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Lecture 29

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McGill University
Biology (Sci)
BIOL 300
Siegfried Hekimi

th BIOL 300 November 16 2012 Lecture 29 Dr. Shock G-protein coupled receptors (GPCRs) are seven-pass TM receptors; these receptors can be hormones, neurotransmitters, light (rhodopsin), odorants and act largely physiologically • While most receptors are dimer, all GPCRs are monomers • On the extracellular side is the ligand- binding domain and on the intracellular part is the domain which will interact with a G-protein G-proteins are trimeric proteins, i.e. they contain three subunits: • The alpha subunit is a GTPase • The beta and gamma subunits are structural, always bound together • The alpha subunit will join them once it is modulated in some way by an effector protein through binding of the ligand to the GPCR • Effectors are usually ion channels or enzymes that product second messengers (like cAMP, cGMP, DAG or IP3) In the resting state, when the hormone is not yet secreted, GPCRs are not ligand-bound and the signal transduction pathway is “off” • In this resting state, the G=protein is in a trimeric form where the alpha subunit is GDP bound Binding of the hormone to the GPCR will cause a conformational change which allows interaction with the G-protein • Both G-alpha and G-gamma are linked to the membrane by covalently attached lipids; a G- alpha mutated to lack the amino acid to which the lipid moiety is attached produces a null mutant (i.e. nonfunctional) When the ligand is bound, the GPCR can bind to the G-alpha which kicks out the GDP and activates the G-alpha subunits • Therefore, for this procedure, the GPCR could be considered as a GEF 1 th BIOL 300 November 16 2012 Lecture 29 Dr. Shock At this point, G-alpha will leave the G- protein because of a conformational change caused by binding of GTP, and will travel through the membrane to its target (the effector) As the hormone leaves the GCPR, G-alpha will bind to its effector, activating it. The GTP will quickly by hydrolyzed and the whole pathway will be shut down until G-alpha re-associated with the rest of the G-protein, and becomes activated by the GPCR once again. Mutagenesis of certain amino acids can produce certain phenotypes (like preventing hydrolysis). This can also be done with certain chemicals • There are chemicals which mimic the structure of non-hydrolyzable GTP which constitutively turn on these pathways by keeping the G- alpha active • Certain bacterial toxins, like the cholera toxin, are used to modify G-alpha in such a way to keep it in its GTP-bound form (i.e. always on) • This particular G-alpha this was used on opened sodium channels in the intestines, which released water, causing diarrhea There are different G-alpha subunits; some of them stimulate their effectors (like raising cAMP), while others inhibit their effectors, called a Gi(lowering cAMP) (don’t need to know table) • Therefore, you can have multiple responses from the same GPCR, or the same response from multiple GPCRs depending on the effectors • Also, G-beta-gamma can also active effectors in rare situations 2 th BIOL 300 November 16 2012 Lecture 29 Dr. Shock Scientists wanted to figure out which part(s) of the GPCR bound to the G-protein; this can be done by comparing two very closely related GPCRs, like the alpha-2 and beta-2 adrenergic receptors • The only difference between these receptors is that the alpha-2 binds an inhibitory G-alpha, reducing the amounts of cAMP in the cell, while the beta-2 binds a G-alpha that activates its effector, raising the amounts of cAMP • Other than this, the two receptors are very structurally similar They carried out a domain-swapping experiment to figure out which small region of the GPCR is responsible for binding G-alpha • This can be done by transfecting these receptors into cells; alpha-2 will have low cAMP and beta-2 will have high cAMP (which can be measured experimentally) • You can then make a clone where you combine half of the alpha-2 receptor with half of the beta-2 receptor and then transfect this into a cell • In this case, cAMP levels are very high, indicating that the part of beta-2 present in this chimeric receptor is responsible for binding G-alpha • This experiment can then be carried out by swapping out smaller and smaller regions, which narrows down the G-alpha binding domain to one intracellular loop between TM domains 5 and 6 This is a powerful and efficient experiment, but can only be used when you have two very similar proteins to compare and swap. The most famous GPCRs are those that modulate adenylyl cyclase, which modulates the amount of cAMP in the cell • This is involved in the fight or flight response, which is mediated by epinephrine (adrenaline), which binds to different GPCRs, most of them modulating adenylyl cyclase • They work by activating adenylyl cyclase to produce more cAMP, which activates protein kinase A (PKA) • Responses include: • Breakdown of glycogen in the liver (for bursts of energy) • Increasing heart contractions • Constricts arteries of non-vital organs (like the skin) to help supply more blood to the important ones • In general, makes you more alert On the top of this diagram, we can see the structure of epinephrine (or adrenaline), which is a ligand for these various GPCRs 3 th BIOL 300 November 16 2012 Lecture 29 Dr. Shock • The 4 drugs seen here are prescription drugs which can be administered in various scenarios; e.g. epinephrine is administered directly to the heart when you go into cardiac arrest to raise blood pressure • The other 3 drugs do not necessarily bind to the GPCR directly; their actions may not even be precisely know, we just know their effects • They all stimulate the fight or flight response • The first drug is very similar to epinephrine, just lacking two hydroxyl groups, and having an additional methyl group; its called ephedrine, commonly marked as sudafed • It’s an over-the-counter drug used to help with symptoms of a stuffy nose by constricting blood flow to the peripheral organs, mainly the nose, to lower inflammation (but also increases heart rate); they are also much weaker than epinephrine • Removing one hydroxyl group from ephedrine produces methamphetamine which is marketed as speed or crystal ice • This is an illegal drug • Chemically, since these two drugs are so similar, it’s actually not that hard to make methamphetamines from ephedrine • It’s much more powerful than ephedrine, but has mostly the same effects • MET was actually used commonly as a dieting drug in the 50s and 60s, but was reclassified in the 70s because people realized that it’s dangerous and has a high potential for addiction • You can get certain methamphetamines for certain rare diseases • Removing a methyl group fro
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