MODULE 27 Test Review PDF

MODULE 27 GPCRs – Definition Ligands: hormones, neurotransmitters, local mediators. One ligand can activate different receptors (e.g., adrenaline activates > 9 receptors, acetylcholine activates 5, serotonin - 15). The receptor polypeptide chain threads across the lipid bilayer seven times (s...

MODULE 27 GPCRs – Definition Ligands: hormones, neurotransmitters, local mediators. One ligand can activate different receptors (e.g., adrenaline activates > 9 receptors, acetylcholine activates 5, serotonin - 15). The receptor polypeptide chain threads across the lipid bilayer seven times (serpentine receptors). Examples: rhodopsin, a light-activated protein in the vertebrate eye, olfactory receptors in the vertebrate nose. ~50% of all known drugs work through G-protein-linked receptors. Trimeric G-Proteins Ligand binding induces a conformational change that activates trimeric GTP-binding proteins (G proteins). The G proteins are at the cytoplasmic face of the plasma membrane and couple the receptors to enzymes or ion channels. The G proteins are composed of α, β, and γ subunits; α and γ subunits have covalently attached lipid anchors. In an unstimulated state, the α subunit binds GDP. In the stimulated state, (1) the α subunit undergoes a conformational change, releases GDP, and binds GTP, and (2) the trimer dissociates into an α subunit and a βγ complex; both have signaling functions. Switching off the G-proteins The α subunit is a GTPase; once it hydrolyzes its GTP to GDP, it re-associates with the βγ complex to form an inactive heterotrimeric G protein. The time during which the α subunit and βγ complex remain apart and active is short; it depends on how quickly the α subunit hydrolyzes bound GTP. The GTPase activity of the α subunit is enhanced by the binding of its target effector protein or a regulator of G protein signaling (RGS). Thus, effectors and RGS proteins are α subunit-specific GTPase activating proteins (GAPs). Types of Heterotrimeric G Proteins Gα subunit type Effect of activated Gα on the target protein Gαs (stimulatory) Stimulation of adenylyl cyclase Gαi (inhibitory) Inhibition of adenylyl cyclase Gαq (activating the PI cascade) Stimulation of phospholipase C Gαt (inhibitory, for transducin) Stimulation of cGMP phosphodiesterase G Proteins That Signal via Cyclic (c) AMP cAMP is made from ATP by adenylyl cyclase, and it is destroyed by cAMP phosphodiesterase to adenosine 5′-monophosphate (5′-AMP). Extracellular signals usually increase cAMP levels by increasing the cyclase activity. Adenylyl cyclase is a transmembrane protein regulated by G proteins and Ca2+. MODULE 27 Receptors that act via cAMP are coupled to a stimulatory G protein (Gs), which activates adenylyl cyclase. Inhibitory G protein (Gi), inhibits adenylyl cyclase. cAMP-dependent Protein Kinase (PKA) cAMP activates ion channels in the plasma membrane or cAMP-dependent protein kinase (PKA). PKA transfers phosphate group from ATP to serine and threonine residues on proteins. Inactive PKA has two catalytic subunits and two regulatory subunits. cAMP binding to the regulatory subunits alters their conformation and causes their dissociation from the catalytic subunits. The released catalytic subunits are active and phosphorylate their targets. G Proteins That Activate the Inositol Phospholipid Pathway G proteins with a G alpha-q subunit activate plasma membrane-bound phospholipase C-β. The phospholipase acts on PIP2 at the inner half (leaflet) of the plasma membrane lipid bilayer. The phospholipase cleaves PIP2 to inositol 1,4,5-trisphosphate (IP3) and diacylglycerol; at this step, the signaling pathway splits into two branches. Two Branches of the Inositol Phospholipid Pathway IP3 diffuses into the cytosol, opens Ca2+-release channels in the smooth ER membrane. The response is terminated by: 1. IP3 dephosphorylation to IP2. 2. IP3 phosphorylation to IP4. 3. Ca2+ being pumped out of the cell. Diacylglycerol (DAG) DAG remains in the plasma membrane due to its hydrophobic properties. DAG could be cleaved to arachidonic acid, a messenger and precursor of eicosanoids that participate in pain and inflammatory responses. DAG also activates the Ca2+-dependent serine/threonine protein kinase C (PKC). The rise in cytosolic Ca2+ induced by IP3 allows the translocation of PKC from the cytosol to the plasma membrane, where it is activated by Ca2+, DAG, and phosphatidylserine. Ca2+: an Intracellular Messenger Many extracellular signals increase cytosolic Ca2+. Ca2+ is low in the cytosol and high in the extracellular space and ER lumen. Low cytosolic Ca2+ is maintained by active transporters in the plasma membrane. When a signal opens Ca2+ channels, Ca2+ enters the cytosol, increasing [Ca2+] by 20-fold. Ca2+ channels: voltage-dependent; IP3-gated Ca2+-release channels releasing Ca2+ from the ER; (3) ryanodine receptors, releasing Ca2+ from the ER to stimulate