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Lecture 22: "GPCR Functions"

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Biology 2382B
Robert Cumming

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Cell Biology Lecture No. 22: GPCR Functions st Monday April 1 , 2013 LECTURE 21 CONT’D Hypothetical Signal Transduction Pathway: -A simple signal transduction pathway involving one kinase and one target protein has the receptor tightly bound to a protein kinase that, in the absence of a bound ligand, is held in the inactive state. Ligand binding triggers a conformational change in the receptor, leading to activation of the appended kinase. The kinase then phosphorylates the monomeric, inactive form of a specific transcription factor, leading to its dimerization and movement from the cytosol into the nucleus, where it activates the transcription of target genes. A phosphatase in the nucleus will remove the phosphate group from the transcription factor, causing it to form the inactive monomer and then move back into the cytosol. Switching Mechanism Of G Proteins: -GTPase switch proteins cycle between active and inactive forms. The switch protein is active when it has bound GTP and inactive when it has bound GDP. Conversion of the active into the inactive form by hydrolysis of the bound GTP is accelerated by GTPase-Accelerating Proteins (GAPs) and other proteins. Reactivation is promoted by Guanine Nucleotide Exchange Factors (GEFs) that catalyze the dissociation of the bound GDP and its replacement by GTP. The ability of a G protein to interact with other proteins and thus transduce a signal differs in the GTP-bound "on" state and GDP-bound "off" state. (a) In the active "on" state, two domains (switch I and switch II) are bound to the terminal gamma phosphate of GTP through interactions with the backbone amide groups of a conserved threonine and glycine residue. When bound to GTP in this way, the two switch domains are in a conformation such that they can bind to and thus activate specific downstream effector proteins. Release of the gamma phosphate by GTPase-catalyzed hydrolysis causes switch I and switch II to relax into a different conformation, the inactive "off" state (in this state they are unable to bind to effector proteins). G Protein-Coupled Receptor System: -The G Protein-Coupled Receptors (GPCRs) system is the most numerous class of receptors in animal cells. A complex of trimeric G-proteins is usually coupled to these receptors, which have 7 membrane- spanning domains. The GPCR system is important because it affects so many processes (e.g. vision, smell, development, metabolism, etc.). Certain pathogens will exploits this system and can cause various diseases, which is why most drugs are designed to target these G Protein-Coupled Receptors. G Protein- Coupled Receptors have the same orientation in the membrane and contain 7 transmembrane domains (α-helical regions), along with extracellular and intracellular domains important for ligand and G-protein interactions respectively. The Trimeric G Protein: -The trimeric G protein is a complex of three protein subunits (G , G , and G ). The G is the subunit that α β γ α binds GTP, while the associated protein subunits G and G βre geneγally regarded as one unit that can dissociate from the G suαunit. The G and Gαsubunitγ are lipid-anchored proteins at the cytoplasmic face of the plasma membrane and G is onlyαactive when bound to GTP (inactive when bound to GDP). This trimeric G protein complex essentially acts as an intermediary molecule between the GPCR and the effector protein. Activation Of Effector Proteins Associated With GPCR: -The G αnd G β/γsubunits of the trimeric G protein complex are tethered to the membrane by covalently attached lipid molecules. Following the ligand (hormone) binding to the GPCR, this induces a conformational change in the now active receptor and allows it to bind with the G subunit. The binding α of the activated GPCR to the G subαnit induces a conformational change in it and triggers the dissociation of GDP from its binding cleft. The later binding of a GTP molecule to the empty cleft on the G αubunit causes the dissociation of the G and Gα β/γsubunits and the free and activated G -GTP αinds to and activates an effector protein. The hydrolysis of GTP terminates this short-lived state as well as signalling and leads to the reassembly of the trimeric G protein complex (returning the system to the resting state). The binding of another ligand molecule causes repetition of the cycle. In some pathways, the effector protein is actually inhibited by the G subαnit and activated by the free G β/γsubunit. Demonstrating Receptor-Mediated Dissociation Of G & G : α γ -Fluorescence Energy Transfer (FRET) is useful in helping demonstrate the receptor-mediated dissociation of G aαd G β/γsubunits, where cAMP acts as an extracellular signalling molecule by binding to GPCR. Cells were transfected with genes encoding two fusion proteins: a G fused to cαan fluorescent protein (CFP) and a G β/γfused to yellow fluorescent protein (YFP). When CFP and YFP are nearby, as in the resting trimeric G protein complex, fluorescence energy transfer can occur between CFP and YFP. As a result, irradiation of resting cells with 440 nm light (which directly excites CFP but not YFP) causes the emission of 527nm light, characteristic of YFP. However, if cAMP binding leads to the dissociation of the G αnd G β/γsubunits, then FRET cannot occur. In this case, irradiation of cells at 440 nm causes emission of 490-nm light, characteristic of CFP. The drop in yellow fluorescence, which results from the dissociation of the G -αFP fusion protein from the G -YFP β/γion protein, occurs within seconds of cAMP addition. Major Classes Of Trimeric G Proteins: -The fact that there are many different types of subunits forming the trimeric G protein complex and that they are alternatively spliced for expression in different tissues shows the broad range of functions attributed to this GPCR system. It’s also important to know that some G subunits are stimulatory when α binding to effector proteins (G ),αshile others are inhibitory (G ). αi LECTURE 22 + Activation Of Muscarinic Acetylcholine Receptors & K Channels In The Heart: -Binding of acetylcholine to GPCR
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