gi, Gs G12, Gs heterologous heterologous Gi, Gs heterologous GTPγS labeling, seco Second messenger analy PTX ñöI1/% ® 2dï ù]− ® ~ ñd2%aÉ%/ ß]]² analysis, siRNA, PTX GTPγS or GTP-AA labelin second messenger a messenger analysis, PTX NanoBRET second messenger analy RTK Activation of Ras The mechanism by which EGF activates Ras. Step 1, EGF binding causes receptor dimerization and autophosphorylation on cytosolic tyrosines. Step 2, the adaptor protein GRB2 binds receptor phosphotyrosine residues via its SH2 domain. GRB2 contains SH3 domains allow binding to GEF protein known as Sos Sos then recruits Ras to the complex. RTK Activation of Ras Step 3, In the last step of Ras activation , Sos promotes GTP exchange for GDP on Ras. The activated Ras-GTP complex then dissociates from Sos but remains tethered to the inner leaflet of the cytoplasmic membrane via a lipid anchor sequence. The active form of Ras then activates the MAP kinase portion of the signaling pathway Ras Activation of MAP Kinase Ras activates MAP kinase via a phosphorylation cascade that proceeds from Ras to Raf kinase, to MEK kinase, and finally to MAP kinase. MAP kinase then dimerizes and enters the nucleus MAP kinase=ERK MAP Kinase Activation of Transcription In the final steps of RTK-Ras/MAP kinase signaling, MAP kinase phosphorylates and activates the p90RSK kinase in the cytoplasm. Both kinases enter the nucleus where they phosphorylate ternary complex factor (TCF) and serum response factor (SRF), respectively. The phosphorylated forms of these TFs bind to serum response element (SRE) enhancer sequences that control genes such as c-fos. c-fos activates the expression of genes that propel cells through the cell cycle. SREs occur in a number of genes that are regulated by growth factors present in serum. Signaling via Phosphatidylinositol 3-phosphates In addition, RTKs can signal via PI 3,4-bisphosphate and PI 3,4,5trisphosphate formed by the enzyme PI-3 kinase. The SH-2 domain of PI-3 kinase binds to phosphorylated IRS-1 bound to the RTK The PI 3-phosphate compounds synthesized by PI-3 kinase activate protein kinase B (PKB). Activation of Protein Kinase B Signaling downstream of PI 3-phosphates is conducted by PKB. PKB is recruited to the membrane via binding to PI 3-phosphates via its PH domain (pleckstrin-homology). There it is phosphorylated and activated by the PDK1 & mTorc2 kinases. PDK1 also is recruited to the membrane via binding to PI 3phosphates. Activated PKB then enters the cytosol, where it phosphorylates target proteins PKB is also known as AKT mTORC2 ligands. The ErbB4 ligands BTC, NRG1β, NRG2β, and NRG3 differentially stimulate ErbB4 coupling to survival and proliferation in CEM/ formation that exposes a dimerization arm in subdomain II, thereby facilitating dimerization of the extracellular region (Burgess et al., Regulation of the signaling outcomes is due to stability of active receptor complex formation. Called kinetic proof reading. This model assumes a time lag between initial receptor ligand binding and phosphorylation of tyrosines and: Recruitment of downstream signal transducers and full receptor activation. Stable complexes- (High affinitybinding EGF) Transducers the receptor becomes fully activated and activate a negative feedback event. One possibility is recruitment of phosphatase which dephosphorylate and inactivate the receptor. Transient signaling Non-stable complexes (Low affinity Binding EREG) fall apart prior to activation of negative feedback. The complexes reform keeping the initial signaling event going. Sustained signaling Fig. 3. Ligand stimulation of ErbB receptor tyrosine phosphorylation creates docking sites for numerous signaling effectors. Putative sites of EGFR, ErbB2, ErbB3, and ErbB4 tyrosine phosphorylation are denoted, as well as signaling effectors predicted or shown to bind to these sites of phosphorylation (Rotin et al., 1992; Cohen et al., 1996; Zrihan-Licht et al., 1998; Zrihan-Licht et al., 1998; Keilhack et al., 1998; Hellyer et al., 2001; Schulze et al., 2005; Kaushansky et al., 2008). The ErbB receptors are not drawn to scale. Fig. 3. Ligand stimulation of ErbB receptor tyrosine phosphorylation creates docking sites for numerous signaling effectors. Putative sites of EGFR, ErbB2, ErbB3, and ErbB4 tyrosine Conformation model for biased signaling by EGFR Ligands Binding of different ligand results in changes of JM coil-coil