Lecture 6 Outline | Neurotransmitter Receptors | PDF

2022-10-25 Neurotransmitter Receptors II: Metabotropic Receptors NROC36H3F © Arruda Carvalho UTSC...

2022-10-25 Neurotransmitter Receptors II: Metabotropic Receptors NROC36H3F © Arruda Carvalho UTSC The Life Cycle of Neurotransmission I. Synthesis (1-2) II. Storage (3) Lecture 3 III.Release Lecture 4 From Molecules to Networks, © 2014, 2009, 2004 Elsevier Inc IV. Receptor Binding (4-5) V. Transmitter Inactivation (6-9) Lecture 3 © Arruda Carvalho UTSC 1  2022-10-25 Lecture Outline Overview of Ionotropic vs. Metabotropic Actions GPCR General Structure GPCR Activation and Action Binding to G proteins Effector Mechanisms: Presynaptic Effector Mechanisms: Postsynaptic GPCR Types and Specific Mechanisms mGluRs mAChRs GABABRs Dopamine and Serotonin Receptors GPCR Desensitization Overview © Arruda Carvalho UTSC © 2018 Arruda Carvalho UTSC winter term Lecture Outline Overview of Ionotropic vs. Metabotropic Actions GPCR General Structure GPCR Activation and Action Binding to G proteins Effector Mechanisms: Presynaptic Effector Mechanisms: Postsynaptic GPCR Types and Specific Mechanisms mGluRs mAChRs GABABRs Dopamine and Serotonin Receptors GPCR Desensitization Overview © Arruda Carvalho UTSC © 2018 Arruda Carvalho UTSC winter term 2  2022-10-25 Ionotropic Receptors Mediate Behaviors, Metabotropic Receptors Modulate Behaviors Nicotininc α- and β-adrenergic receptors acetylcholine (ACh) Muscarinic ACh receptors receptors GABAB receptors γ-aminobutyric acid A (GABAA) and Glycine Subset of glutamate and receptors serotonin receptors Dopamine receptors 5-HT3 Receptor Receptors for neuropeptides, P2X Purinergic odorant receptors, rhodopsin Receptors AMPA/Kainate and NMDA receptors Principles of Neural Science ©McGraw Hill © Arruda Carvalho UTSC How does the Activation of these Second Messenger Cascades Impact Synaptic transmission? Ionotropic Metabotropic Principles of Neural Science ©McGraw Hill © Arruda Carvalho UTSC 3  2022-10-25 Metabotropic Receptors Regulate a Variety of Channel Types: Resting channels Voltage-gated channels (action potential, Ca2+ influx for neurotransmitter release) Ligand-gated channels © Arruda Carvalho UTSC Ionotropic Receptors Mediate Behaviors, Metabotropic Receptors Modulate Behaviors ACh, Glutamate, Serotonin, and GABA Bind to and activate both ionotropic and metabotropic receptors. Thus, they each induce both fast responses (milliseconds), and slow-onset and longer-duration responses (from tenths of seconds to hours). Neuropeptides Produce their effects largely by binding only to metabotropic Receptors © Arruda Carvalho UTSC 4  2022-10-25 Metabotropic Receptors Regulate a Variety of Channel Types Principles of Neural Science ©McGraw Hill © Arruda Carvalho UTSC Lecture Outline Overview of Ionotropic vs. Metabotropic Actions GPCR General Structure GPCR Activation and Action Binding to G proteins Effector Mechanisms: Presynaptic Effector Mechanisms: Postsynaptic GPCR Types and Specific Mechanisms mGluRs mAChRs GABABRs Dopamine and Serotonin Receptors GPCR Desensitization Overview © Arruda Carvalho UTSC © 2018 Arruda Carvalho UTSC winter term 5  2022-10-25 GPCR General Structure Rosembaum et al., Nature, 2009 © Arruda Carvalho UTSC GPCR General Structure N- Terminus Single polypeptide: Seven membrane-spanning helical segments wrapped through the membrane Amino acid loops connect the transmembrane domains C- Terminus From Molecules to Networks, © 2014, 2009, 2004 Elsevier Inc © Arruda Carvalho UTSC 6  2022-10-25 Ligand Binding Domain GPCR General Structure b Adrenergic receptor Replacing the Asp in TM3 with a Glu reduces transmitter binding by >100-fold, and with a Ser, reduces