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Transcript
BIOL 201: Cell Biology and Metabolism WEEK 11 Lipid Soluble Hormones: Hormones are molecules that are made by cells that act through specific receptors and confer information to a target cell so that it responds correctly to some stimulus Different Types of Intercellular Signaling: Endocrine Signaling: Have a cell that secretes a hormone, which is released into circulation and acts on cells far away (global signals) Paracrine Signaling: Have a cell that secrets a hormone which acts on a nearby cells Autocrine Signaling: Cell releases a hormone that acts on itself. Typical of a lot of tumor cells that produce growth factors, inducing there growth Signaling between Attached Receptors: The receptors interact between two cells, and are important for organizing a cellular response in all the cells in a given tissue Intracellular Signal Response Systems in Cells: Receptor signaling pathways are so few, but yet carry out most of the diversity that is seen in tissues in humans A number of the receptor signaling pathways are conserved among eukaryotic organisms Pathways work from receptors all the way to changes in transcription o Respond to the extracelluar environment, go to the nuclease and change the genetic output Going to focus on the G-Protein Coupled receptors Lipid Soluble Hormones – Steroids: They don’t require receptors on the surface of the cell, but because they can go through the cell membrane, can interact with intracellular receptors (can be in the cytoplasm or in the nucleus) When these molecules interact with the receptors, there ultimate change will be at the level of the genetic output Steroid Hormone Structure: They are all based on a cholesterol backbone They are very hydrophobic They interact with receptors that have similar structures Have major effects on growth and behavior Nuclear Hormone Receptors: Maintain same structure: o Variable N-Domain, always followed by DNA binding domain, then followed by ligand domain The top 3 are initially found in the cytoplasm (Plus androgen) The bottom 3 are always located in the nucleus (Plus Vitamin D) Steroid Hormone Receptors always homodimerzie Nuclear Hormone Receptors act as heterodimerzes The Zinc Finger Motif: When it forms a homodimer, forms a point of symmetry Steroid Hormone Receptors: The way the steroid hormone receptors bind to the DNA, they need to bind to Reverse Palindromes Nuclear Hormone Receptors: These receptors interact with direct repeats in the DNA Mechanism of Steroid Hormone Activity: o The hormone receptors are always found in the cytoplasm when they are not activated o There is an inhibitor protein HSP 90. It binds to the ligand-binding domain of the receptors in the cytoplasm, until the hormone comes in. When it comes in, it binds to the LBD, and causes a conformational change in the receptor protein which kicks out the HSP 90 o This then allows the receptor to go into the nucleus o When its in the nucleus, finds homodimers, binds to the DNA, and the activation domains will induce RNA Pol II transcription complex to turn on the genes Mechanism of Nuclear Hormone Activity: Under no ligand circumstances, the receptors bind to their specific genes but block the transcription of those genes As soon as the hormone comes in and acts with its appropriate receptor, it turns on the transcription of the gene by increasing the levels of Histone acytliation (when there is no hormone, recruits histone deacetlase, keeping the chromatin condensed) RAR Receptor Function in Development: Retinoic Acid can lead to posterior changes in the spinal chord to take on a more anterior structure if taking at the inappropriate times (causing deformities) Environmental Mutagens Mimic RA Activation: Tadpoles and frog development are sensitive to environmental conditions Turns out that the sunlight leads to the production of Methoprenoic Acid by acting on an insecticide that found its way into the water leading to deformities o This can bind to Retinoic acid receptors because they have similar structures, leading to growth G-Protein Coupled Receptors and cAMP: Dissociation Constants (K0) And Binding Assays: An easy of way to quantify a number of receptors on a cell. Take the known ligand and combine it with a radioactively labeled form of the ligand With the mixture, can introduce into a cellular environment, wash away the stuff that didn’t bind, and get an idea of the number of counts that bind to the cell. Will give you an idea of how much