dimer Sinclair et al. Sinclair et al. Page 32 The ligands (AR, ER,TGFa and EPI) that induce the TGFa type coil-coil dimer are more likely to induce differentiation Page 32 Author Manuscript than ligands (EGF, HBtor Tyrosine Kinase (RTK); Epidermal Growth Factor Receptor (EGFR); Cancer; EGF) that induce EGFmembrane Domain; Juxtamembrane Domain; Ligand-Induced Dimerization; Signal duction; Allostery type coil-coil dimer s Author M DUCTION Figure 1. Monitoring growth factor-dependent assembly at the TM-JM junction using bipartite tetracysteine display Modular Structure of Nuclear Receptors The N-terminal region serves as an activation domain in some receptors. The ligand-binding domain is located in the C-terminal sequence region. This region serves as a hormonedependent activation domain in some receptors, and in other receptors, as a repression domain in the absence of ligand The second subdomain helix makes non-specific contacts with the DNA backbone. The peptide loop in DNA binding Dimer formation region has the least sequence and size cons between nuclear receptors. Like the NTD, thi Contributes to DNA binding specificity Cysteines Bind to zinc NR DNA binding domains. (A) Cartoon representation of NR DBDs indicating important motifs. This domain contains subdomains, each containing one zinc finger. The firstDBDs subdomain residues Figure 2. NRtwo DNA binding domains. (A) Cartoon representation of NR indicating important motifs. This domain con interact with the DNA major groove to make base- specific interactions on genomic response subdomains, each containing one zinc finger. The first subdomain residues interact with the DNA major groove to make b elements. The second subdomain participates in DBD dimerization and makes non- specific contacts specific interactions on genomic response elements. The second subdomain participates in DBD dimerization and makes with the DNA backbone. specific contacts with the DNA backbone. Some NRs, like LRH-1 and GCNF, also contain C-terminal extensions (CTEs) t base-specific contacts with the minor groove. Cartoon representation ofthe folded GRgroove. DBD highlighting the important reg NR contain C-terminal extensions (CTEs) that make(B) base-specific contacts with minor (PDB: 3FYL). Zinc atoms are represented as spheres.. | These models suggested that LBDs have several conformations: Not bound to Ligand, bound to coactivator and bound to corepressor Multiple steps for activation Nuclear receptor The size of the ligand binding pocket varies among the different receptors, being for instance very large in PPARg, which allows binding of very differently sized ligands (273). Several differences are evident when comparing unliganded and ligand-bound receptors. The liganded structures are more compact than the unliganded noted that these motifs represent consensus idealized sequences and that naturally occurring HREs can show significant variation from the consensus. Although some monomeric receptors can bind to a single hexameric motif, most receptors bind as homo- or heterodimers to HREs composed typically of two core hexameric motifs. Hormone binding domain contains 12 alpha helices No ligand Plus ligand Dynamic stabilization model H12 is not in one fixed position, but rather is dynamic and dependent on ligand. FIG. 4. Schematic drawing of the nuclear receptor ligand-binding domain (LBD). On the left, the LBD from the crystal structure of the unliganded RXRa is shown. On the right, the ligand-bound LBD of the RARg is shown. Cylinders represent a-helices that are numbered from 1 to 12. Note the different position of the COOH-terminal helix 12 that contains the core AF-2 domain in both situations. [From Wurtz et al. (294), reprinted by permission from Nature, Macmillan Magazines Ltd.] Can bind Coactivators or corepressors Ligand-activation - a switch from an active repressor to a full activator Coactivator-link: LxxLL a short helix (green) on SRC Corepressor-link: longer helix (red) on NcoR Multiple modes of transcription activation of estrogen receptors Non-Genomic Genomic Signaling Estrogen/er Non-Genomic signaling via GPR30 Estrogen independent signaling EGF activation of ERFR results in activation Map kinase pathway that activates ERK. ERK phosphorylates serine 118 in AF1 Domain of ER This results in interaction of AF1 domain with Coactivators which cause Transcription and growth of breast cancer cells