muscle cell contraction or Ca2+ signaling in non-muscle cells. MODULE 27 Ca2+ Oscillations in a Cell Response Ca2+-sensitive fluorescent molecules visualize cytosolic Ca2+. The initial Ca2+ signal is localized to discrete cell regions; if the extracellular signal persists, the localized signal propagates as a Ca2+ wave in the cytosol. A Ca2+ “spike” is often followed by a series of spikes; these oscillations persist until the receptors are active. The initial release of Ca2+ stimulates more Ca2+ release, a process known as Ca2+-induced Ca2+ release; however, when a limit is reached, Ca2+ inhibits further release. Cells sense the frequency of Ca2+ spikes via Ca2+-sensitive proteins that change their activity as a function of Ca2+ spike frequency. Ca2+/Calmodulin-dependent Protein Kinases (CaM-Kinases) Cytosolic Ca2+ signals are transduced by Ca2+-binding proteins. Calmodulin protein: two or more Ca2+ ions are required to bind and activate the protein. Calmodulin is a regulatory subunit in enzymes or binds proteins to alter their activity. Ca2+/calmodulin-dependent protein kinases (CaM-kinases) phosphorylate Ser or Thr. CaM-kinase II functions as: 1. A molecular memory device because it autophosphorylates and stays active even after the Ca2+ signal has decayed. 2. A frequency decoder of Ca2+ oscillations; the enzyme activity increases as a function of pulse frequency. G Proteins Regulating Ion Channels Example: Acetylcholine released by the vagus nerve reduces the rate and strength of heart muscle cell contraction. Binding of acetylcholine to muscarinic acetylcholine receptors (GPCRs) activates Gi protein by catalyzing the binding of GTP to the α subunit. The released βγ subunit opens K+ channels in the heart muscle plasma membrane. The increased K+ permeability hyperpolarizes the membrane, which reduces the frequency of heart muscle contraction. Cyclic Nucleotide-gated Ion Channels Binding of an odorant to an olfactory GPCR activates a trimeric G protein (Gαolf and Gβγ). Gαolf activates adenylyl cyclase (AC3) to make cAMP. cAMP opens cAMP-gated cation channels and an influx of Na+ and Ca2+ depolarizes the cell. The initial depolarization is amplified by the activation of Ca2+-dependent Cl− channels. The depolarization of the olfactory receptor neuron initiates a nerve impulse to the brain. cAMP also activates protein kinase A (PKA) that regulates intracellular events, including the transcription of cAMP-regulated genes. MODULE 27 Cyclic Nucleotide-gated Ion Channels (continued) The retina has rod and cone types of photoreceptors. Rods are specialized for night non-color vision and respond to light with changes in membrane potential. The rods contain cGMP-gated Na+ channels; in the dark, cGMP causes Na+ influx. Light changes the conformation of GPCR rhodopsin to activate the G protein transducin. cGMP phosphodiesterase is activated, cGMP is hydrolyzed, cGMP-gated Na+ channels close, Na+ influx is reduced, and the photoreceptor cell is hyperpolarized. Desensitization of GPCRs After exposure to a high concentration of ligands for a prolonged period, GPCRs terminate activity in three ways: 1. The receptors are altered and no longer interact with G proteins (receptor inactivation). 2. The receptors are temporarily moved inside the cell (receptor sequestration). 3. The receptors are destroyed in lysosomes after internalization (receptor downregulation). The desensitization depends on the phosphorylation of the receptor by G-protein-linked receptor kinases (GRKs). GRKs phosphorylate a GPCR only after the receptor has been activated by a ligand. Once a receptor is phosphorylated by GRKs, it binds to an arrestin protein that inactivates the receptor by preventing the interaction with G proteins. Putting It Together GPCRs regulate plasma-membrane-bound enzymes or ion channels via hetero(trimeric) G proteins; activated G proteins disassemble into α subunits and βγ complexes. Some GPCRs (with G alpha-s or G alpha-i) regulate adenylyl cyclase and alter the intracellular concentration of cAMP. Some GPCRs (with G alpha-q) activate a phosphoinositide-specific phospholipase C that hydrolyzes PIP2 to IP3 and diacylglycerol; IP3 releases Ca2+ from the ER; diacylglycerol, phosphatidylserine, and Ca2+ activate protein kinase C (PKC). A rise in cAMP and Ca2+ levels stimulates protein kinase A (PKA) and Ca2+/calmodulin-dependent protein kinases (CaM-kinases), respectively. PKC, PKA, and CaM-kinases phosphorylate proteins at Ser or Thr residues. The GPCR responses are terminated when their ligand is removed, then the GTP bound to the Gα subunit is hydrolyzed to GDP, IP3 is dephosphorylated by a phosphatase (or phosphorylated by a kinase), cyclic nucleotides are hydrolyzed by phosphodiesterases, Ca2+ is pumped out of the cytosol, and phosphorylated proteins are dephosphorylated. Desensitization of GPCRs is through phosphorylation by GRKs, allowing for arrestin binding.