Cell Communication 1: GPCRs 2024 Lecture Notes PDF

In this lecture, we will investigate cellular signal, concentrating on G- Protein Coupled Receptors – their activators, G-proteins, and downstream effectors Signals come in a variety of types. A signal can range from light (which activates the GPCR receptors in the rods and cones in your eye), to...

In this lecture, we will investigate cellular signal, concentrating on G- Protein Coupled Receptors – their activators, G-proteins, and downstream effectors Signals come in a variety of types. A signal can range from light (which activates the GPCR receptors in the rods and cones in your eye), to small molecules (dopamine, morphine, etc.), to proteins (Wnt signaling involved in development), to mechanical signals. Signaling pathways can be divided into three components 1.In order to detect a signal, cells must have an appropriate receptor that detects the stimulus and converts it into a biological signal the cell can use 2.Often, the signal is relayed within the cell by a pathway of sequentially acting proteins to the appropriate intracellular target in a process referred to as  transduction 3.Finally, the cell responds by mounting a response – the response can be a change in the activities of cellular proteins, a change in the pattern of genes it expresses, or both. The textbook provides us with a useful analogy for transduction A cell phone receives radio signals from the broadcast towers and converts those signals into sounds your ear can perceive and vice versa when you speak. The phone can also get signals from your voice, from Bluetooth, wifi, and it can detect light using its camera, or vibrations/movement, or mechanical action as you press  the screen or buttons. All these inputs illicit different responses. Likewise, a signal received by a cell (in this case a ligand binding to a membrane receptor) must be converted into an intracellular biochemical pathway that does something within the cell to change its state Eukaryotic cells depend on multiple extracellular signals and there are a few general principles that we must consider as we discuss signaling pathways Individual cells respond only to signals they can detect Different cells may be primed to respond differently to the same signal by the expression of different/distinct sets of receptors that can detect the signal, and the expression/regulation of distinct downstream effector proteins/molecules in the pathway. A single cell may have none, a few, or thousands of receptors for a particular signal Combinations of signals acting together can elicit distinct cellular responses As shown in the figure, most cells require signals to promote their survival – if these are deprived then the cell will activate programmed cell death and die (cancer cells are a notable exception and can survive independently of these signals) Some cells require signals to grow and divide Some cells respond to various signals by changing their cell fate during development – you’ll learn about these pathways in the second half of the course One signal molecule may induce different response in different target cells Example: acetylcholine is a neurotransmitter, its structure is shown in (D) Acetylcholine signals to pacemaker cells in the heart to decrease the rate of your heartbeat Acetylcholine signals to cells in your salivary gland to trigger  secretion of components of saliva by regulated exocytosis The acetylcholine receptor protein in both cell types is identical, but the cells have a completely different physiological response Acetylcholine binds to a different receptor (a ligand-gated ion channel) on skeletal muscle cells (a ligand- gated ion channel) to trigger muscle contraction Therefore: the signal alone is not the message; the information depends on how the target cell receives and interprets the signal Another key concept is that signals can act on different time scales Some signals exert their effect rapidly (on the order of milliseconds to minutes) These signaling pathways are fast because they alter the activities of proteins that are present within the cell Some signals exert their effects slowly (minutes to hours) These pathways involve changes in gene expression; they are slower because of the time required for transcription of new mRNAs and the time required for translation of these new proteins It’s also important to consider that intercellular communication occurs over varying distances depending on the mechanism of the pathway Contact-dependent signaling can occur between two cells that are in direct physical contact with one another In this case, the signaling cell expresses a ligand molecule that is bound to the