Fall 2010's Biochemistry Notes

Links:

exam 2
https://docs.google.com/document/d/1v-8292bbokdMypiTyumz0QJ9CW0ScJdeyyCSzgNGTOY/edit?hl=en_US&authkey=CL67zqUE

exam 3
https://docs.google.com/leaf?id=0B99-sSwVe231YWRmYzEzOWItNmNiOS00ZWQzLTgxODYtZjhiOWI3ZWYzOGQ1&hl=en_US&authkey=CPf78pwM

exam 4
https://docs.google.com/leaf?id=0B99-sSwVe231YjY1ZWQyMjctZjFmZS00MDBkLTkyNDgtZDFiYzA1YmJlMmQy&hl=en_US&authkey=CJjumagB

Japanese Cartoons in Biology

Remember Sonic Hedgehog protein?  Well, how about the Raichu probe?

Raichu stands for Ras interacting chimeric unit, where someone has attached a CFP to Raf to make a chimera protein (fusion protein).  Then, this Raf-CFP can can interact with Ras engineered with YFP.  Normally, Raf only binds to Ras-GTP, not Ras-GDP.  Bioengineers invented Raichu to be able to detect and localize Ras-GTP in action, using FRET.  When they use fluorescence to zap the cell with CFP's excitation wavelength, CFP is excited and sends out light at the CFP emission wavelength.  Conveniently, CFP's emission wavelength is YFP's excitation wavelength, so if Ras-CFP has a GTP and binds Raf-YFP, then your initial light excites CFP which excites YFP so you will see the color of YFP's emission, not CFP's emission. If you see that, that means Ras is bound very closely to Raf.

Raichu actually stands for Ras interacting protein chimeric unit, but it's not surprising they left out the "p" when making the acronym.  :P

Apoptosis video

Nice video for apoptosis, covers and LABELS all the proteins we learned about:

