BMB 462 Lecture 7 Outline of Last Lecture I. Continuation of Primary Active Transporta. P-Type Continuedb. F-Type and V-Type ATPasec. ABC TransportersII. Secondary Active Transporta. Lactose Permeaseb. Na+/Glucose SymporterIII. Signal Transductiona. General Featuresb. General Processc. Types of Receptorsd. Example: Beta- Adrenergic ReceptorOutline of Current Lecture I. Continued Analysis of the Beta-Adrenergic Receptora. Review of Structureb. Mechanismc. TerminationII. Different Signal Transduction CascadesIII. G Protein InhibitorsIV. Multivalent ProteinsV. Insulin Regulation of Gene ExpressionCurrent LectureConcepts to remembers from previous courses/lectures:- The structure of ATP (adenosine, sugar, and 3 phosphate groups)I. Continued Analysis of the Beta-Adrenergic Receptora. Review of StructureThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.i. Beta-Adrenergic Receptor has 7 transmembrane helices, a hormone binding pocket facing the extracellular environment, and is associated with the G protein GS1. GS has 3 subunits – alpha, beta, and gammab. Mechanismi. A hormone binds to the hormone-binding pocket, causing a conformational change that makes the receptor protein shift.a. GDP leaves the associated GS protein, allowing GTP to bind,which activates the switch1. Once the hormone binds, and the conformation shift occurs, Gαβγ dissociates from the receptor protein2. Gα is palmitoylated (it is attached to a 16:0 fatty acid) while Gγ is prenylated (it is attached to an isoprene); Gγ and Gβ remain associateda. Linking the G proteins to lipids cause the subunits to diffuse only in the plane of the membrane3. The lipid-linked proteins have a hydrophobic lipid portion in the membrane that keeps them associated with the membranea. When the hormone binds to the receptor, you activate the G protein (which has GTP bound to it) and the α subunit dissociates from the βγ subunits i. Βγ subunits stay associated to the membrane, which means the 3 subunits can only move in a 2D plane (they can’t move out into the cytosol) which increase the probability of beneficial interactions 1. The α subunit is more likely to hit Adenylyl cyclase and interact with it if it’s associated with the membrane than diffusing through the cytosol4. Lipid rafts further cluster components (i.e. signal transduction components) so the GS protein doesn’t have to diffuse around the entire cells to find thema. i.e. Adenylyl cyclase is likely close to the GS protein-receptor complex, so the G protein can find it more quickly.c. Signal Termination – if a signal is activated, the cell needs ways to terminate it:i. G proteins – these proteins are very important in many signal transduction pathways1. They are a large family of small proteins that bind GTP and slowly hydrolyze it to GDP + Pi (they’re also known as GTPases)a. The most famous example is Ras, a protononcogene that converts to oncogene and causes cancer.2. G proteins serve 2 functions:a. Switch – releasing GDP and binding GTP turns G proteins on (makes them active). They are turned off by a timing mechanismb. Timer – GTPase activity hydrolyzes GTP to release Pi; GDP isnow bound, making the protein inactive3. Most G proteins are pretty bad at releasing GDP to bind GTP; they need help from GEFs (Guanisine nucleotide Exchange Factors; a receptor) which aid in releasing GDP4. Kcat = # of reactions/second an enzyme can catalyze; this value is usually 1000s-100,000s of reactions/seconda. The Kcat for G proteins, however, is about 5 reactions/minute; they are really bad enzymes, which is good because it gives them a period of time when they’re active before they shut themselves off.i. Activity is moderated by GAPs (GTPase Activator Proteins) that speed up GTP hydrolysis; this allows the cell to control how long the switch is on.ii. Phosphodiesterases1. Phosphate is attached to the 3’ and 5’ Carbons of active cAMP; in order to stop the signal, you have to remove the Pi from cAMP2. Phosphodiesterases hydrolyze the 2nd messenger cNMP (i.e. cAMP or cGMP) back to NMPa. It uses water to dephosphorylate to NMP in order to stop the signaling moleculeb. There are some specific phosphodiesterases for cAMP, and some for cGMP*Many drugs work by inhibiting different components of signal transduction. This resultsin changing what’s going on in the cell (which is useful therapeutically)cGMP is really important in controlling smooth muscle contractionsIncreasing cGMP increases male arousal (Viagra inhibits phosphodiesterase, which increases the levels of cGMP; it was originally going to be a medicine to regulate blood pressure)- Continuing types of signal termination -iii. Desensitization1. In addition to just terminating the signal and shutting off the pathway, you sometimes need to regulate pathway sensitivitya. Desensitization is more refined than turning the complete pathway offb. i.e. In eyes: when you go into bright light the eyes desensitize and don’t absorb as much light; when you go into the dim room, the eyes re-sensitize so they can absorbmore lightc. i.e. When there is a lot of adrenaline in the blood, and still binding to receptors:i. GSα & GSβγ are almost never bound. But GSβγ recruits the βARK protein, a receptor kinase1. βARK phosphorylates the receptor which gives it a lower affinity to epinephrine; epinephrine is now bound for a shorter period of time, which means less signaling2. After phosphorylation, βARR (β-Arrestin) is attracted to and binds to the phosphorylated C-terminus (it needs a Pi present to bind)a. This creates a receptor-arrestin complex which gets endocytosed into the cell so that there are fewer receptors available on the cell surface to bind the signalb. This results in a reduced sensitivityii. To restore sensitivity, the receptor-arrestin complexslowly dephosphorylates so βARR is no longer bound and the receptor can return to the plasma membraneII. Different Signal Transduction Cascades (Mix and Match components)a. Other G proteins: there are potentially over 1000 types; the cell creates this by mixing and matching componentsi. GSα – activates adenylyl cyclaseii. GIα – inhibits adenylyl cyclase (and is associated with another complex)iii. GQα – activates phospholipase C.iv. Transducin (receptor cells in eyes) – activates phosphodiesterases to break down cGMPb. Other 2nd Messengersi.
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