Lesson 3 Cell Signaling & Motility: G Proteins, Signal Transduction: Curations and Articles of reference as supplemental information: #TUBiol3373
Curator: Stephen J. Williams, Ph.D.
Updated 7/15/2019
Lesson 3 Powerpoint (click link below):
cell signaling and motility 3 finalissima sjw
Four papers to choose from for your February 11 group presentation:
Structural studies of G protein Coupled receptor
Shapiro-2009-Annals_of_the_New_York_Academy_of_Sciences
G protein as target in neurodegerative disease
Today’s lesson 3 explains how extracellular signals are transduced (transmitted) into the cell through receptors to produce an agonist-driven event (effect). This lesson focused on signal transduction from agonist through G proteins (GTPases), and eventually to the effectors of the signal transduction process. Agonists such as small molecules like neurotransmitters, hormones, nitric oxide were discussed however later lectures will discuss more in detail the large growth factor signalings which occur through receptor tyrosine kinases and the Ras family of G proteins as well as mechanosignaling through Rho and Rac family of G proteins.
Transducers: The Heterotrimeric G Proteins (GTPases)
An excellent review of heterotrimeric G Proteins found in the brain is given by
Heterotrimeric G Proteins by Eric J Nestler and Ronald S Duman.
from Seven-Transmembrane receptors – Scientific Figure on ResearchGate. Available from: https://www.researchgate.net/figure/Examples-of-heterotrimeric-G-protein-effectors_tbl1_11180073 [accessed 4 Feb, 2019] and see references within
See below for the G Protein Cycle
<a href=”https://www.researchgate.net/figure/32-The-G-protein-cycle-In-the-absence-of-agonist-A-GPCRs-are-mainly-in-the-low_fig2_47933733″><img src=”https://www.researchgate.net/profile/Veli_Pekka_Jaakola/publication/47933733/figure/fig2/AS:669499451781133@1536632516635/32-The-G-protein-cycle-In-the-absence-of-agonist-A-GPCRs-are-mainly-in-the-low.ppm” alt=”.3.2: The G protein cycle. In the absence of agonist (A), GPCRs are mainly in the low affinity state (R). After agonist binding, the receptor is activated in the high affinity state (R*), and the agonist-GPCR-G protein complex is formed. GTP replaces GDP in Gα. After that the G protein dissociates into the Gα subunit and the Gβγ heterodimer, which then activate several effector proteins. The built-in GTPase activity of the Gα subunit cleaves the terminal phosphate group of GTP, and the GDP bound Gα subunit reassociates with Gβγ heterodimer. This results in the deactivation of both Gα and Gβγ. The G protein cycle returns to the basal state. RGS, regulator of G protein signalling.”/></a>
From Citation: Review: A. M. Preininger, H. E. Hamm, G protein signaling: Insights from new structures. Sci. STKE2004, re3 (2004)
For a tutorial on G Protein coupled receptors (GPCR) see
https://www.khanacademy.org/test-prep/mcat/organ-systems/biosignaling/v/g-protein-coupled-receptors
cyclic AMP (cAMP) signaling to the effector Protein Kinase A (PKA)
from https://courses.washington.edu/conj/gprotein/cyclicamp.htm
Cyclic AMP is an important second messenger. It forms, as shown, when the membrane enzyme adenylyl cyclase is activated (as indicated, by the alpha subunit of a G protein).
The cyclic AMP then goes on the activate specific proteins. Some ion channels, for example, are gated by cyclic AMP. But an especially important protein activated by cyclic AMP is protein kinase A, which goes on the phosphorylate certain cellular proteins. The scheme below shows how cyclic AMP activates protein kinase A.
Updated 7/15/2019
Additional New Studies on Regulation of the Beta 2 Adrenergic Receptor
We had discussed regulation of the G protein coupled beta 2 adrenergic receptor by the B-AR receptor kinase (BARK)/B arrestin system which uncouples and desensitizes the receptor from its G protein system. In an article by Xiangyu Liu in Science in 2019, the authors describe another type of allosteric modulation (this time a POSITIVE allosteric modulation) in the intracellular loop 2. See below:
Mechanism of β2AR regulation by an intracellular positive allosteric modulator
Xiangyu Liu1,*, Ali Masoudi2,*, Alem W. Kahsai2,*, Li-Yin Huang2, Biswaranjan Pani2, Dean P. Staus2, Paul J. Shim2, Kunio Hirata3,4, Rishabh K. Simhal2, Allison M. Schwalb2, Paula K. Rambarat2, Seungkirl Ahn2, Robert J. Lefkowitz2,5,6,†, Brian Kobilka1
Positive reinforcement in a GPCR
Many drug discovery efforts focus on G protein–coupled receptors (GPCRs), a class of receptors that regulate many physiological processes. An exemplar is the β2-adrenergic receptor (β2AR), which is targeted by both blockers and agonists to treat cardiovascular and respiratory diseases. Most GPCR drugs target the primary (orthosteric) ligand binding site, but binding at allosteric sites can modulate activation. Because such allosteric sites are less conserved, they could possibly be targeted more specifically. Liu et al. report the crystal structure of β2AR bound to both an orthosteric agonist and a positive allosteric modulator that increases receptor activity. The structure suggests why the modulator compound is selective for β2AR over the closely related β1AR. Furthermore, the structure reveals that the modulator acts by enhancing orthosteric agonist binding and stabilizing the active conformation of the receptor.