http://www.youtube.com/watch?v=hqhxnWty5jc

Book Notes: Cancer Part 2

• cancer critical genes: small subset of genes whose mutation usually causes cancer
• proto-oncogenes: genes who, when mutant, are overactive and drive cells to become cancerous
• tumor suppressor genes: genes who prevent cancerous cells (when mutant, they are inactive and allow cells to become cancerous)
• DNA maintenance genes: genes who, when mutant, are inactive and allow cells to become genomically unstable, which may or may not lead to cancer
• to test possible proto-oncogenes, you insert a copy of the oncogene (mutated version)
• if cells become cancerous, then the inserted gene really was an oncogene
• to test possible tumor suppressor genes, you have to knockout both copies of the gene in the cell
• as long as cells have one copy of a tumor suppressor gene, they can withhold the cell from abnormal growth
• tumor viruses: viruses who don't intentionally cause cancer, but by inserting their own genome into the DNA, causes a mutation that leads to cancer
• they are usually retroviruses, who carry RNA that is reverse transcribed into DNA and then inserted
• DNA decoding of transformed cells helped determine oncogenes
• tumor DNA is fragmented and put with fibroblasts that already divide a lot (but are still normal)
• if one of the fragment contains an oncogene, some of the cells will start forming masses: transformed!
• sequencing the genome of these transformed cells compared to the non transformed cells showed the oncogene
• Ras was the first, and is mutated in 1 of every 5 cancers
• a point mutation prevents it from hydrolyzing GTP, so it is hyperactive and never shuts off
• dominant effect: only one copy of the Ras gene needs to be mutated this way in order to produce an uncontrolled effect
• studying retinoblastoma, eye cancer, helped decode tumor suppressor genes
• there were 2 types of eye cancer, one hereditary, one just random.  The hereditary version impacted both eyes (so it must be a genomic problem in all the eye cells)
• they found a deletion on chromosome 13, where one copy of the Rb protein was missing
• it's hereditary because families inherit the one good copy and one bad copy, so as soon as the one good copy is mutated, they get retinoblastoma
• the non hereditary is random and usually in one eye because there must be 2 separate events that mutate both copies of Rb in one cell
• Rb regulates cell cycle, so mutated Rb allows inappropriate and uncontrolled cell proliferation
• usually tumor suppressor genes are identified by screening many cancers and finding out what gene is repeatedly missing or defective
• tumor suppressor genes can be inactivated in many ways
• chromosome deletion, point mutation, segregation/recombination errors, etc, etc
• loss of heterozygosity: where one copy of a cancer-critical gene is lost or deactivated, so now the cell has a predisposition to cancer (being cancer-prone)
• epigenetics: methylation of a tumor suppressor gene causes it to be stored in heterochromatin and silenced for all the progeny
• proto-oncogenes can be overactivated in many ways
• point mutation, deletion, gene amplification, chromosomal rearrangement that ends up altering a gene or the regulatory sequence of the gene
• 3 methods of cancer-critical gene identification
1. CGH (comparative genomic hybridization): DNA fragments from normal cells and from cancers cells are fluorescently labelled
• the labelled fragments hybridize to complementary sequences on a DNA microarray (where each spot corresponds to a known sequence of the genome)
• where the tumor complement binds, it means that many copies of the gene exist (gene amplification)
• where the normal complement binds, it means there was a deletion
• where there is no light, it means that the two copies of the genome exist and are paired up (blocking any probes from hybridizing)
2. DNA microarray can also be used with mRNA probes
3. RNAi: siRNA can be engineered to knockout a specific gene by destroying the mRNA of that gene or inhibiting the translation
• then you observe whether the knockout cell becomes cancerous or not
• best test involves doing knockouts or causing overexpression of a gene in actual mice
• mice that develop tumors soon after prove the possibility of that gene being a cancer-critical gene
• so what are the pathways in which these cancer-critical genes actually cause cancer?
1. cell proliferation pathways: just about any and every signaling protein can be affected, usually with proteins that activate or relay signals becoming hyperactive (proto-oncogenes) -- i.e. Ras, EGF receptor
• the inhibitors involved in pathways tend to be the tumor suppressor genes -- i.e. Rb