radiolabled ligand is bound to the receptors. It is an indicator of all the known labeled ligand bound Get Total Binding. However, a lot of binding is Nonspecific Binding o Subtraction of Total Binding and Nonspecific Binding give a curve for Specific Binding: Can say how many moles of receptor are on the surface Must repeat the experiment with lots of none-labeled ligand This count will give the affinity of the ligand to receptor R + L RL: Largely depending of the affinities of the two molecules: If half the binding sites are occupied, the [Free Receptors] = [RL]. Therefore, the [L] = to KD Signaling Through Cell Surface Receptors: Most signaling happens when a hormone is released, it eventually interacts with a receptor that is specific. This receptor then illicits a response in the cell. Has to transfer the signal into the cell. Does this by interacting with intracellular molecules (Second Messenger Molecules, where the first messenger is the ligand) Activation of the receptor give rise to short term and long term responses Afterwards, the receptor can’t always be activated. Therefore, there is a means to regulate the receptors activity, getting rid of the ligand in the receptor Second Messengers – Intracellular Mediators of External Signals: They are activated downstream of the receptor-ligand binding. They vary a lot: o Cyclic AMP, Cyclic GMP, Diacylglycerol, Inositol 1,4,5Trisphosphate G-Protein Coupled Receptors (GPCR): Starts with the binding of epinephrine to its receptor The receptor for epinephrine is made up a membrane spanning (7) receptor. Its NTerminal is in the extracallular space, C-Terminal domain in the cytosol Has loops on intracellular space: They interact with Heterotrimeric G-Proteins o They are important for downstream transduction signaling There are non-covalent interactions of the amino acids in the transmembrane domain Mechanism of Action of GPCR: The G-Proteins consists of 3 subunits: o G-Alpha o G-Beta o G-Gamma Epinephrine interacts with the G-Protein coupled receptor. Immediately, G-Alpha recognizes a major conformation change that is in the receptor itself G-Alpha is in a GDP bound state. When it interacts with the activated receptors, it undergoes its own conformational change, kicking out GDP from the binding site o Activated receptor acts as a Guanine Exchange Factor This allows GTP to interact with G-Alpha. When GTP binds, the G-Protein falls apart, whereas Beta/Gamma stick together. The 2 complexes move away. GAlpha finds an Effector Protein (Adenylyl Cyclase for epinephrine). Interacts with this protein and modifies it so that there is a change in its activity The Effector Protein will carry out the appropriate downstream event that are typical of this receptor (activating the formation of second messengers (cAMP for epinephrine) or directly effect ion channels) o Also acts as a GAP Helps G-Alpha protein to hydrolyze GTP back to GDP, therefore inactivating GAlpha, so that it can recomplex with G-Beta/Gamma, forming the Heterotrimeric Complex Different effector proteins for all the different hormones/receptors Cyclic AMP – Regulation of Protein Kinase A: Cyclic AMP is formed from the activity of Adenylyl Cylase: Converts AMP to Cyclic AMP This can then interact with Protein Kinase A, and activates it o PKA has 2 Catalytic subunits and two regulatory subunits. For every regulatory subunits that it has, 2 cAMP binds. Therefore, when cAMP binds it releases the Catalytic subunits, which are the activated Protein Kinase A PKA then phosphorylates a number of intracellular targets Protein Kinase A Affects Gene Expression: PKA makes its way into the nucleus, and phosphorylates key transcription factors (CREB), which gives rise to long term changes by changing transcription of its specific genes G-Protein Activity Regulation: The receptor acts as a GEF for the G-alpha The Effector protein acts as a GAP for G-Alpha G-Protein Switching Mechanism: There are two key amino acid residues that interact with the gamma phosphate of GTP: Gly-60 and Thr-35. They change the conformation of the protein Bidirectional G-Protein Effects: End up altering the conformation of the Heterotrimeric G-Protein such that you end up stimulating Adenylyl Cyclase (Stimulatory G-Alpha) In some cases, get a different situation, where you don’t have a Stimulatory active G-Alpha, but an Inhibitory active G-Alpha. Instead of G-Alpha stimulating the effector protein, it inhibits it G-Protein Function and Glycogen Metabolism: GPCR – Other Effector Proteins: GPCR can also