binding by >10,000-fold Kobilka, Annu Rev Neurosci 1992 © Arruda Carvalho UTSC GPCR General Structure b Adrenergic receptor mACh receptor Variations in these 5 amino acid positions grant the specificity between binding of different transmitters to individual GPCRs. From Molecules to Networks, © 2014, 2009, 2004 Elsevier Inc © Arruda Carvalho UTSC 7  2022-10-25 GPCR General Structure M3 mACh receptor Conserved amino acids for all members of GPCR family Ligand binding amino acids From Molecules to Networks, © 2014, 2009, 2004 Elsevier Inc © Arruda Carvalho UTSC Lecture Outline Overview of Ionotropic vs. Metabotropic Actions GPCR General Structure GPCR Activation and Action Binding to G proteins Effector Mechanisms: Presynaptic Effector Mechanisms: Postsynaptic GPCR Types and Specific Mechanisms mGluRs mAChRs GABABRs Dopamine and Serotonin Receptors GPCR Desensitization Overview © Arruda Carvalho UTSC © 2018 Arruda Carvalho UTSC winter term 8  2022-10-25 A Shared Logic of GPCR Activation Principles of Neural Science ©McGraw Hill © Arruda Carvalho UTSC G protein identity will determine the effector mechanism of GPCR activation Table 15-3 Molecular Biology of the Cell (© Garland Science 2008) Principles of Neural Science ©McGraw Hill © Arruda Carvalho UTSC 9  2022-10-25 Common G protein Effector Targets Principles of Neural Science ©McGraw Hill © Arruda Carvalho UTSC Lecture Outline Overview of Ionotropic vs. Metabotropic Actions GPCR General Structure GPCR Activation and Action Binding to G proteins Effector Mechanisms: Presynaptic Effector Mechanisms: Postsynaptic GPCR Types and Specific Mechanisms mGluRs mAChRs GABABRs Dopamine and Serotonin Receptors GPCR Desensitization Overview © Arruda Carvalho UTSC © 2018 Arruda Carvalho UTSC winter term 10  2022-10-25 Ligand-induced GPCR Conformational Changes Activate G proteins Inactive State Active State Hanlon and Andrew, J Cell Sci 2015 Agonist binding stabilizes the active conformation and shifts the equilibrium toward the active state Antagonist binding blocks activation through (1) binding to the inactive state, impeding transition to active state (negative antagonism), or (2) binding to both active and inactive conformations, stabilizing the ratio and preventing a further transition into the active state (neutral antagonism) © Arruda Carvalho UTSC Ligand-induced GPCR Conformational Changes Activate G proteins M3 mACh receptor X X Specificity and efficiency of G protein coupling X Transduction of ligand-induced conformational Main point of G protein coupling changes to the area of i3 essential for G-protein coupling and activation From Molecules to Networks, © 2014, 2009, 2004 Elsevier Inc © Arruda Carvalho UTSC 11  2022-10-25 Ligand-induced GPCR Conformational Changes Activate G proteins Illustration of the conserved rearrangement of residue contacts between inactive- and active-state structures in β 2AR. Dotted circles around 6x37 and 7x53 denote the movement of TM6 and TM7 upon activation Venkatakrishnan et al., Nature, 2016 “Despite the diversity in activation pathways between receptors, the pathways converge near the G-protein coupling region. This convergence is mediated by a highly conserved structural rearrangement of residue contacts between transmembrane helices 3, 6 and 7 that releases G-protein-contacting residues” © Arruda Carvalho UTSC Lecture Outline Overview of Ionotropic vs. Metabotropic Actions GPCR General Structure GPCR Activation and Action Binding to G proteins Effector Mechanisms: Presynaptic Effector Mechanisms: Postsynaptic GPCR Types and Specific Mechanisms mGluRs mAChRs GABABRs Dopamine and Serotonin Receptors GPCR Desensitization Overview © Arruda Carvalho UTSC © 2018 