• cancer cells might eliminate Rb, or by inhibiting the inhibitor of the inhibitor of Rb (in times of stress, p16 blocks cyclinD/Cdk4 from inhibiting Rb, who inhibits E2F from continuing the cell cycle, SO, if a mutation inhibits p16, cyclinD/Cdk4 is free to inhibit Rb so that E2F happily progresses the cycle)
2. cell growth pathways: cancer needs to be able to grow uncontrollably, otherwise uncontrolled division will just lead to smaller and smaller cells
• PI3/Akt pathway important for growth, cancers activate this pathway in the absence of growth signals so they just grow all the time
• overactive Akt helps with protein synthesis, glucose uptake, and lipid synthesis
• so it's usually PTEN phosphatase who is messed up, because he's the inhibitor of Akt
3. apoptosis pathways: apoptosis usually activates in response to DNA damage and mutations, so because cancers need mutations, they must shut off apoptosis so they can keep proliferating
• Bcl2 is a protein that normally inhibits apoptosis
• in a B-lymphocyte cancer, the Bcl2 gene is translocated to an active promoter, so Bcl2 is overexpressed
• this keeps apoptosis from activating
4. DNA damage pathways: but really the star player is p53
• known as the guardian angel gene, p53 is super critical because it happens to be involved in 2 of the above 3 pathways, and in DNA damage systems
• to start, p53 is normally kept at very low levels: continuously made and continuously degraded
• when a cell is stressed or the genome is damaged, p53 levels shoot up
• active p53 (1) stops any cell dividing to buy some time (2) activates DNA repair systems (3) or activates apoptosis when nothing can be done
• this is the protection that prevents cells with just a few mutations from blowing over into cancer
• p53 is a gene regulatory protein, it activates expression of other proteins that carry out the above-listed functions
• mutation of p53 usually happens in its DNA-binding domains, so it can't bind to genes and activate transcription, and the cell can (1) divide uncontrollably (2) avoid apoptosis (3) have genetic instability w/o DNA repair and (4) ignore stresses like radiation and drug treatment
• DNA tumor viruses are the lysogenic viruses, who integrate their genome into the host genome and sit quietly
• virus genome contains stuff that take control of reproduction machinery to generate more viruses
• accidents can occur that activate some components of the virus genome that activate DNA replication and division, leading to tumor formation
• e.g. papillomavirus produces a protein E7 that blocks Rb, and an E6 protein that inhibits p53
• Metastasis has 2 steps: invasion and re-establishment
• EMT (epithelial to mesenchymal transition): normal organism development process where epithelial cells change from adhesive and stable to less adhesive and migratory
• this happens when E-cadherin is lost and cells can't form junctions, so they can go out exploring and getting into blood vessels
• colonization: cancer cells must acquire the ability to proliferate in a new environment in order to do this
• that's why most cancer cells that escape the primary tumor don't actually form new metastases
• CASE STUDY: COLORECTAL CANCER
• colon epithelium has rapid turnover rate, so it is constantly growing and dividing (at a controlled rate) to replace old cells
• renewal depends on special crypts (pockets) of stem cells in the epithelium
• benign tumors look like small protruding masses called polyps, the first sign of abnormal growth
• the most common mutations happen to 3 proteins: K-Ras, p53, Apc
• Apc is an inhibitor of the Wnt pathway but causing degradation of B-catenin (recall B-catenin can go in to the nucleus to activate transcription)
• but tumors tend to form faster with a hyperactive B-catenin rather than an inactive APC
• the high frequency of APC mutations comes from APC being involved in other pathways as well and getting mutated affects the mitotic spindle formation, so chromosome segregation is messed up
• one form of colon cancer is hereditary, due to a mutation in DNA mismatch repair
• the inherited predisposition involves having one messed up copy of the repair gene and one good copy
• once a mutation inactivates the one good copy, cells will start building mutations, increasing chances for cancer
• tumors often have specific order of mutations
• in most cases, Apc is one of the first to go
• mutations w/o Apc's guardianship will build up
• then Ras falls apart
• cells now proliferate w/o substrate or neighbor inhibition
• then p53 is messed up
• now cells can mutate faster and avoid apoptosis
