BISC 421 1st Edition Lecture 10 Outline of Current LectureI. Neurotransmitters and their receptors continuedCurrent LectureNeurotransmiters and Their Receptors (continued) •Two diferent categories of receptors: these are the G protein coupled receptors•Many of these receptors use the same neurotransmitter-‐ main thing to take away from this •You have metabotropic and ionotropic receptors for the same neurotransmitterLargest family of cell surface receptors. 5% of the genes in the nematode C. elegans. Over 1000 involved in the sense of smell alone. Signaling molecules range from proteins to small peptides, amino acid derivatives, and fatty acids. Despite their diversity, all have the same general structure. Span the membrane 7 times. Called serpentne receptors.N-terminus is extracellularC-terminus is intracellularC2, C3, and C-terminal tail interact with G-proteins.Their activation does not directly lead to changes in membrane potential.Work via altering the activity of effector proteins. Ion channels, enzymes, etc.Activate intracellular proteins called trimericG-proteins.Bind guanine nucleotides.•The receptors themselves: bind the ligand. These are GPCRs and one of the largest families of receptors•5% of C. elegans genes are for G protein coupled receptors-‐ very large group•Wide arrange of signaling molecules-‐ many ligands that can bind these, many diferenttypes•Despite diversity all pretty much have the same shape•Given the name serpentine receptors because one extra transmembrane domain (cross7 times) – but DO NOT make channels•A lot of the conservation in sequence and structure is in the transmembrane regions because it doesn’t need to be diferent-‐ what is important for transmembrane domainsis that they are hydrophobic•What makes them diferent is the amino terminus and the c terminus regions of theprotein. Amino terminus is the binding region (green part). C terminus is the G protein binding site (blue). This is where the diversity in the structure lies depending on the types of proteins that are binding•By a ligand binding it will not directly lead to a change in membrane potential, but theyare modulatory proteins-‐ efector proteins•Efector proteins can be enzymes, can modify ion channels, etc•Modify by transduction: ligand binds and activated G proteins binding to receptor on intracellular side and these G proteins are trimeric and bind guanine nucleotidesComposed of 3 subunits: D E, and J.Both GD and GJ are linked to the membrane covalently attaching to membrane lipids. During signaling, the E and J subunits are attached and referred to as the GEJ subunit.In the resting state, GD has a GDP bound and is associated with GEJ. Receptor activation binds to GD and allows the release of GDP. GTP then binds.•Structure of the trimeric G protein: alpha beta and gamma proteins come together•Both alpha and gamma are linked to the membrane but not transmembrane•Within the alpha subunit is a guanine nucleotide binding region-‐•Whole role of the receptor is to determine whether the alpha subunit is bound to GTPor GDP•Three basic players: receptor, trimeric G protein, and efector (enzyme, ion channel etc)o 1. Inactive receptor with ligand binding domaino 2. Once receptor binds to ligand, becomes activated and recruits trimeric G protein (in inactive state bound to GDP). Now these units bind and cause conformation change in the protein which makes it difficult for alpha subunit to bind GDP, dissociating GDPo 3. Now ready to bind GTPo 4. Binds GTP which activated G protein and triggers dissociation of alpha subunit from beta gamma and alpha subunit is free to find the efector it is supposed to efecto 5. Binds efector protein and activates it•As long as bound to GTP it will stay active, if GTP hydrolyzed to GDP then becomes inactive. Determination of how long active is how long bound to GTPFluorescent techniques have measured the time course of G-protein activation. Fluorescence resonance energy transfer. (FRET) GD-subunit fused with CFP. GE-subunit fused with YFP. When in close proximity, emitted light from CFP excites YFP. As these protein move apart, the fluorescence decreases.•How fast does this happen?•Relatively slow-‐ happens in the course of seconds•Can use fluorescence to look at proteins associated•Know that activation of G protein separates the alpha protein from the beta gamma •FRET technique can show how the proteins in a cell are movingChimeric proteins have been used to determine which domains of the receptors interact with the G- proteins. D2 and E2-adrenergic receptors. Each couples with a different GD- subunit. Demonstrated that the C3 loop mediates the association with the G- proteins. Similar approaches suggest that the C2 loop, between TM3 & 4 may also play a role in binding.•Where in the protein is there interaction?•Two types of g protein coupled receptors – both adrenergic-‐ a2 and B2 have diferent functions because bind to diferent proteins-‐ one Ga protein is meant to inhibit adenylyl cyclase and the other one stimulates it•Made chimeric proteins-‐ took amino part of a2 and linked it to carboxy part of B2-‐ whathappened-‐ found that bound to Gas-‐ realized which part binds it • Made another chimeric farther to the carboxy side, found that region that is efecting must be in the yellow region (the region that is binding to the G protein)Example of differential effects of neurotransmitters and distinct receptors. Cardiac ACh receptors. Muscarinic ACh receptors. Binding of ACh slows the rate of contraction. Coupled to Gi. In this case, the GD-subunit is not the regulator. The GEJ-subunit complex is GEJ directly interacts with a K+-channel and activates it.•One example of where a g protein can afect a K+ channel excitability• Muscarinic ACh receptors are coupled to a Galphai but upon binding of the ACh to the receptor it activates the G protein to activate the K+ channels in the muscle making it more difficult to contract (activation of K+ will inhibit muscle contraction)• Small molecule neurotransmitterso Small vesicleso Amino acidsGlutamate-‐ excitatory Acetylcholine Glycine-‐ inhibitory  GABA-‐ inhibitoryo ATPo Catecholamineso Histamineo Dopamineo epinephrine•Peptideneurotransmitterso Methiolnine• All of the diferent neurotransmitters and their post synaptic efect (most common)•ACh-‐ excitatory but