Abstract
Drugs targeting the orthosteric, primary binding site of G protein–coupled receptors are the most common therapeutics. Allosteric binding sites, elsewhere on the receptors, are less well-defined, and so less exploited clinically. We report the crystal structure of the prototypic β2-adrenergic receptor in complex with an orthosteric agonist and compound-6FA, a positive allosteric modulator of this receptor. It binds on the receptor’s inner surface in a pocket created by intracellular loop 2 and transmembrane segments 3 and 4, stabilizing the loop in an α-helical conformation required to engage the G protein. Structural comparison explains the selectivity of the compound for β2– over the β1-adrenergic receptor. Diversity in location, mechanism, and selectivity of allosteric ligands provides potential to expand the range of receptor drugs.
Recent structures of GPCRs bound to allosteric modulators have revealed that receptor surfaces are decorated with diverse cavities and crevices that may serve as allosteric modulatory sites (1). This substantiates the notion that GPCRs are structurally plastic and can be modulated by a variety of allosteric ligands through distinct mechanisms (2-7). Most of these structures have been solved with negative allosteric modulators (NAMs), which stabilize receptors in their inactive states (1). To date, only a single structure of an active GPCR bound to a small-molecule positive allosteric modulator (PAM) has been reported, namely, the M2 muscarinic acetylcholine receptor with LY2119620 (8). Thus, mechanisms of PAMs and their potential binding sites remain largely unexplored.
Fig 1. Structure of the active state T4L-B2AR in complex with the orthosteric agonist BI-167107, nanobody 689, and compound 6FA. (A) The chemical structure of compound-6FA (Cmpd-6FA). (B) Isoproterenol (ISO) competition binding with 125I-cyanopindolol (CYP) to the β2AR reconstituted in nanodisks in the presence of vehicle (0.32% dimethylsulfoxide; DMSO), Cmpd-6, or Cmpd-6FA at 32 μM. Values were normalized to percentages of the maximal 125I-CYP binding level obtained from a one-site competition binding–log IC50 (median inhibitory concentration) curve fit. Binding curves were generated by GraphPad Prism. Points on curves represent mean ± SEM obtained from five independent experiments performed in duplicate. (C) Analysis of Cmpd-6FA interaction with the BI-167107–bound β2AR by ITC. Representative thermogram (inset) and binding isotherm, of three independent experiments, with the best titration curve fit are shown. Summary of thermodynamic parameters obtained by ITC: binding affinity (KD = 1.2 ± 0.1 μM), stoichiometry (N = 0.9 ± 0.1 sites), enthalpy (ΔH = 5.0 ± 1.2 kcal mol−1), and entropy (ΔS =13 ± 2.0 cal mol−1 deg−1). (D) Side view of T4L-β2AR bound to the orthosteric agonist BI-167107, nanobody 6B9 (Nb6B9), and Cmpd-6FA. The gray box indicates the membrane layer as defined by the OPM database. (E) Close-up view of Cmpd-6FA binding site. Covering Cmpd-6FA is 2Fo– Fc electron density contoured at 1.0 σ (green mesh).From Science 28 Jun 2019:
Vol. 364, Issue 6447, pp. 1283-1287
Fig 3. Fig. 3 Mechanism of allosteric activation of the β2AR by Cmpd-6FA.
(A) Superposition of the inactive β2AR bound to the antagonist carazolol (PDB code: 2RH1) and the active β2AR bound to the agonist BI-167107, Cmpd-6FA, and Nb6B9. Close-up view of the Cmpd-6FA binding site is shown. The residues of the inactive (yellow) and active (blue) β2AR are depicted, and the hydrogen bond formed between Asp1303.49and Tyr141ICL2 in the active state is indicated by a black dashed line. (B) Topography of Cmpd-6FA binding surface on the active β2AR (left, blue) and the corresponding surface of the inactive β2AR (right, yellow) with Cmpd-6FA (orange sticks) docked on top. Molecular surfaces are of only those residues involved in interaction with Cmpd-6FA. Steric clash between Cmpd-6FA and the surface of inactive β2AR is represented by a purple asterisk. (C) Overlay of the β2AR bound to BI-167107, Nb6B9, and Cmpd-6FA with the β2AR–Gscomplex (PDB code: 3SN6). The inset shows the position of Phe139ICL2 relative to the α subunit of Gs. (D) Superposition of the active β2AR bound to the agonist BI-167107, Nb6B9, and Cmpd-6FA (blue) with the inactive β2AR bound to carazolol (yellow) (PDB code: 2RH1) as viewed from the cytoplasm. For clarity, Nb6B9 and the orthosteric ligands are omitted. The arrows indicate shifts in the intracellular ends of the TM helices 3, 5, and 6 upon activation and their relative distances.