• each tumor will mutate its own variation of similar genes (like messing up the Ras regulator as opposed to Ras itself), but the control mechanisms need to be disabled in a proper order
• even different patients with the same type of cancer will have different mutations, which makes cancer treatment very difficult
• cancer prevention or early diagnosis of a primary tumor is the best method of avoiding cancer
• treatment is difficult because you must remove ALL the bad cells, or else even just one cancer stem cell will regenerate a tumor
• surgery makes it difficult to get every tiny metastasis, and drug/chemical treatments also hurt good cells
• traditional cancer treatment exploits genetic instability
• radiation and drugs kill cancer cells because they can't repair the DNA damage, while normal cells will pause cycling (so you lose hair) but will fix the damage
• unfortunately, cancer cells can evolve resistance to treatment (like bacteria!)
• new treatments exploits CAUSE of genetic instability
• first, consider that there are multiple ways to repair DNA damage
• a single strand break is fixed by one system
• if that system is down, the stalled replication fork that results when DNA Pol encounters the ssbreak can be fixed by homologous recombination
• second, consider that cancer cells tend to lose some of the repair systems in order to become cancer
• for example, let's say a cancer cell can't repair a stalled fork, but it can fix ssbreaks
• third, consider that we can devise drugs to impair repair systems
• in the example with the cancer cell that can't repair stalled forks, what if we give it a drug that inhibits the repair of ssbreaks?
• now the tumor can't repair ssbreaks, and it already couldn't repair stalled forks, so it will die....
• BUT the normal cells live, because unlike the cancer cell, they CAN repair stalled forks, even if the drug also affects them and inhibits the repair of ssbreaks
• these treatments take advantage of the fact that normal cells have multiple options, while cancer cells have very few repair/survival options because of their mutations
• cancer cells have still another advantage: genomic heterogeneity
• even though they are all descended from the same ancestor, they have such a high rate of mutation that much of the population have different variations in their genome
• this means you can't just use one type of treatment to eradicate a whole tumor
• multidrug resistance: phenomenon where a cell, after surviving just one drug treatment, somehow is resistant to a bunch of other drugs that it's never met before
• this happens when a gene called Mdr1 is amplified (has multiple copies now)
• Mdr1 makes a transporter that pumps out drugs, overexpression of the protein makes sure no drugs hang around and damage the cell: they just get pumped out
• new therapies are being developed due to our increasing knowledge
• targeting the one hyperactive protein that a cancer may be dependent upon (oncogene addiction: the cell is addicted to/dependent on the oncogene)
• send in antibodies that either carry toxins or enzymes that cleave pro-drugs into toxins at the cancer site (because antibodies bind so specifically, you use anti-cancer antibodies)
• we can also engineer small molecules that target the one hyperactive protein of oncogene addiction
• e.g. CML (chronic myelogenous leukemia) is associated with Philadelphia chromosome
• this chromosome snaps the genes Abl and Bcr and rejoins them so they fuse into Bcr-Abl
• the fusion protein combines the Tyr kinase of Abl and the active expression of Bcr to make a hyperactive kinase
• this prevents apoptosis of unnecessary lymphocytes, so they build up in the blood
• a special molecule called Gleevec blocks Bcr-Abl, and effectively eliminates CML in early diagnosis patients
• less effective with later patients b/c cancers have usually evolved resistance to Gleevec binding
• probably going to have to do a cocktail to fully eradicate cancerous cells
• another approach is to target the tumor blood vessels
• there are drugs that inhibit the VEGF receptor
• also, endothelial cells that are about to make new blood vessels have distinguishable characteristics that we can engineer molecules to attack them specifically
• the final method is to enhance the existing immune system to target tumors
• we can tag cancer cells with antibodies to attract macrophages to gobble them up
• the future of treatment is probably going to involve a simultaneous combinations of drugs and molecules, because no cell will have resistance to all of them without some time to evolve (i.e. if we used the drugs one by one, every time, there would always be some cells resistant the single drug used)

