Intracellular Signaling and the Cell Membrane BMS100_PHL1-22v3_F2022 PDF

Intracellular Signaling and the Cell Membrane • Receptors in the cell membrane are key in detecting extracellular signals and modifying cell function based on those signals ▪ transduction = the intracellular events that transform the extracellular signal into an intracellular signal • Additional si...

Intracellular Signaling and the Cell Membrane • Receptors in the cell membrane are key in detecting extracellular signals and modifying cell function based on those signals ▪ transduction = the intracellular events that transform the extracellular signal into an intracellular signal • Additional signaling events involving the membrane: ▪ source of some 2nd messengers ▪ site where some 2nd messengers accumulate ▪ site where ionic 2nd messengers are regulated ▪ important site where regulatory proteins and enzymes localize and integrate signaling What happened there? • Concentration of the first messenger increased (ligand) • Binds to the cell membrane receptor • The cell membrane receptor becomes activated, resulting in activation an intracellular protein associated with the receptor • That protein activated a mechanism (in this case an enzyme) that increased the intracellular concentration of the active form of a second messenger • The second messenger binds to and activates another protein… and that protein will activate or inactivate other biochemical signaling cascades that have some sort of effect ▪ Effectors Intracellular Signaling – A Model Inactive system: extracellular signal ends 1st messenger Inactivation As time goes on: • ligand releases from receptor • receptor-associated effectors inactivate • 2nd messenger is either metabolized or removed 2nd messenger “inactivator” Inactive 2nd messenger What happened there? • Concentration of the first messenger decreased • Cell membrane receptor is no longer activated • The protein associated with the receptor is becomes inactive ▪ Often inactivates itself – a long-lasting signal may need frequent activation of the receptor • The protein or enzyme no longer produces or increases the concentration of the second messenger… • … and another mechanism decreases or inactivates the second messenger • Since the concentration of the 2nd messenger drops, it no longer activates the protein it binds to ▪ Effectors are no longer activated Typical Components • An extracellular signal activates a membrane-bound receptor ▪ the extracellular signal is known as the 1st messenger • Receptor activation → increased cytosolic or membrane concentrations of a 2nd messenger ▪ Wide range of 2nd messengers ▪ increased 2nd messenger concentration can be due to simple, direct processes or they can be a complicated series of biochemical events ▪ Single activated receptor → many molecules of 2nd messenger = amplification • Signal termination is due to a number of processes ▪ Inactivation of receptor (i.e. no 1st messenger) or receptorassociated effectors ▪ degradation or removal of 2nd messenger ▪ negative feedback Caveats to the Model • This model is not perfectly accurate for any 2nd messenger system ▪ It is oversimplified ▪ There are components that are not strictly accurate because they have been generalized • What this model does offer: ▪ A way to categorize the steps in very important but somewhat complicated signaling events ▪ A way to quickly see the similarities and differences across different biological strategies for intracellular signaling Types of Cell Membrane Receptors Molecular Biology of the Cell, 6th ed. Alberts et. al. p. 818, fig 15-6 The G-Protein-Coupled Receptors (GPCR) Molecular Biology of the Cell, 6th ed. Alberts et. al. p. 818, fig 15-6 G-Protein-Coupled Receptors • Largest family of cell membrane receptors • 800 unique G-protein-coupled receptors (GPCRs) ▪ Almost half of medications act on these receptors or their pathways • All act in a very similar way ▪ Receptor activation → activation of a protein that binds to a guanine nucleotide • Hence “G-protein” • The activated G-protein will modify the activity