Allosteric sites may not face the same evolutionary pressure as do orthosteric sites, and thus are more divergent across subtypes within a receptor family (24–26). Therefore, allosteric sites may provide a greater source of specificity for targeting GPCRs.
- D. M. Thal, A. Glukhova, P. M. Sexton, A. Christopoulos, Structural insights into G-protein-coupled receptor allostery. Nature 559, 45–53 (2018). doi:10.1038/s41586-018-0259-zpmid:29973731CrossRefPubMedGoogle Scholar
- D. Wacker, R. C. Stevens, B. L. Roth, How Ligands Illuminate GPCR Molecular Pharmacology. Cell 170, 414–427 (2017).
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Additional information on Nitric Oxide as a Cellular Signal
Nitric oxide is actually a free radical and can react with other free radicals, resulting in a very short half life (only a few seconds) and so in the body is produced locally to its site of action (i.e. in endothelial cells surrounding the vascular smooth muscle, in nerve cells). In the late 1970s, Dr. Robert Furchgott observed that acetylcholine released a substance that produced vascular relaxation, but only when the endothelium was intact. This observation opened this field of research and eventually led to his receiving a Nobel prize. Initially, Furchgott called this substance endothelium-derived relaxing factor (EDRF), but by the mid-1980s he and others identified this substance as being NO.
Nitric oxide is produced from metabolism of endogenous substances like L-arginine, catalyzed by one of three isoforms of nitric oxide synthase (for link to a good article see here) or release from exogenous compounds like drugs used to treat angina pectoris like amyl nitrate or drugs used for hypertension such as sodium nitroprusside.
The following articles are a great reference to the chemistry, and physiological and pathological Roles of Nitric Oxide:
46. The Molecular Biology of Renal Disorders: Nitric Oxide – Part III
Curator and Author: Larry H Bernstein, MD, FACP
https://pharmaceuticalintelligence.com/2012/11/26/the-molecular-biology-of-renal-disorders/
47. Nitric Oxide Function in Coagulation – Part II
Curator and Author: Larry H. Bernstein, MD, FCAP
https://pharmaceuticalintelligence.com/2012/11/26/nitric-oxide-function-in-coagulation/
48. Nitric Oxide, Platelets, Endothelium and Hemostasis
Curator and Author: Larry H Bernstein, MD, FACP
https://pharmaceuticalintelligence.com/2012/11/08/nitric-oxide-platelets-endothelium-and-hemostasis/
49. Interaction of Nitric Oxide and Prostacyclin in Vascular Endothelium
Curator and Author: Larry H Bernstein, MD, FACP
50. Nitric Oxide and Immune Responses: Part 1
Curator and Author: Aviral Vatsa PhD, MBBS
https://pharmaceuticalintelligence.com/2012/10/18/nitric-oxide-and-immune-responses-part-1/
51. Nitric Oxide and Immune Responses: Part 2
Curator and Author: Aviral Vatsa PhD, MBBS
https://pharmaceuticalintelligence.com/2012/10/28/nitric-oxide-and-immune-responses-part-2/
56. Nitric Oxide and iNOS have Key Roles in Kidney Diseases – Part II
Curator and Author: Larry H Bernstein, MD, FACP
57. New Insights on Nitric Oxide donors – Part IV
Curator and Author: Larry H Bernstein, MD, FACP
https://pharmaceuticalintelligence.com/2012/11/26/new-insights-on-no-donors/
59. Nitric Oxide has a ubiquitous role in the regulation of glycolysis -with a concomitant influence on mitochondrial function
Curator and Author: Larry H Bernstein, MD, FACP
Biochemistry of the Coagulation Cascade and Platelet Aggregation: Nitric Oxide: Platelets, Circulatory Disorders, and Coagulation Effects
Nitric Oxide Function in Coagulation – Part II
Nitric oxide is implicated in many pathologic processes as well. Nitric oxide post translational modifications have been attributed to nitric oxide’s role in pathology however, although the general mechanism by which nitric oxide exerts its physiological effects is by stimulation of soluble guanylate cyclase to produce cGMP, these post translational modifications can act as a cellular signal as well. For more information of NO pathologic effects and how NO induced post translational modifications can act as a cellular signal see the following:
Nitric Oxide Covalent Modifications: A Putative Therapeutic Target?
58. Crucial role of Nitric Oxide in Cancer
Curator and Author: Ritu Saxena, Ph.D.
https://pharmaceuticalintelligence.com/2012/10/16/crucial-role-of-nitric-oxide-in-cancer/
Note: A more comprehensive ebook on Nitric Oxide and Disease Perspectives is found at
Cardiovascular Diseases, Volume One: Perspectives on Nitric Oxide in Disease Mechanisms
available on Kindle Store @ Amazon.com
http://www.amazon.com/dp/B00DINFFYC
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