pg. 1230 - 1240

Book Notes: Cancer Part 1

• normal cells work cooperatively, and obey signals and controls on division and growth so the organism can survive
• cancer cells work "selfishly", growing and dividing out of control, nabbing up nutrients and creating blood vessels so they can survive
• know the difference between BENIGN and MALIGNANT (is it invasive)
• several types of cancer by tissue of origin
• carcinoma: from epithelium, most common because epithelium is usually exposed to stresses and dangers (serves as protective barrier, making it vulnerable to damage and mutation)
• sarcoma: from muscle/connective tissue
• leukemia/lymphoma: from white blood cells, lymphocytes, and nerve cells
• cancer cells tend to be all clones of the primary tumor
• patients with CML, a leukemia with the mutation Philadelphia chromosome, show that the chromosome break happens at exactly the same place for all cells in the tumor, but it happens a few hundred basepairs different between patients
• this implies cells of one tumor all derive from one ancestral cancerous cell and are all clones
• cancer results from a genetic mutation, which may be from chemical carcinogens or radiation/UV light
• however, you need more than one mutation in ONE CELL LINE to have cancer
• organisms have a high overall rate of mutation, but that rate is including all the cells of a human (there are several trillion cells in the body, a human has about several trillion mutations over the lifetime, so it's like maybe one mutation per cell per day, and remember that repair machinery usually fixes the mutation)
• cancer usually happens in old age because that's when you've likely built up a lot of mutations and the tumor has had some time to grow into a visible mass
• e.g. leukemia didn't show up until 5 years after the atomic bombs razed Hiroshima, and lung cancer takes 20 years of heavy smoking before surprise!
• rule of thumb: minimum 5 mutations to become cancerous
• creating a mutant cancerous cell takes microevolution
• mutation rate: how fast does this kind of cell get mutations?
• number of reproducing individuals: how many cells in this tissue divide at a time?
• rate of reproduction: how fast can a cell divide and how many times can it divide?
• selective advantage: does the mutation kill the cell or give it a bonus or do nothing?
• you need several rounds of division (epithelial cells divide constantly) with advantageous mutations carrying through each step until you shake loose the body's regulatory systems
• cancer also involves epigenetic mutations
• epigenetics involving histone modification is a way of controlling which genes are active and which are silent
• mutations happen with the enzymes that modify histones, the proteins that interpret the histone code, etc et
• these mutations are also passed on to daughter cells
• because cancer cells proliferate uncontrollably, they are also genetically unstable
• it's usually because the cancers have a mutation in DNA repair/maintenance genes
• the increase in mutation rate plus their rapid division rate makes their evolution rate really fast--cancers with genetic instability speed rapidly towards malignancy
• cancer cells can also accumulate bad mutations that end up killing it, so it is the cancer cell with the right mutations that will persist into tumors and metastases
• the core mutations of cancer cells tend to be in control of cell death, control of cell differentiation, or both (for optimal cancer growth :D )
• any mutation or external condition that promotes cell growth can and will help cancerous cells
• normal tissue growth controlled by apoptosis, especially when apoptosis serves as a control to destroy mutated cells
• cells that don't do apoptosis when they're supposed to keep growing and passing on mutations, potentially forming cancer
• stem cells will produce daughter cells that proliferate for a bit before committing to differentiation
• if the differentiation step is blocked, daughter cells just keep dividing and dividing
• other core mutations are DNA damage response and other stress responses
• the DNA damage response goes hand in hand with the cell cycle checkpoints: mutations in either system won't be able to stop a damaged cell from continuing to divide and pass on the mutations
• being able to survive through these mutations makes them more undefeatable
• the final barrier to cancers is replicative cell senescence, when the telomeres run out, the cell would normally do apoptosis
• cancers can avoid the chkpt when telomeres run out and just keep dividing without telomeres (meaning every round shrinks the chromosome by a bit)
• cancers can also have a mutation that reactivates telomerase or a mutation that creates something similar to telomerase
• new theory suggests tumors are organized with cancer stem cells at the top and limited dividing lower cancerous daughter cells
• if the regular cancer daughter cell is implanted in a mouse, it can't generate a new tumor because it has limited dividing capacity
• only a small percent of cells in a tumor (<1) can propagate indefinitely
• where do cancer stem cells come from?
• (1) from real stem cells and (2) from normal proliferating cells that developed a mutation that makes them propagate indefinitely like a stem cell
• cancer stem cells divide slowly, so treatments that target rapidly dividing cells won't harm them, and then the tumor mass will regrow
• metastasizing cancer is the most dangerous, but it requires cancer cells to overcome some barriers first
• 1st, the tumor cells must invade neighboring normal tissue and keep spreading
• 2nd, the tumor cells must find a blood or lymphatic vessel and get in
• 3rd, the tumor cells must grab onto a new site while floating through the vessel and form a small clump (micrometastases)
• 4th, the clump must develop into a large tumor for stability
• many cells are usually able to get into the vessel, but very few can attach and colonize in a new location
• even forming micrometastases is no guarantee of continued survival
• metastasis usually requires a LOT of mutations in all the right places
• a large tumor also needs a supply of nutrients
• a tumor over 1-2 mm will need to induce angiogenesis: formation of new blood vessel
• normal and tumor cells secrete angiogenic signals in response to hypoxia (lack of nutrients and oxygen)
• these signals activate transcription and secretion of VEGF
• VEGF attract endothelial cells and stimulate growth of a blood vessel
• induced vessels are usually disorganized and go random places with dead ends
• this leaves a lot of area still in hypoxia, which causes natural selection for cells that survive in tough conditions, which makes cancer even tougher to eradicate
• stroma: the surrounding connective tissue, even tumor cells talk with them like normal cells
• stroma contains fibroblasts, white blood cells, etc, for support
• cancer cells send signals to the stroma
• the stroma responds with signals that stimulate growth and secrete proteases to loosen the ECM for invasion
• tumor and stroma evolve together (experiment where a tumor is plucked out and put next to normal fibroblasts showed the tumor can't survive)
• possible source of treatment: inhibit deranged stroma to kill tumors
• sum up of characteristic cancer behaviors:
1. survive and proliferate in weird conditions (not attached to substrate, etc, etc)
2. insensitive to anti-proliferation signals
3. avoid apoptosis
4. avoid stress and damage responses
5. induce help from stroma
6. induce angiogenesis
7. invade and proliferate far far away
8. genetically unstable
9. have stable telomeres