of an enzyme ▪ The activated G-protein is on a “timer” • Has intrinsic GTP-ase activity • When GTP hydrolyzed → GDP then the G-protein is inactive G protein-Coupled Receptors - Structure • Receptor = integral transmembrane protein ▪ spans the membrane 7 times • 3 protein subunits: α, β, γ ▪ Unstimulated: α is bound to GDP, and βγ is bound to α ▪ Stimulated: α subunit releases GDP, replacing it with GTP and the α subunit disengages from the βγ subunits ▪ When the α unit hydrolyzes GTP → GDP, it becomes inactivated again ▪ The βγ subunit can also sometimes activate signals Molecular Biology of the Cell, 6th ed. Alberts et. al. p. 818, fig 15-6 Gs GPCR 1. A ligand binds to a receptor associated with a Gs Gprotein 2. Gs releases GDP and binds GTP at the alpha subunit ▪ The βγ subunit detaches from the G-protein 3. Gs binds to and activates adenylyl cyclase ▪ Membrane-bound enzyme that converts ATP to cAMP 4. cAMP binds to protein kinase A (PK A) ▪ Binds to inhibitors of PK A, which then detach, and allow the active parts of PK A to work 5. PK A phosphorylates a multitude of effector proteins Gs GPCR • Common motif in signal transduction – activation of signals happens when inhibitory influences are “released” ▪ Release of inhibitors from PK A ▪ Allowing calcium to move down its concentration gradient, from storage to cytosol • Next class we will focus on regulation of gene transcription Molecular Biology of the Cell, 6th ed. Alberts et. al. p. 836, fig 15-27 cAMP? • Here we see the conversion of ATP to cAMP ▪ Enzyme – adenylyl cyclase • Inactivation of cAMP results when it is converted to 5’-AMP ▪ Enzyme – cyclic AMP phosphodiesterase ▪ The “turning off” process Molecular Biology of the Cell, 6th ed. Alberts et. al. p. 834, fig 15-25 G-protein mechanisms • There are 2 other G-protein mechanisms that we will look at in more detail: ▪ Gi, Gq • These signaling mechanisms are present very many cell types and modulate a huge number of cellular responses • They all follow a very similar pattern, though: G-protein activated → 2nd messenger increases or decreases → modulation of an effector that responds directly to the 2nd messenger (2nd messenger effector) → the 2nd messenger effector modulates the activity of other effectors ▪ Exception – sometimes βγ subunits activate effectors on their own, without “using” a 2nd messenger GSubunit Subunit Activity protein (impact on 2nd messengers) Gs Gi Gq Gt Biochemical Effects A Few Biologic Impacts α Stimulates adenylyl cyclase → cAMP production cAMP activates PK A → phosphorylation of effectors Glycogenolysis, thyroid hormone synthesis… many α Inhibits adenylyl cyclase → decreased cAMP production Decreased PK A activation βγ Activates K+ channels* More negative cell membrane potential α Activates phospholipase C → IP3 and DAG production IP3 → calcium release from ER DAG → activation of PK C α Activates cGMP phosphodiesterase → decreased cGMP Decreased cGMP → closes Na+ channels → more negative cell membrane potential *No 2nd messenger involved Inhibition of above Reduction of heart rate Detection of light – rod photoreceptors in retina The Gq GPCR • Unique – uses Ca+2 and IP3 and DAG as 2nd messenger systems ▪ IP3 is a 2nd messenger that has one effect → it causes release of Ca+2 from where it’s stored in the endoplasmic reticulum (ER) ▪ Ca+2 has many effects – it can bind to and activate a number of proteins → modulation of a very large range of effectors ▪ Good example of a Ca+2–binding protein: calmodulin Molecular Biology of the Cell, 6th ed. Alberts et. al. p. 841, fig 15-33 Ca+2 –calmodulin interactions • A “resting” cell (no calcium signal) has 0.1 micromolar of Ca+2 in the cytosol, and 1 – 3 mmol of calcium in the endoplasmic reticulum and in the extracellular space ▪ The concentration gradient for is very high → it “wants” to enter the cytosol ▪ A cell that is “activated” can reach Ca+2 concentrations of 10 micromolar or more (100X increase) • When the concentration of Ca+2 increases in the cytosol, then it will bind to calcium-binding proteins in the cytosol → an effect Ca+2 