Enzyme Linked Receptors and and Signalling Through Proteolysis PDF

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notes on lectures about enzyme linked receptors, inlcudes notes on lectures about signalling through proteolysis. lectures by Sandip Patel...

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Enzyme Linked Receptors and and Signalling Through Proteolysis

Week 5 Guanylyl cyclases Receptor guanylyl cyclases are activated by natriuretic peptides. ANP(atrial) and BNP(brain) both of these involved in sodium homeostasis (as you can tell from the name natriuretic) structural similarity to tyrosine kinases also in dimers have an extracellular domain that binds their ligand have an enzymatic domain (cyclase) like the RTK this means that like the RTK there is activation of the enzyme upon binding of the signalling molecule These receptors work through the 2nd messenger cyclic GMP activation of these receptors produces cGMP from GTP this is analogous to adenylyl cyclases forming cAMP from ATP seen here GTP can be involved in switching or it can be involved in forming a second messenger

As mentioned before, receptors can be present on the inside of a cell

shown is the idea that receptors can also be on the inside of a cell and are not limited to just being on the cell surface

There is a second form of guanylyl cyclase that is soluble and are within the cell these can also produce cyclic GMP through its catalytic domains which take GTP only thing it is lacking is the extracellular domain that would anchor it to the cell surface with this domain lacking these are therefore soluble activated by nitric oxide NO which binds to the heme binding domain and causes conformational changes that result in the production of cGMP NO is a gas which is different to all the signalling molecules mentioned before due to it being a gas it can readily pass through the plasma mem NO is very unstable and short lived meaning the signalling using NO is very localised as it only happens at the site where NO is produced therefore cGMP can be produced 1 of 2 ways  firstly by receptor guanylyl cyclases (activated by natriuretic peptides)  the 2nd way is through soluble guanylyl cyclases (activated by NO Signalling needs to be transient so how is this second messenger turned off? cGMP is degraded by phosphodiesterase back down to GMP

again this is analogous to cAMP which is degraded by its phosphodiesterase back down to AMP

Further analogies between cGMP and cAMP a further analogy can be seen between these 2 when we look at how they are mediated in the previous lectures it was mentioned that cAMP was mediated through the protein kinase PKA which is composed of regulatory and catalytic subunits and once cAMP binds to it these catalytic subunits dissociate these go on to phosphorylate substrate proteins cGMP effects mediated by its own protein kinase: protein kinase G PKG which itself is composed of a regulatory and catalytic domains once cGMP binds to this the catalytic domain is activated and can go on to phosphorylate things the only difference with this and PKA is that the regulatory and catalytic domains on PKG are all on the same subunit and do not dissociate cGMP/NO signalling regulates smooth muscle relaxation shown is a nerve being stimulated and producing acetylcholine and which triggers the production of NO as it is a gas it can easily escape the endothelial cell and enter the neighbouring smooth muscle

cell once the NO enters the smooth muscle cell it activates the soluble guanylyl cyclase causing production of the second messenger cGMP cGMP binds to its protein kinase activating its catalytic activity which causes the smooth muscle cell to relax does this through regulating processes which decrease the Ca ion conc within the cell (as learned before increasing calcium levels causes contraction, conversely decreasing levels causes relaxation)

Effects of inhibiting the phosphodiesterase as a reminder the phosphodiesterase is what degrades the cGMP here the phosphodiesterase is PDE5 inhibiting this enzyme's activity with something like a drug would increase the levels of cGMP in the cell as there is nothing to degrade it this would therefore increase the activity of the cGMP protein kinase which means that the Ca ion concs would further decrease this leads to the relaxation of the smooth muscle cell this process is what viagra does - inhibits PDE5 causing the relaxation of the smooth muscle in the blood vessels of the penis allowing for blood flow which leads to an erection Receptor serine/threonine kinases as the name suggests these receptors have serine/threonine activity - phosphorylating these substances like other receptors mentioned - these have a extracellular binding domain which allows the ligand to bind this initiates a cascade that first begins with the activation of the kinase domain

generally 2 types of receptor I and II Best studied are receptors for Transforming growth factor β superfamily