Arruda Carvalho UTSC winter term 12  2022-10-25 General Mechanisms of GPCR Modulation of Synaptic Transmission Presynaptic GPCRs: Neurotransmitter Release Ga Phosphorylation of proteins involved in vesicle recruitment, docking, and fusion - can cause either inhibition or facilitation of synaptic transmission Principles of Neural Science ©McGraw Hill © Arruda Carvalho UTSC General Mechanisms of GPCR Modulation of Synaptic Transmission Presynaptic GPCRs: Neurotransmitter Release Ga: Modulation of neurotransmitter release by PKA and PKC targets Brown and Sihra, Handb. Exp. Pharmacol. 2008 © Arruda Carvalho UTSC 13  2022-10-25 Ga: Modulation of neurotransmitter release by PKC targets 1. PKC phosphorylation of SNAP-25 PKC phosphorylation of SNAP-25 (S187) increases the association between SNAP-25 and Syntaxin, increasing SNARE complex formation, and thus facilitating exocytosis From Molecules to Networks, © 2014, 2009, 2004 Elsevier Inc © Arruda Carvalho UTSC General Mechanisms of GPCR Modulation of Synaptic Transmission Presynaptic GPCRs: Neurotransmitter Release Ga: Modulation of neurotransmitter release by PKA and PKC targets Brown and Sihra, Handb. Exp. Pharmacol. 2008 © Arruda Carvalho UTSC 14  2022-10-25 Ga: Modulation of neurotransmitter release by PKA targets 2. PKA phosphorylation of Syntaphilin Syntaphilin competes with Syntaphilin SNAP-25 for Syntaxin binding PKA PKA phosphorylation of P Syntaphilin releases Syntaxin for SNARE assembly From Molecules to Networks, © 2014, 2009, 2004 Elsevier Inc © Arruda Carvalho UTSC General Mechanisms of GPCR Modulation of Synaptic Transmission Presynaptic GPCRs: Neurotransmitter Release Ga: Modulation of neurotransmitter release by PKA and PKC targets Brown and Sihra, Handb. Exp. Pharmacol. 2008 © Arruda Carvalho UTSC 15  2022-10-25 Ga: Modulation of neurotransmitter release by PKA targets 3. PKA phosphorylation of Snapin Synaptotagmin binds to Ca2+ and SNARE complex to promote fusion Snapin Snapin binds to SNAP-25 and enables its binding to Synaptotagmin © Arruda Carvalho UTSC Ga: Modulation of neurotransmitter release by PKA targets 3. PKA phosphorylation of Snapin Synaptotagmin binds to Ca2+ and SNARE complex to promote fusion Snapin PKA phosphorylation of Snapin increases its affinity for SNAP-25, promoting release Snapin binds to SNAP-25 and enables its binding to Synaptotagmin From Molecules to Networks, © 2014, 2009, 2004 Elsevier Inc © Arruda Carvalho UTSC 16  2022-10-25 Summary of Effects of PKA and PKC on Transmitter Release PKA PKC Phosphorylation of synapsin 1, releasing its tether to actin and promoting vesicle Phosphorylation of SNAP-25, availability promoting SNARE formation Phosphorylation of RIM – alteration of Munc 18 – Regulation of fusion pore Rab3 and Munc13 binding to promote accelerating exocytosis release Phosphorylation of SNAP-25, promoting SNARE formation Phosphorylation of syntaphilin (which competes with SNAP-25 for syntaxin-1A binding) releases syntaxin-1A for SNARE assembly Snapin phosphorylation increases SNAP25-synaptotagmin binding promoting release © Arruda Carvalho UTSC GPCR-Mediated Effects are Reversed by Phosphatases Principles of Neural Science ©McGraw Hill © Arruda Carvalho UTSC 17  2022-10-25 General Mechanisms of GPCR Modulation of Synaptic Transmission Presynaptic GPCRs: Neurotransmitter Release Gbg: 1. Inhibition of voltage-dependent calcium channels (presynaptic nerve terminals) 2. Direct interaction with of one or more of the components of the exocytotic machinery 3. Regulation of GIRK channels Betke et al, Prog Neurobio 2012 © Arruda Carvalho UTSC General Mechanisms of GPCR Modulation of Synaptic Transmission Presynaptic GPCRs: Neurotransmitter Release Gbg 1. Inhibition of voltage-dependent calcium channels (VDCCs) As the presynaptic membrane is depolarized, VDCCs become activated resulting in an influx of calcium into the cell which can then bind to Synaptotagmin to trigger exocytotic events Gbg-mediated inhibition of VDCCs will decrease neurotransmitter release Brown and Sihra, Handb. Exp. Pharmacol. 