extracellular surface of its plasma membrane  The target cell expresses a receptor protein that is also bound to its membrane This ligand may only interact with its receptor when the two cells are adjacent to each other Cell signaling also occurs on distance scales of less than a cell diameter to a few cell diameters away We’ve already seen an example of this when neurons communicate with each other by regulated exocytosis of neurotransmitters at synapses Paracrine signals are released into the extracellular fluid where they act locally on neighboring cells Cell-cell signaling may also act over long distances Endocrine signaling occurs when endocrine glands secrete hormones into the bloodstream Hormones are distributed throughout the body, target cells with appropriate receptors can be far-removed Autocrine signaling is when a cell secretes a signal that binds to a receptor on the same cell and activates a signaling pathway.  Signal transduction pathways have evolved to perform multiple functions: Transduce the signal into a molecular form that can carry out the response Relay the signal from point of reception to point of action in the cell Amplify the received signal Integrate multiple signals that in combination can cause distinct responses Feedback from a pathway can regulate an upstream component of that pathway Distribute the signal to coordinate several responses in parallel Remember phosphorylation and GTP-binding proteins (G-proteins)? Amino acid residues that have a hydroxyl (-OH) group are the main targets for phosphorylation by kinases Proteins can be phosphorylated on three different amino acid residues: serines, threonines, and tyrosines (one set of kinases target serines and threonines, another set targets tyrosines).  G-proteins – cycle between active and inactive conformations depending on whether GTP or GDP is bound, respectively. GTP binding is controlled by GEFs (guanine exchange factors), and the ability to intrinsically hydrolyze GTP to GDP leads to the G-protein to turn off. The hydrolysis can be accelerated by GTPase Activating Proteins (GAPs). GTP-binding proteins are GTPases that come in two prime varieties – monomeric (small) GTPases, and larger heterotrimeric GTPases. The small GTPases (monomeric) and the Galpha subunit have a common GTPase domain, however the Galpha subunit also has a second domain called the “helical domain” G proteins have an endogenous GTPase activity GTP bound = ON, GDP bound = OFF The GTPase activity of G proteins is usually regulated by accessory factors Guanine nucleotide exchange factors (GEFs) induce G proteins to release GDP and to bind to GTP to become active While many GTP-Binding proteins  have intrinsic GTPase activity, GTPase activating factors (GAPs) can bind a G protein and enhance its rate of GTP hydrolysis, thereby serving to regulate the time a GTP-binding protein is active. These GTP and GDP-bound states drive distinct structural conformations of the G-protein (allosteric changes) that regulate what the G-protein can bind in its “on’ and “off” states. There are three main classes of receptors we will discuss. For this lesson, we will focus on GPCRs. The second class of receptors are G-protein coupled receptors (GPCRs) Largest family of cell surface receptors in animals (>700 in humans) GPCRs detect odorants, light, many other types of signals These receptors have 7 transmembrane α-helices Extracellular-facing N-terminus and extracellular loops that may  bind signals Intracellular-facing C-terminus and intracellular loops that interact with downstream effectors The second class of receptors are G-protein coupled receptors (GPCRs) Largest family of cell surface receptors in animals (>700 in humans) GPCRs detect odorants, light, many other types of signals These receptors have 7 transmembrane α-helices Extracellular-facing N-terminus and extracellular loops that may  bind signals Intracellular-facing C-terminus and intracellular loops that interact with downstream effectors Ligand binding to the outside of the receptor triggers a conformational change on residues on the cytosolic side. Different GPCRs engage ligands in different ways, but all transduce a conformational change that to the cytosolic side of the GPCR. This enables the signal to be relayed to the inside of the cell, across the lipid bilayer. Small molecules can be agonists, which activate the GPCR and transduce a signal to the cell interior. Or they can be antagonists, which compete for agonists. Antogonists bind the GPCR, but do not activate it – they keep the GPCR in the off state. They are effectively competitive inhibitors. A specific GPCR can have multiple agonists and antagonists. Harry Potter is