pg. 1205 - 1223

Book Notes: Signaling Part 5

• pathways involving gene regulatory proteins (who activate gene expression) depend on regulated proteolysis to control them
• Notch, Wnt, and Hedgehog are all these pathways, and they are especially involved in animal development
• Notch is famous for affecting neural cell development
• lateral inhibition: if one cell develops into a neuron, it signals to its neighbors to NOT being nerve cells and instead become epidermal
• this occurs because the developing neuron displays the Delta signal protein on its surface
• neighbors all have Notch receptors that bind Delta and receive the signal to develop into epidermis
• Notch signaling can also be used for promoting neighbors to become the same cell type in other cases
• when Notch binds Delta, a membrane-bound protease cleaves Notch's tail
• the tail zooms off into the nucleus and activates a set of genes by binding and converting a transcriptional repressor into an activator
• Notch is actually cleaved 3 times in its lifetime: (1) during synthesis, it is cleaved by Golgi to form heterodimer, (2) during ligand-binding, when the extracellular head is cleave off, and (3) after ligand-binding, when the cytoplasmic tail is cleaved off
• cleavage is irreversible, meaning Notch is a one-time use receptor
• Notch and Delta are glycoproteins, and their specific match-up depends on how their sugar sidechains are modified (glycosylation is important!)
• Wnt is famous for its versatility: it plays a role in catenins (i.e. jxns), cell polarity, etc, etc
• Wnt has 3 well-known pathways:
1. Wnt/beta-catenin pathway: Wnt affects the gene regulatory protein beta-catenin
2. planar polarity pathway: coordinate polarization in plane of epithelium
3. Wnt/Ca2+ pathway: stimulates increase of calcium level
• Wnt is a secreted molecule that binds to Frizzled receptors
• Wnt-bound Frizzled recruits Dishevelled, which then relays the signal down one of the 3 pathways
• for the beta-catenin pathway, Wnt binds to both Frizzled and an LRP (LDL-receptor-related protein)
• any beta-catenins not involved in jxn-forming is bound by degradation complex, which keeps it away from the nucleus and promotes its degradation
• 2 kinases (GSK3 and CKIgamma) phosphorylate C tail of LRP, which inactivates axin (part of the degradation complex), so free beta-catenin accumulates and migrates to the nucleus
• without Wnt, the set of genes controlled by Wnt are silent
• LEF1/TCF proteins bound to Groucho (a co-repressor)
• when Wnt activates, beta-catenin comes in and displaces Groucho as the co-partner
• Beta-catenin serves as a co-ACTIVATOR for the genes
• c-Myc is one of the proteins expressed by Wnt signaling, and it stimulates growth and proliferation (potential cancer!!!)
• Hedgehog is similar to Wnt: is secreted, modified by lipids, trigger switch from repression to activation, and leads to growth/proliferation response
• 3 transmembrane proteins mediate responses to the Hedgehog proteins:
• Patched: multipass with some of it in intracellular vesicles, and some on the cell surface to bind Hedgehog
• Smoothened: 7pass transmembrane
• iHog: 4-5 immunoglobulin domains and 2-3 fibronectin domains on cell surface, serve as co-receptors with Patched
• Patched normally sequesters Smoothened 
• Hedgehog binding to iHog partnered with Patched inhibits Patched by causing endocytosis and degradation of Patched
• result is that Smoothened is free and phosphorylated and continues the signaling pathway
• Cubitus interruptus (Ci): gene regulatory protein that is a target of Hedgehog
• in normal conditions, Ci is proteolysed into smaller proteins that hang in the nucleus as repressors
• this proteolysis is dependent on phosphorylation by PKA and GSK3 and CK1  (all Ser/Thr kinases)
• when Hedgehog binds, Smoothened runs free and recruits Costal2, a scaffold protein, and Fused, another Ser/Thr kinase
•  Costal2 normally holds the proteolytic kinases together to process Ci, but binding to Smoothened blocks this ability
• uncleaved Ci also goes to the nucleus, but acts as an activator
• uncleaved Ci can also express the Patched gene = more Patched inhibits further Hedgehog signaling

