* Neurons can change their state (e.g. which receptors, channels, neurotransmitters are opened, modulated, or expressed) depending on what is going on in their local environment
* They receive signals from other neurons (neurotransmitters) and other cells (hormones, growth factors, and trophic factors)
* They have specialized machinery that can transduce these signals to changes in their physiological state.
Note:
Today we take a broad overview of signal transduction pathways that work to change the physiological state of neurons. Many of the pathways and second messengers should be familiar to you from basic cell biology.
* Ligand gated ion channels (channel linked receptors/ionotropic receptors)– e.g. nAChR, AMPA receptors
* Enzyme linked receptors– typically have extracellular binding site for signals. Has intracellular domain with catalytic activity regulated by signal. Most are protein kinases that phosphorylate intracellular proteins. e.g. tyrosine kinase
* G-protein coupled receptors– 7-transmembrane spanning receptors that signal through trimeric G-proteins intracellularly. The proteins can alter the function of many downstream proteins. e.g. muscarinic AChR, metabotropic glutamate receptors
* Intracellular receptors– activated by cell permeant or lipophilic signaling molecules like steroid hormones. Signal binds directly to an intracellular protein which then activates transcription
For enzyme linked receptors the signal binds extracellularly, which activates the intracellular enzymatic domain of the same protein catalyzing the production of a product from a substrate.
For g protein coupled receptors, the signal binds to the receptor, then the g-protein binds and becomes activated.
For intracellular receptors, the signaling molecule passes through lipid membrane, binds to the intracellular receptor and activates the receptors which can then enter the nucleus to regulate transcription.
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## Downstream of activated receptors: G-proteins
* G-proteins– GTP binding proteins
* G-proteins generally couple the active receptor to downstream targets. Called G-proteins because they hydrolyze GTP
* Two types of G-proteins:
* Heterotrimeric G- proteins, composed of an α,β, γ subunits. Multiple members of each class. α subunit binds and hydrolyses GTP
G proteins couple receptor activation to downstream effects for G-protein coupled receptors.
They hydrolyze guanine triphosphate to guanine diphosphate so that downstream proteins can become phosphorylated and activated.
There are two types…
heterotrimeric composed of three distinct subunits. It is the alpha subunit that binds to the guanine nucleotides GDP and GTP.
binding of GDP allows the alpha subunit to bind to the beta and gamma subunits to form an inactive trimer. Binding of the extracellular signal to the receptor allows the g-protein to bind the receptor and GDP to be replaced with GTP. Then the alpha subunit with GTP is free to dissociate from the trimer and bind downstream effector molecules to mediate a host of responses inside the cell.
The monomeric GTPases also relay signals from membrane receptors to intracellular targes like the cytoskeleton. Ras is the first small G protein discovered (rat sarcoma tumors). Helps regulated cell differentiation and proliferation, relaying signals from receptor kinases.
Rate of GTP hydrolysis is important property of G-protein mediated signaling and can be regulated by proteins like GAPs (or GTPase activating proteins) that replace GTP with GDP to return G proteins to their inactive form.
* - Guanosine-5'-triphosphate (GTP) is a purine nucleoside triphosphate.
* - Effector enzymes for activated G-proteins include adenylyl cyclase, guanylyl cyclase, phospholipase C, and others.
* - In some cases G-proteins can directly modulate ion channels. mAChR that slow heart rate from vagus nerve stimulation are thought to be due to beta/gamma G protein subunits binding to and modulating K channels. Alpha subunits of g proteins can lead to rapid closing of voltage-gated Ca and Na channels.
<figure><img src="figs/MolBiolCell-4e-Fig-15-28_bb1fb6c.png" height="300px"><figcaption>Molecular Biology of the Cell 4e Fig. 15.28</figcaption></figure>
* Ion channels– can be directly-activated by both the βγ subunits (can gate some types of K⁺ channels) or by α subunits (can cause closing of voltage sensitive Na⁺ and Ca²⁺ channels)
* Enzymes that produce 2nd messengers– e.g. adenylyl cyclase, guanylyl cyclase, and phosopholipases
* Each 2nd messenger does different things
* Wide diversity of physiological responses
Note:
In some cases G-proteins can directly modulate ion channels. mAChR that slow heart rate from vagus nerve stimulation are thought to be due to beta/gamma G protein subunits binding to and modulating K channels. Alpha subunits of g proteins can lead to rapid closing of voltage-gated Ca and Na channels.
Effector enzymes for activated G-proteins include adenylyl cyclase, guanylyl cyclase, phospholipase C, and others.
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## Effector pathways associated with G-protein coupled receptors
There are many types of alpha, beta, and gamma g-protein subunits allowing a specific and diverse range of downstream responses.
This shows three examples of different heterotrimeric g proteins bound to 3 types of receptors with 3 different cellular responses.
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## Second messengers: calcium
* Maintained at low concentrations inside cytosol
* Binds to many proteins and regulates their activity
* Calmodulin– binds Ca²⁺ and then can activate calmodulin dependent protein kinases
* IP3 receptors– channel that lets calcium out of ER
Note:
Maybe the most common intracellular messenger in neurons.
One target of calcium is calmodulin, a calcium binding protein abundant in the cytosol of all cells. Calcium binding to this protein initiates downstream effects by binding to targets like protein kinases.
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## Proteins involved in delivering and removing calcium to the cytoplasm
ATPase called the calcium pump (Ca-proton pump). Works on cell membrane and also pumps calcium into intracellular organelles like ER and mitochondria.
Na/Ca exchanger that replaces intracellular Ca with extracellular sodium ions.
VGCCs
calcium binding effector proteins like calmodulin mediate downstream effectors of calcium.
calcium binding buffer proteins serve as calcium buffers (calbindin, common in strongly expressed in some neuron subtypes). Can blunt the magnitude and kinetics of calcium signals.