neither an agonist nor an antagonist. Or is he? Maybe in some fan fiction. The opiod receptor has a lot of fame and press these days – as it is the target for endogenous opiods like beta-endorphin, but also the target for exogenous or synthetic opiods like morphine, opium, oxycodone. Antagonists can be life-saving. Like the use of maloxone. It has high affinity for opiod receptors and can compete off opiates, but keep the receptor inactive when naloxone is bound. Be a protagonist and pick up some antagonist at your pharmacy – you might be able to save a life. Another example of a GPCR: the beta-adrenergic receptor. Agonists include epinephrine. Antagonists include propranolol. This receptor, once activateD, initiates the fight or flight response (adrenaline) GPCRs are major drug targets. GPCRs signal through Gproteins. Unlike small G-proteins like ran or ras, these are heterotrimeric G- proteins with an alpha, beta, and gamma subunit. The G-alpha protein is the GTP-binding protein, which undergoes GTP-dependent conformational changes. There are a number of Galpha subunits – the major classes of Galpha subunits are shown above. The G-beta- gamma subunits form an obligate (always found together) heterodimer. Together with the Galpha subunit, they can form a heterotrimer. The heterotrimer only forms when Galpha is in the GDP state. The GPCR binds the heterotrimeric G-protein. Once the GPCR is activated, it induces guanine exchange on the G-alpha subunit. GDP is released. As the cytosolic concentration of GTP is higher than GDP, GTP binds the G-alpha subunit. This causes a conformational change in the G- alpha subunit, causing dissociation of the Gbeta-gamma subunit. Galpha-GTP and the free Gbeta- gamma subunits are free to engage and activate or inhibit downstream effector molecules until the Galpha subunit hydrolyzes GTP to GDP, and the Galpha-beta-gamma heterotrimer reforms. GPCR GEF activity enables GDFP dissociation and GTP binding to the G-alpha subunit. The Galpha then dissociates from the Gbeta- gamma heterodimer, and each can go on to activate downstream effectors. Signaling downstream of GPCRs occurs through multiple molecules GPCRs communicate directly with heterotrimeric G-proteins Heterotrimeric G proteins are composed of α, β, and γ subunits – only the α subunit binds to GTP/GDP In its GDP-bound state, all three subunits associate in an inactive conformation When Gα binds to GTP, it  dissociates from Gβγ and adopts an active conformation When a ligand binds to the extracellular surface of the GPCR, the cytosolic surface acts as a GEF for Gα causing it to release GDP, bind to GTP, and become activated Gα and Gγ are covalently attached to lipids in the membrane so they diffuse within the bilayer The active subunits diffuse within the plane of the membrane to activate downstream signaling molecules (more about these in a bit) There are many Galpha subunits – each with specific downstream effectors. Of note, a key downstream effector is adenylyl cyclase (AC). AC is activated by Galpha-s (Stimulate) and inhibited by Galpha-I (Inhibitory). There are also many Gbeta and Ggamma proteins – leading to a combinatorial set of heterotrimeric G-proteins that can be formed. Galpha subunits have a catalytic site – ie, they have all the residues needed to hydrolyze GTP to GDP. This is an intrinsic timer so that downstream activation is limited by the time the G-alpha subunit is in the GTP state. Give this some thought…. Other proteins, called GTPase activating proteins (GAPs), can bind and stimulate the Galpha subunit’s intrinsic GTPase activity. They do this by binding the Galpha subunit in a state akin to its conformation when GTP hydrolysis is in the transition state. Ie, it lowers the activation energy for the Galpha to hydrolyze the GTP! What are the signaling targets downstream of heterotrimeric G- proteins? The Gβγ subunit can activate ligand-gated ion channels Acetylcholine slows the heart by acting through pacemaker cells Receptor activation ==> dissociation of Gα and Gβγ Gβγ opens K+ channels to make he membrane harder to to activate by depolarization and slows heart rate What are the signaling targets downstream of heterotrimeric G- proteins? Many G proteins activate membrane-bound enzymes that produce small messenger molecules First messengers are heterotrimeric G-proteins Second messengers are these molecules synthesized at the membrane that diffuse into the cytosol to act on other downstream signaling proteins cAMP and IP3 and DAG are second messengers – ie. downstream signaling molecules. The first second messenger molecule we’ll discuss is cAMP cAMP is generated from ATP by adenylyl cyclase and degraded to AMP by