affect ion channels using an alternate mechanism Acetylcholine binds, causing conformational change. This is transduced by G-Beta/Gamma They translocated, and their effector proteins are Ion Channels, activating and opening them GPCR – Other Effector Proteins: Another mechanism is involved in vision This happens in Rod cells (help in low light vision) There are a number of molecules: Rhodopsin Opsin combined with 11-cis-Retinal form Rhodopsin (the protein that responds to photons) o Will respond to even a single photon. The photon causes the conformational change. It turns 11-cis-Retinal to an all-trans-Retinal o This conformational change causes a major change in Rhodopsin. The change in Rhodopsin is transduced by another Heterotrimeric G-Protein complex; G-Alphat (Very specific) o Following activation of the Rhodopsin receptor, end up chaning the conformational. G-Alphat becomes activated, transloacting through the membrane, interacting with Cyclic GMP Phosphodiesterase o CGMPP: Converts Cyclic GMP (second messenger) back to GMP. In doing this, it inactivates the second messenger o Under normal circumstances, the membrane in the cells are depolarized in dark situations. When you shine light on them, leads to lower level of cGMP. This shuts of the cGMP gated ion channels, hyperpolarizing the cell. The brain then interprets this as light Intracellular Amplification: One single molecule of activated Rhodopsin on the membrane can single up to 500 molecules of G-Alphat Once this is activated, can make major changes in the cell It is a typical way of how G-Protein Coupled Receptors leads to rapid and major changes within the cell from a little amount of hormones Functional Role of G-Protein Regulators: Want to ensure that you can turn off all the amplification signaling after the stimulus is terminated The GAP activity is a major regulator There are molecules that help get back to the ground state Very hard to get back into the ground state if these are knocked out Cyclic AMP – Synthesis and Breakdown; Phosphodiesterases are another way that help get back to the ground state Turn the Second messenger back to a deactivated form. Work on cAMP or CGMP o cAMP to AMP by cAMP phosphodiesterase Protein Kinase A is Localized to the Nuclear Membrane of Heart Muscle Cells: In the heart, bring PKA in close proximity to the phosphodiesterase so that you can: o Maintain PKA close to where it acts o To help bring back to ground state quickly (Turns it off quickly) G-Protein Regulators: GPCR gets activated, and there are Protein Kianses that recognize that it gets activated. These phosphorylate the C-Terminal loop on the cytosol site. This is recognized as a binding site for Beta-Arrestin. It interacts with this region and AP2 and Clathrin, which are important for endocytosis The receptors become endocytosis so they are no longer involved in signaling (can then either go back or be degraded) It is all dependent on Beta-Arrestin Second Messengers – Intracellular Mediators of External Signals: Diacylglycerol (DAG): Membrane bound Inositol 1,3,5-triphosphate (IP3): It is fully diffusible Formation: Phosphatidylinositol goes through phosphorylations that make it become very energetic. Gives rise to PIP2. It is a major substrate of Phospholipase C. This cleaves it to DAG and IP3 Ca2+ are another second messenger o Induces secretion of cargo from hormone containing vesicles in neuroendocrine cells o Enhances neurotransmitter release from neurons o Binds to EF/Hand protein Calmodulin to affect protein targets Binds to 4 molecules of calcium, causing a major conformational change, allowing it to interact with protein effectors DAG and IP3 effects are associated with changing the levels of intracellular Ca Regulation of Calcium Levels in Cells and G-Protein Activation: Get an interaction with a hormone and a receptor causes a conformational change in Phospholipase C (the Effector Protein) through G-Alpha. This leads to DAG and IP3 IP3 will open up ion channels, changing the conformation, causing Ca to leave the Endoplasmic Reticulum When the intracellular levels of Ca increase, the calcium binds to Protein Kinase C. This will go to the membrane, and interact with the DAG that is up there. This interaction will activate PKC. The PKC will then interact with a number of important targets leading to: cell growth, cell division and changes in cytoskeleton This is seen a lot in the Pituitary Calcium and Fertilization: When sperm fertilizes the egg, leads to a spike in calcium, filling the occytes