Channels that allow Ca to be released from the the interior of the ER like the inositol trisphosphate receptors (IP3). These are regulated by IP3, a second messenger.
Another one intracellular releasing channel is the ryanodine receptor. These are activated by cytoplasmic Ca and for at least muscle cells, membrane depolarization.
<figure><img src="figs/MolBiolCell-4e-Fig-15-40_8aee979.png" height="400px"><figcaption>Molecular Biology of the Cell 4e Fig. 15.40</figcaption></figure>
<figure><img src="figs/MolBiolCell-4e-Fig-15-31_f75639e.png" height="300px"><figcaption>Molecular Biology of the Cell 4e Fig. 15.31</figcaption></figure>
<figure><img src="figs/MolBiolCell-4e-Fig-15-35_466d627.png" height="400px"><figcaption>Molecular Biology of the Cell 4e Fig. 15.35</figcaption></figure>
Second messengers regulate neuronal functions by modulating the phosphorylation of intracellular proteins. This addition and removal of phosphate groups rapidly and reversibly modulates protein function.
Phosphorylation is carried out by protein kinases.
Phosphate groups are removed by phosphatases.
Protein substrates of kinases and phosphataes include enzymes, neurotransmitter receptors, ion channels, structural proteins.
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## Regulation of cellular proteins by phosphorylation
* PKA– cAMP dependent protein kinase. Ser/thr kinase. Tetramer of 2 regulatory and 2 catalytic subunits. cAMP binds the regulatory subunits causing the release of catalytic subunits
* CaMKII– Ca²⁺/calmodulin-dependent protein kinase. Ser/thr kinase, very abundant in brain. 12 or so subunits. Downstream targets: many ion channels, other signal transduction proteins, tyrosine hydroxylase. Thought to be involved in learning/memory
* PKC– Ser/thr kinase activated by DAG and Ca²⁺. DAG causes PKC to move from the cytosol to the membrane where it binds Ca²⁺ and gets activated
<figure style="margin:15px 0;"><figcaption class="big">binding of cAMP to regulatory subunits free up the catalytic subunits</figcaption><img src="figs/Neuroscience5e-Fig-07.09-1R_copy_9cfd048.jpg" height="100px"><figcaption>Neuroscience 5e Fig. 7.9</figcaption></figure>
<figure style="margin:15px 0;"><figcaption class="big">binding of calmodulin opens up protein to activate catalytic domain</figcaption><img src="figs/Neuroscience5e-Fig-07.09-2R_copy_d343a3d.jpg" height="100px"><figcaption>Neuroscience 5e Fig. 7.9</figcaption></figure>
<figure style="margin:15px 0;"><figcaption class="big">DAG causes PKC to change its localization which leads it to be active</figcaption><img src="figs/Neuroscience5e-Fig-07.09-3R_copy_e5e5f7e.jpg" height="100px"><figcaption>Neuroscience 5e Fig. 7.9</figcaption></figure>
<figure><img src="figs/MolBiolCell-4e-Fig-15-32_607bbe8.png" height="300px"><figcaption>Molecular Biology of the Cell 4e Fig. 15.32</figcaption></figure>
* Protein tyrosine kinases– Two types receptor tyrosine kinases (Eph receptors, growth factor receptors) and cytoplasmic kinases (many oncogenes). Cytoplasmic tyrosine kinases are particularly important for cell growth and differentiation
* MAP kinases– mitogen activated kinases. Are often intermediate kinases, become activated by kinases and kinase other proteins. Often found downstream of receptor tyrosine kinases
Note:
Mitogen activated protein kinases (MAP kinases)
* first identified as having a role in cell growth
* also called extracellular signal regulated kinases (ERKs).
* normally inactive in neurons, but activated when phosphorylated by other kinases
* part of kinase cascades.
* activation can be triggered by extracellular growth factors that bind receptor tyrosine kinases that activate monomeric G proteins like ras.
<figure><img src="figs/MolBiolCell-4e-Fig-15-56_d3306b4.png" height="300px"><figcaption>Molecular Biology of the Cell 4e Fig. 15-56</figcaption></figure>
* >estrogen receptors are largely located in the cytosol. Hormone binding to the receptor triggers a number of events starting with migration of the receptor from the cytosol into the nucleus, dimerization of the receptor, and subsequent binding of the receptor dimer to specific sequences of DNA known as hormone response elements.
>upstream activating sequence or upstream activation sequence (UAS) is a cis-acting regulatory sequence. It is distinct from the promoter and increases the expression of a neighbouring gene.
-upstream from minimal promoter TATA box, binding site for transactivators
-a cis acting regulatory sequence (like IRES)
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## CREB
* CREB (cAMP response element binding protein). An important transcription factor
* Normally bound to DNA but not active. Phosphorylation activates it and it activates transcription. CREB is important for transcription of tyrosine hydroxylase, neuropeptides, neurotrophins and channel proteins
* Important for learning and memory, mothering instincts, synaptic plasticity
Can result from strong synaptic stimulation at cerebellar purkinje neurons or from weak synaptic stimulation in the hippocampus.
* Both parallel fibers and climbing fibers must be simultaneously activated for LTD to occur. With respect to calcium release however, it is best if the parallel fibers are activated a few hundred milliseconds before the climbing fibres.
LTD is thought to result mainly from a decrease in postsynaptic receptor density,
likely from phosphorylation of AMPA receptors by PKC and their elimination from the synapse and involves mapk cascade
* Hippocampal/cortical LTD can be dependent on NMDA receptors, metabotropic glutamate receptors (mGluR), or endocannabinoids.[4]
* LTP involves
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## Regulation of tyrosine hydroxylase by protein phosphorylation