cAMP phosphodiesterase Caffeine inhibits phosphodiesterase – your cup of morning coffee is partially stimulating your brain by keeping cAMP around in your neurons for longer cAMP mediates its downstream effects by activating cAMP- dependent protein kinase (PKA) for fast AND slow signaling Example of signaling by cAMP and PKA: glycogen utilization by skeletal muscles Muscles and liver store glucose as a polysaccharide called glycogen (polymer of glucose) that’s broken down rapidly to provide glucose as a short-term energy supply The hormone epinephrine is secreted by the adrenal gland in response to stress or physical  exertion Epinephrine binds to a GPCR on muscles to activate heterotrimeric G- proteins that, in turn, activate adenylyl cyclase Adenylyl cyclase converts ATP to cAMP cAMP activates PKA which goes on to phosphorylate another kinase called phosphorylase kinase Phosphorylase kinase phosphorylates a THIRD enzyme named glycogen phosphorylase Glycogen phosphorylase causes glycogen breakdown into glucose that the muscle will use to power contraction Multi-step signal transduction cascade, but FAST because no new gene expression is required cAMP will then be broken down to AMP by cAMP phosphodiesterase (not shown) to terminate the signal Another example of signaling by cAMP and PKA: changes in gene expression In many cell types, a rise in cAMP responses involve changes in gene expression that occur over minutes to hours In this case, once activated by binding to cAMP, PKA enters the nucleus to phosphorylate transcription factors This activation leads to  expression of sets of genes This mechanism is important for many different processes including hormone production in endocrine cells and the formation of long-term memories in certain neurons in the brain Thus, cAMP acts as a second messenger in both short- and long- term signaling pathways Another example of signaling by cAMP and PKA: changes in gene expression In many cell types, a rise in cAMP responses involve changes in gene expression that occur over minutes to hours In this case, once activated by binding to cAMP, PKA enters the nucleus to phosphorylate transcription factors This activation leads to  expression of sets of genes This mechanism is important for many different processes including hormone production in endocrine cells and the formation of long-term memories in certain neurons in the brain Thus, cAMP acts as a second messenger in both short- and long- term signaling pathways Remember: Ca+2 concentration is low in the cytosol Pumps in the plasma membrane pump some calcium out of the cell BUT pumps in the ER and in mitochondria also transport Ca+2 into the ER lumen and into the matrix, respectively ER calcium is used as an internal “reservoir” to be used in specific signaling pathways The other most common enzyme activated by heterotrimeric G- proteins is phospholipase C (PLC) PLC signals through the production of TWO downstream second messengers GPCR is activated by ligand è heterotrimeric G-proteins activated by GPCR è PLC is activated by Gβγ PLC binds to a membrane lipid – inositol phospholipid and cleaves  it into: 1.Diacylglycerol which stays associated with the membrane, and 2.Inositol 1,4,5-trisphosphate (IP3) which is release from the membrane and diffuses into the cytosol Diacylglycerol recruits a kinase named protein kinase C (PKC) to bind to the cytosolic face of the plasma membrane IP3 binds to a ligand-activated Ca+ ion channel in the ER membrane causing it to open and release Ca+2 into the cytosol Ca+2 binds to PKC on the membrane and ACTIVATES it to phosphorylate its own set of downstream targets to propagate the signal The targets of PKC vary depending on the type of cell The other most common enzyme activated by heterotrimeric G- proteins is phospholipase C (PLC) PLC signals through the production of TWO downstream second messengers GPCR is activated by ligand è heterotrimeric G-proteins activated by GPCR è PLC is activated by Gβγ PLC binds to a membrane lipid – inositol phospholipid and cleaves  it into: 1.Diacylglycerol which stays associated with the membrane, and 2.Inositol 1,4,5-trisphosphate (IP3) which is release from the membrane and diffuses into the cytosol Diacylglycerol recruits a kinase named protein kinase C (PKC) to bind to the cytosolic face of the plasma membrane IP3 binds to a ligand-activated Ca+ ion channel in the ER membrane causing it to open and release Ca+2 into the cytosol Ca+2 binds to PKC on the membrane and ACTIVATES it to phosphorylate its own set of downstream targets to propagate the signal The targets of PKC vary depending on the type of cell In the next lecture, we’ll talk about enzyme coupled receptors.