2008 © Arruda Carvalho UTSC 18  2022-10-25 General Mechanisms of GPCR Modulation of Synaptic Transmission Presynaptic GPCRs: Neurotransmitter Release Gbg 2. Inhibition of SNARE complex direct interaction with syntaxin, SNAP-25 and VAMP/ synaptobrevin Betke et al, Prog Neurobio 2012 Prevents the tight zippering of the SNARE complex necessary to drive membrane fusion, and thus can inhibit vesicle exocytosis © Arruda Carvalho UTSC General Mechanisms of GPCR Modulation of Synaptic Transmission Presynaptic GPCRs: Neurotransmitter Release Gbg 3. Regulation of G protein-gated inward-rectifier K+ channel (GIRK) channels M2 Gbg directly interacts with GIRK channels, opening the channel and leading to hyperpolarization GIRK and other inward-rectifier channels pass current more readily in the inward than the outward direction, although in physiological situations K+ current is always outward. As such, GIRK activation leads to K+ efflux which hyperpolarizes the membrane Principles of Neural Science ©McGraw Hill © Arruda Carvalho UTSC 19  2022-10-25 Lecture Outline Overview of Ionotropic vs. Metabotropic Actions GPCR General Structure GPCR Activation and Action Binding to G proteins Effector Mechanisms: Presynaptic Effector Mechanisms: Postsynaptic GPCR Types and Specific Mechanisms mGluRs mAChRs GABABRs Dopamine and Serotonin Receptors GPCR Desensitization Overview © Arruda Carvalho UTSC © 2018 Arruda Carvalho UTSC winter term General Mechanisms of GPCR Modulation of Synaptic Transmission Postsynaptic GPCRs: Excitability 1. PKA-mediated closure of K+ channels PKA phosphorylation leads to closure of the serotonin-sensititive (S)- type K+ channel, decreasing K+ efflux from the cell, thereby leading to depolarization and a decrease in resting membrane conductance. Principles of Neural Science ©McGraw Hill © Arruda Carvalho UTSC 20  2022-10-25 Long-Term Modulation of Synaptic Transmission by GPCRs Postsynaptic GPCRs: Excitability 2. Changes in transcription and chromatin structure, e.g. PKA activation of CREB Principles of Neural Science ©McGraw Hill © Arruda Carvalho UTSC Transcription factors involved in memory formation: CREB Barco et al., Exp Op Therap Targets 2003 © Arruda Carvalho UTSC 21  2022-10-25 Lecture Outline Overview of Ionotropic vs. Metabotropic Actions GPCR General Structure GPCR Activation and Action Binding to G proteins Effector Mechanisms: Presynaptic Effector Mechanisms: Postsynaptic GPCR Types and Specific Mechanisms mGluRs mAChRs GABABRs Dopamine and Serotonin Receptors GPCR Desensitization Overview © Arruda Carvalho UTSC © 2018 Arruda Carvalho UTSC winter term GPCR Family Tree From Molecules to Networks, © 2014, 2009, 2004 Elsevier Inc © Arruda Carvalho UTSC 22  2022-10-25 Lecture Outline Overview of Ionotropic vs. Metabotropic Actions GPCR General Structure GPCR Activation and Action Binding to G proteins Effector Mechanisms: Presynaptic Effector Mechanisms: Postsynaptic GPCR Types and Specific Mechanisms mGluRs mAChRs GABABRs Dopamine and Serotonin Receptors GPCR Desensitization Overview © Arruda Carvalho UTSC © 2018 Arruda Carvalho UTSC winter term Metabotropic Glutamate Receptors (mGluRs) 1. mGluRs exist as functional dimers in the membrane in contrast to the single-subunit forms of most other GPCRs – Maximal activity requires binding of 2 Glu molecules. 