TGF β receptors TGF β receptors are activated by ligands of the TGF β superfamily all of the TGF β superfamily are secreted proteins Examples: TGF β Activin Bone morphogenetic protein BMP these secreted proteins are generally active during early development Activated TGF β receptors signal through smad proteins upon first binding of the ligand the type II receptors phosphorylate the neighbouring type I receptors the type I receptors then go on to phosphorylate the smad proteins which are associated with the type I receptor

Different TGF beta ligands phosphorylate distinct smad proteins TGF and its receptor phosphorylate smad2 and smad3 whereas BMP and its receptor phosphorylate smad1, smad5 and smad8 after this there is convergence the phosphorylated smad proteins then go on to associate with smad 4

this newly formed complex then goes on to enter the nucleus and activate their target genes this is a transcriptional pathway this is a slow process compared to the electrical signalling in the nerve cells and again is key during early development

TGF β signalling is important during mesoderm induction as mentioned in previous lectures FGF signalling (in tyrosine signalling) is involved in forming the early layers such as the mesoderm TGF beta is also active during this process which is key to development FGF and TGF beta work together in the formation of this layer

Signalling through proteolysis pathway is known as wnt signalling named after the activating stimulus which is known as wnt which is a secreted protein like the TGF beta superfamily this secreted protein can signal through 3 distinct pathways activated by the wnt ligands  Canonical pathway.  Planar cell polarity PCP pathway.  Ca2 ! pathway. however we are only going to consider the canonical pathway The main proteins in the Canonical Wnt signalling Wnt stimulus binds to the receptor of Frizzled and Lrp5/6

one of the key effector proteins in this pathway is dishevelled at the bottom of the cascade is a transcription factor known as beta catenin so therefore this is a slow event like the serine/threonine kinases also involved in embryo development and regulating gene expression

Canonical wnt signalling seen on the surface are the transmembrane protein receptors where the stimulus (wnt) is binding this then initiates a cascade that ends at the nucleus where it is seen beta catenin goes into the nucleus and regulates gene expression what is different about this pathway is that beta catenin in the resting state is being degraded

Canonical pathway - resting state this is in the absence of the wnt ligand as mentioned beta catenin is actively degraded this happens through its formation of a complex with 4 proteins knowns as the 'destruction complex' the 4 proteins are  Axin  APC  GSK3  CK1α in this complex beta catenin is phosphorylated which leads to its active degradation (proteolysis) therefore there is no transcription of genes

the protein 'groucho' also prevents transcription during this resting state which binds to the genes and acts as a inhibitor Canonical pathway - active wnt binds to frizzled but it also binds to LRP5/6 which is a coreceptor this binding activates dishevelled dishevelled then recruits axin (which is part of the destruction complex) therefore there is no degradation of beta catenin as axin has been taken from the destruction complex so levels of beta catenin increases beta catenin then goes into the nucleus and displaces groucho to activate transcription turning the genes on Importance of wnt signalling there is a gradient of wnt ligands within the embryo which produces signalling of different extents within the embryo it is important in defining parts of the embryo and what they will subsequently become wnt signalling also important for adults as it is responsible in remodelling the nerve circuits

Hedgehog signalling - also involves proteolysis like the wnt pathway - also named after the stimulus (hedgehog) and is also a secreted protein so quite similar to the wnt pathway 3 types of hedgehog proteins:  sonic  indian  desert key proteins of the hedgehog pathway

hedgehog is the ligand which binds to the receptor patched the key effector protein is smoothened also involves a transcription factor cubitus interruptus or Gli in mammalian systems same as the wnt pathway these t.factors are actively degraded in the absence of the ligand but accumulates and regulates gene expression when the signalling pathway is activated Hedgehog signalling regulates polarity of segments in the embryo shown in the slide is the wild type embryo to the left looks normal with its various spaced out segments once the gene for hedgehog is disrupted (on the right) the embryo looks much more compact and loses its various segment

Practical calcs: Osmolarity is dependent upon the number of impermeant molecules in a solution, not on the identity of the molecules. For example, a 1M solution of a nonionizing substance such as glucose is a 1 Osmolar solution; a 1M solution of NaCl = 2 Osm; and a 1M solution of Na2SO4 3 Osm. So in our example, the osmolarity of the 0.9% NaCl solution is 0.15M * 2 = 0.3 Osm. Osmolarity is calulated in 2 steps: Ex. for 0.3% NaClMolarity of NaCl = 0.3/100ml) / (molecular weight of NaCl) Osmolarity = 2 * Molarity of NaCl...