and calmodulin • Each calmodulin binds to four Ca+2 ions before it becomes activated • Once calmodulin is activated, it can bind to effectors ▪ i.e. calmodulin kinases Molecular Biology of the Cell, 6th ed. Alberts et. al. p. 841, fig 15-33 The Gq GPCR Molecular Biology of the Cell, 6th ed. Alberts et. al. p. 838, fig 15-29 Gq GPCR 1. A ligand binds to a receptor associated with a Gq Gprotein 2. Gq-alpha activates phospholipase C 3. Phospholipase C cleaves a membrane lipid into IP3 and diacylglycerol (DAG) ▪ Membrane lipid = PIP2 ▪ IP3 is water soluble – it enters the cytosol ▪ DAG is lipid soluble – it stays within the cell membrane and diffuses throughout it Gq GPCR 4. IP3 activates a Ca+2–release channel in the ER → movement of Ca+2 from ER into the cytosol 5. Both Ca+2 and DAG work together to activate membranebound protein kinase C (PK C) ▪ Why do you think that PK C needs to be membranebound? 6. PK C (the 2nd-messenger-activated effector) can modulate the activity of many other effectors… ▪ … and Ca+2 can also bind to other molecules such as calmodulin Gi GPCR • This is a ubiquitous inhibitory G-protein that downregulates the activity of Gs ▪ Gi-α inactivates adenylyl cyclase ▪ Gi-βγ opens a K+ channel • Opening of K+ channels brings the cell closer to its Nernst potential for K+ ▪ -90 mV is the Nernst potential for K+ ▪ This is very negative membrane potential – it tends to cause most cells to be “less” activated, as we will see later Receptors coupled to ion channels • Recall that: ▪ Due to the activity of the Na+/K+-ATPase and K+ channels, the cell membrane is inside-negative with a low cytosolic concentration of sodium and a high concentration of K+ ▪ Therefore, sodium wants to diffuse into the cell if a channel for sodium opens • What would be the impact on the membrane potential if sodium enters the cell? Molecular Biology of the Cell, 6th ed. Alberts et. al. p. 818, fig 15-6 Receptors coupled to ion channels • Recall that: ▪ Calcium concentrations inside the cell are almost 10,000 times lower than outside the cell at rest ▪ What would happen to the cytosolic calcium concentration if you opened a channel that allowed calcium to cross the cell membrane? Molecular Biology of the Cell, 6th ed. Alberts et. al. p. 818, fig 15-6 Receptors coupled to ion channels • Many receptors can open ion channels after they bind a ligand (first messenger) ▪ If the channel allows sodium to enter → the membrane becomes more “inside-positive” • This is known as depolarization ▪ If the channel allows more potassium to leave → the membrane becomes more “inside-negative” • This is known as hyperpolarization ▪ If the channel allows calcium to enter the cytosol → binding to calmodulin • Making the membrane more inside-positive or more insidenegative impacts the activation of certain membraneassociated proteins ▪ We will discuss the impact in the post-learning Molecular Biology of the Cell, 6th ed. Alberts et. al. p. 818, fig 15-6 The Enzyme-Coupled Receptors Molecular Biology of the Cell, 6th ed. Alberts et. al. p. 818, fig 15-6 Enzyme-coupled receptors • Transmembrane proteins with ligand-binding domain on outer surface of the plasma membrane ▪ Usually only 1 transmembrane domain • Cytosolic domain has either: ▪ Intrinsic enzyme activity • Most common class is receptor tyrosine kinases • We will focus on these for this video ▪ A direct association with an enzyme • For next class Receptor Tyrosine Kinases • Intrinsic kinase activity – i.e. the receptor phosphorylates itself on specific residues of the intracellular face of the receptor • Binding of ligand dimerizes the receptor and activates a tyrosine kinase within the receptor ▪ Phosphorylation by the receptor on its own tyrosine residues activates the receptor → further signaling • Ligand examples ▪ Insulin ▪ Growth factors ▪ Cytokines Receptor Tyrosine Kinases (RTKs) 1. Ligand binds to receptor monomers → 2. Receptor dimerizes and each half phosphorylates the tyrosine residues on the other half 3. Signaling proteins then bind to the phosphorylated receptor and also become activated → signal cascade Molecular Biology of the Cell, 6th ed. Alberts et. al. p. 855, fig 15-44 RTKs – signaling options • Phospholipase C ▪ Same as for Gq • Ras cascade: 1. Ras is a small, intracellular G-protein that is not physically associated with any one receptor – when it encounters an activated RTK, it binds to GTP → activation 2. Ras activates Raf – another small plasma membraneassociated G-protein 3. Activated Raf → activation of MAP kinases • These can phosphorylate transcription factors, enzymes… many effectors 4. Ras inactivates itself by cleaving GTP → GDP (it’s a Gprotein) The RTK → Ras → MAP K cascade Molecular Biology of the Cell, 6th ed. Alberts et. al. p. 855, fig 15-47 The RTK → Ras → MAP K cascade Molecular Biology of the Cell, 6th ed. Alberts et. al. p. 855, fig 15-47 The RTK → Ras → MAP K cascade The Ras-Raf-MAP kinase pathway is the pathway most commonly associated with RTK activation • ** Note – no typical 2nd messengers are produced in this pathway Key pathway for many growth factors Molecular Biology of the Cell, 6th ed. Alberts et. al. p. 855, fig 15-47 RTKs – signaling options PI-3-Kinase (PI3K) → Akt system • Unique signaling mechanism that is key to insulin signaling ▪ Also a wide range of other hormones/growth factors • Basic pathway: 1. RTK is activated, and this causes activation of nearby phosphoinositide-3-kinase (PI3K) • Ras can also directly activate PI3K 2. PI3K attaches an additional phosphate to PIP2 to form PIP3 • Remember – PIP2 is a membrane lipid (about 5% of membrane lipids) 3. PIP3 accumulates and forms “lipid” rafts in the membrane • PIP3 is the 2nd messenger in the system Membrane phospholipids and signaling All of these are variablyphosphorylated phospholipids in the cell membrane • PLC converts PIP2 to IP3 and DAG ▪ Gq activation • PI3K converts many phospholipids to PIP3 RTKs – signaling options • Basic pathway cont… 4. Akt and PDK1 accumulate and cluster together at the site of the PIP3 rafts • Akt and PDK1 are both kinases that are present in the cytosol • PDK1 becomes activated by PIP3 5. When PDK1 is activated, it activates Akt by phosphorylating it • PDK1 = phosphoinositide-dependent kinase 1 6. Akt is the effector – it influences a huge range of intracellular targets • It is also regulated (turned on or off) by many other cellular signals PI3K → PDK1 → Akt • Note how PIP3 brings PDK1 and Akt together at the membrane, as well as acting as a second messenger • https://www.cellsignal.com/contents/science -cst-pathways-pi3k-akt-signalingresources/pi3k-akt-signaling-interactivepathway/pathways-akt-signaling • Interactive diagram that highlights the massive impact of Akt activation… ▪ And also illustrates how Akt is the target of many, many other signals ▪ Take a quick look at the cellular effects (the text not in “bubbles”) • FYI Nitric oxide – a unique messenger • Nitric oxide (NO) is a key mediator that relaxes smooth muscle in a wide variety of blood vessels and visceral organs • Very small, hydrophobic gas that diffuses easily and quickly through cell membranes ▪ Thus can mediate signals within the cell it is produced… ▪ OR can diffuse to another cell • Produced enzymatically by the action of nitric oxide synthase (NOs) on L-arginine ▪ Increases in cytosolic calcium can activate NOs • Nitric oxide is degraded rapidly – less than a minute – since it reacts with oxygen and water ▪ It’s a second messenger – one of the only ones that can diffuse across the cell membrane and impact other cells ▪ Only local effects – it’s a quickly-degraded free radical Nitric oxide-mediated signaling 1. Cytosolic calcium increases ▪ How can we increase cytosolic calcium? 2. Increased intracellular calcium activates NOs 3. NOs produces NO from L-arginine 4. NO binds to and activates guanylyl cyclase (GC) → production of cGMP from GTP ▪ cGMP is also a second messenger 5. Elevations in cytosolic cGMP activates a protein kinase (usually PKG) → changes in cellular activity due to PKG activity ▪ In many cases, results in disengagement of myosin from actin in smooth muscle → smooth muscle relaxation