2. Binding site for glutamate resides in the large N-terminal extracellular domain (ECD) Rondard and Pin, Cur Opinion Pharm 2015 © Arruda Carvalho UTSC 23  2022-10-25 Metabotropic Glutamate Receptors (mGluRs) Rondard and Pin, Cur Opinion Pharm 2015 The extracellular ligand binding domain (ECD) of class C GPCRs consists of a bilobate Venus flytrap (VFT) domain that contains the binding site interconnected through a cystein-rich domain (CRD) to the 7TM domain © Arruda Carvalho UTSC mGluR Activation VFT CRD Model of mGluR activation. Schematic illustration of the major steps in mGluR activation leading to G protein activation. Glutamate or other agonists binding induce Venus flytrap (VFT) domain closure and their reorientation, leading to the formation of a specific interface between cystein-rich domains (CRDs), and finally to the rearrangement at the 7TM dimer interface that is followed by the activation of only one of the two 7TMs Rondard and Pin, Cur Opinion Pharm 2015 © Arruda Carvalho UTSC 24  2022-10-25 Metabotropic Glutamate Receptors (mGluRs) i2 3. Second intracellular loop (i2), as opposed to i3, along with the C-terminal https://www.hellobio.com/mglu-receptor-review/ domain underlie G-protein coupling © Arruda Carvalho UTSC Metabotropic Glutamate Receptors (mGluRs) Basic Neurochemistry: Molecular, Cellular and Medical Aspects. 6th edition. Siegel GJ, Agranoff BW, Albers RW, et al. © Arruda Carvalho UTSC 25  2022-10-25 Metabotropic Glutamate Receptors (mGluRs) Basic Neurochemistry: Molecular, Cellular and Medical Aspects. 6th edition. Siegel GJ, Agranoff BW, Albers RW, et al. Class I mGluRs: Predominantly localized in postsynaptic membrane terminals, mainly activate Gq signaling © Arruda Carvalho UTSC mGluR1 activation leads to a slow EPSC Hartmann et al., Neuron 2014 1) AMPAR-dependent fast EPSC (fEPSC) 2) mGluR1/TRPC3-dependent slow EPSC (sEPSC) © Arruda Carvalho UTSC 26  2022-10-25 mGluR1 sEPSC: Mechanism 2. TRPC3 activation drives ion influx and an increase in Ca2+ levels, leading to… 3. Changes in membrane potential that incur in the activation of 1. mGluR1-mediated voltage-gated increase in DAG levels calcium channels leads to the activation of Transient Receptor Potential Channel 3 (TRPC3), a Na+ and Ca2+ permeable ion channel Mod from Becker, Cerebellum, 2017 Slow depolarizing potential! © Arruda Carvalho UTSC Metabotropic Glutamate Receptors (mGluRs) Basic Neurochemistry: Molecular, Cellular and Medical Aspects. 6th edition. Siegel GJ, Agranoff BW, Albers RW, et al. Class II mGluRs: Pre and postsynaptic, couple to Gi/o proteins, inhibit AC, attenuation of the Ca2+ influx and a reduction in neurotransmitter release © Arruda Carvalho UTSC 27  2022-10-25 Metabotropic Glutamate Receptors (mGluRs) Basic Neurochemistry: Molecular, Cellular and Medical Aspects. 6th edition. Siegel GJ, Agranoff BW, Albers RW, et al. Class III mGluRs: Couple to Gi/o proteins. Presynaptic terminals of glutamatergic and GABAergic neurons, thus modulating both excitatory and inhibitory neurotransmission. Modulatory activity depends on the brain region and/or cell-type involved. © Arruda Carvalho UTSC Lecture Outline Overview of Ionotropic vs. Metabotropic Actions GPCR General Structure GPCR Activation and Action Binding to G proteins Effector Mechanisms: Presynaptic Effector Mechanisms: Postsynaptic GPCR Types and Specific Mechanisms mGluRs mAChRs GABABRs Dopamine and Serotonin Receptors GPCR Desensitization Overview © Arruda Carvalho UTSC © 2018 Arruda Carvalho UTSC winter term 28  2022-10-25 Muscarinic ACh Receptors Present pre- and postsynaptically © Arruda Carvalho UTSC Muscarinic ACh Receptors M1R, M3R, M5R Lebois et al., Neuropharm 2017 M1 is the major postsynaptic mAChR, with M3 and M5 expressed at significantly lower levels. M1Rs are highly expressed in the cerebral cortex, hippocampus and striatum Activation of presynaptic M1Rs leads to activation of Gq, resulting in increase in intracellular Ca2+ levels, which facilitates presynaptic ACh release Activation of postsynaptic M1Rs elevates intracellular Ca2+ levels and leads to slow postsynaptic depolarization and an increase in neuronal excitability © Arruda Carvalho UTSC 29  2022-10-25 Muscarinic ACh Receptors M2R and M4R Lebois et al., Neuropharm 2017 M2 and M4 are the major presynaptic mAChRs in the brain. They couple through Gi to decrease cAMP levels and suppress transmitter release at cholinergic and glutamatergic synapses (dampening excitability) © Arruda Carvalho UTSC Lecture Outline Overview of Ionotropic vs. Metabotropic Actions GPCR General Structure GPCR Activation and Action Binding to G proteins Effector Mechanisms: Presynaptic Effector Mechanisms: Postsynaptic GPCR Types and Specific Mechanisms mGluRs mAChRs GABABRs Dopamine and Serotonin Receptors GPCR Desensitization Overview © Arruda Carvalho UTSC © 2018 Arruda Carvalho UTSC winter term 30  2022-10-25 GABAB receptors Two subunits, GABA B1 and GABA B2, form a heterodimer GABA B1 is responsible for ligand binding in N- terminal VFT domain. GABA B2 does not bind GABA, but (1) interacts with GABA B1 to stabilize its agonist-bound conformation, thereby increasing agonist affinity, and (2) is responsible for G protein coupling Xu et al., Front Pharmacol 2014 © Arruda Carvalho UTSC GABAB receptors Activation of Gi/o protein leads to: Presynaptic Inhibition of Neurotransmitter Release: Gα subunits inhibit adenylyl cyclase to reduce cAMP levels Gβγ subunits inhibit (1) voltage-dependent Ca2+ channels and (2) SNARE complex proteins, preventing release © Arruda Carvalho UTSC 31  2022-10-25 General Mechanisms of GPCR Modulation of Synaptic Transmission Presynaptic GPCRs: Neurotransmitter Release Gbg 1. Inhibition of voltage-dependent calcium channels (VDCCs) As the presynaptic membrane is depolarized, VDCCs become activated resulting in an influx of calcium into the cell which can then bind to Synaptotagmin to trigger exocytotic events Gbg-mediated inhibition of VDCCs will decrease neurotransmitter release Brown and Sihra, Handb. Exp. Pharmacol. 2008 © Arruda Carvalho UTSC General Mechanisms of GPCR Modulation of Synaptic Transmission Presynaptic GPCRs: Neurotransmitter Release Gbg 2. Inhibition of SNARE complex direct interaction with syntaxin, SNAP-25 and VAMP/ synaptobrevin Betke et al, Prog Neurobio 2012 Prevents the tight zippering of the SNARE complex necessary to drive membrane fusion, and thus can inhibit vesicle exocytosis © Arruda Carvalho UTSC 32  2022-10-25 GABAB receptors Activation of Gi/o protein leads to: Postsynaptic Slow hyperpolarization: Gβγ subunits activate GIRK channels, leading to slow hyperpolarization (termed the slow inhibitory postsynaptic potential) © Arruda Carvalho UTSC GABAB receptors Activation of Gi/o protein leads to: Presynaptic Postsynaptic Inhibition of Neurotransmitter Release: Slow hyperpolarization: Gα subunits inhibit adenylyl cyclase to reduce Gβγ subunits activate GIRK channels, cAMP levels leading to slow hyperpolarization (termed the slow inhibitory postsynaptic potential) Gβγ subunits inhibit (1) voltage-dependent Ca2+ channels and (2) SNARE complex proteins, preventing release © Arruda Carvalho UTSC 33  2022-10-25 Lecture Outline Overview of Ionotropic vs. Metabotropic Actions GPCR General Structure GPCR Activation and Action Binding to G proteins Effector Mechanisms: Presynaptic Effector Mechanisms: Postsynaptic GPCR Types and Specific Mechanisms mGluRs mAChRs GABABRs Dopamine and Serotonin Receptors GPCR Desensitization Overview © Arruda Carvalho UTSC © 2018 Arruda Carvalho UTSC winter term Dopamine Metabotropic Receptors D1-Like D2-like G protein Gs Gi/o Subtypes D1 D5 D2 D3 D4 Effect ↑AC, ↑PLC, ↑AC ↓ AC ↓Ca2+ ↓ AC ↓ AC ↓Ca2+ ↑L-type Ca2+ channels Location Frontal Cortex, Cortex, Caudate, NAc, Frontal Cortex, Caudate, HPC, Putamen, NAc, olfactory midbrain, amygdala, Putamen, NAc, striatum olfactory tubercle, cardiovascular hypothalamus tubercle cortex system Pre- and postsynaptic. The main distinction between two classes is that D1-like receptors activate AC through interactions with Gs, whereas D2-like receptors inhibit AC and other effector molecules by interacting with Gi/Go. © Arruda Carvalho UTSC 34  2022-10-25 Serotonin Metabotropic Receptors 5-HTRs are mainly localized in postsynaptic terminals; however, 5-HT1AR, 5-HT1BR, 5-HT1DR, 5-HT1FR, 5-HT2AR and 5-HT4R, are also expressed in presynaptic terminals © Arruda Carvalho UTSC Lecture Outline Overview of Ionotropic vs. Metabotropic Actions GPCR General Structure GPCR Activation and Action Binding to G proteins Effector Mechanisms: Presynaptic Effector Mechanisms: Postsynaptic GPCR Types and Specific Mechanisms mGluRs mAChRs GABABRs Dopamine and Serotonin Receptors GPCR Desensitization Overview © Arruda Carvalho UTSC © 2018 Arruda Carvalho UTSC winter term 35  2022-10-25 GPCR Desensitization 1. Heterologous desensitization (no ligand binding necessary) PKA and PKC phosphorylate sites on the third intracellular loop and possibly the C-terminal cytoplasmic domain. This phosphorylation inhibits binding to Gs 2. Homologous desensitization (ligand bound) G-protein receptor kinase (GRK)-mediated phosphorylation of the GPCR enables binding of β-arrestin, which in turn prevents Gs coupling © Arruda Carvalho UTSC GPCR Desensitization 2. Homologous desensitization (ligand bound) β-arrestin binding (following GRK phosphorylation) enhances rate of GPCR internalization © Arruda Carvalho UTSC 36  2022-10-25 GPCR Desensitization GPCR ligand binding leads to homologous desensitization through two mechanisms: 1. Short-term desensitization, often through phosphorylation by GPCR kinases (GRKs), interaction with β-arrestin and uncoupling of the interaction between the GPCR and the G protein 2. Endocytosis of the GPCR via clathrin-coated pits. GPCR- binding leads to a conformational change in b- arrestin that exposes an AP2 Ramachandran et al., Nat Rev Drug Discov, 2012 binding domain, triggering endocytosis. Following endocytosis, the GPCR is dephosphorylated via protein phosphatases. In the sorting endosome, the GPCRs are either targeted to recycling vesicles (to be reinserted into the membrane) or to lysosomes for degradation © Arruda Carvalho UTSC Lecture Outline Overview of Ionotropic vs. Metabotropic Actions GPCR General Structure GPCR Activation and Action Binding to G proteins Effector Mechanisms: Presynaptic Effector Mechanisms: Postsynaptic GPCR Types and Specific Mechanisms mGluRs mAChRs GABABRs Dopamine and Serotonin Receptors GPCR Desensitization Overview © Arruda Carvalho UTSC © 2018 Arruda Carvalho UTSC winter term 37  2022-10-25 Overview Mod from Rosembaum et al., Nature, 2009 © Arruda Carvalho UTSC Overview Hanlon and Andrew, J Cell Sci 2015 © Arruda Carvalho UTSC 38  2022-10-25 Lecture Outline Overview of Ionotropic vs. Metabotropic Actions GPCR General Structure GPCR Activation and Action Binding to G proteins Effector Mechanisms: Presynaptic Effector Mechanisms: Postsynaptic GPCR Types and Specific Mechanisms mGluRs mAChRs GABABRs Dopamine and Serotonin Receptors GPCR Desensitization Overview © Arruda Carvalho UTSC © 2018 Arruda Carvalho UTSC winter term 39