pg. 946 - 952

Book Notes: Signaling Part 4

• G proteins do more than play with cAMP and Ca2+
• the a subunit of G12 activates GEF for the Rho GTPase
• others bind and open/close ion channels to change membrane potential
• others phosphorylate channels or change levels of cNMP (e.g. cAMP) that affect channels
• olfactory receptors utilize cAMP-gated ion channels
• odor particle binds to olfactory receptor
• the receptor activates the G protein Golf (G subscript olf for olfactory)
• Golf activates adenylyl cyclase = lots of cAMP
• cAMP binds to cation channels that allow Na+ to flood in
• neuron containing the olfactory receptor is depolarized and sends a potential down the axon to the brain
• each neuron makes only 1 type of receptor that senses only a small set of odors
• visual receptors utilize cGMP-gated ion channels
• rod photoreceptors are cells with a rodlike outer and inner segment, followed by the body, followed by the axon and synapse
• the outer segment looks like a pancake stack with lots of rhodopsin molecules in them
• the plasma membrane coating the stacks have lots of cGMP-gated cation channels
• in the dark, cGMP remains bound to the channels, keeping them open
• in the light, the cis chromophore in the rhodopsin absorbs a photon and isomerizes into trans
• the changed chromophore causes the protein opsin to change conformation and activates the G protein transducin (Gt)
• transducin a subunit activates cGMP PDE, which removes cGMP
• visual system resets itself VERY QUICKLY so that we can see the change in light in the next instant
• rhodopsin kinase phosphorylates the tail of activated rhodopsin, to prevent further activation of Gt
• arrestin binds to Pi-rhodopsin to fully inhibit rhodopsin activity
• RGS acts as GAP to make Gt hydrolyze the GTP
• light also causes calcium channels to close, so calcium levels fall, so guanylyl cyclase hurries to make more cGMP, returning to normal "dark" mode
• negative feedback allows system to adapt to continuous light, and be sensitive to CHANGES in light (seeing camera flash in broad daylight)
• all the steps of intracellular signaling pathways are potential SIGNAL AMPLIFICATION steps
• but for rapid sensing, cells must be able to both amplify a signal quickly and destroy the response just as quickly
• therefore, any and every protein/molecule in the pathway can be a target for regulation
• GPCRs are usually desensitized in 3 ways
• receptor inactivation: receptor interaction with G protein is blocked
• e.g. GRK (GPCR kinase) like RK that phosphorylates GPCR after the receptor has been activated by ligand-binding
• then (as with rhodopsin) an inhibitor like arrestin binds and blocks the G protein interaction
• receptor sequestration: move receptor inside the cell to block access to ligand signal
• arrestin can also couple the receptor to endocytosis machinery and clathrin
• later dephosphorylation returns the receptor to the surface
• receptor down-regulation: receptor is destroyed post-activation
• endocytosis of receptor in some cases lead to the receptor ubiquitylation and lysosomal degradation
• EZCR (enzyme-coupled receptor): transmembrane receptor with a cytosolic domain that is an enzyme or associates with another enzyme (note, the abbreviation is my own invention)
• exist 6 classes of EZCRs
1. receptor Tyr kinase: phosphorylate Tyr residues on itself and some signaling proteins
2. Tyr kinase associated receptor: recruit cytoplasmic Tyr kinase
3. receptor Ser/Thr kinase: phosphorylate Ser/Thr on itself and some gene regulatory proteins
4. His kinase associated receptor: phosphorylate itself on His and transfers the Pi to a signaling protein
5. receptor guanylyl cyclase: catalyzes production of cGMP
6. receptorlike Tyr phosphatase: remove Pi from Tyr of signaling proteins (ligands are unknown)
• RTK (receptor Tyr kinase): bind hormones like insulin, growth factors, and ephrins (cell-surface ligands as opposed to free floating signals)
• ephrins and EPHrin receptors both make responses in the receptor cell and the ligand cell: "bidirectional signaling"
• ligand binding causes RTKs to dimerize and cross-phosphorylate each other
• the phosphorylated Tyr residues serve as docking sites for specific intracellular signaling proteins
• these signaling proteins are either activated by docking or by receiving phosphorylation from the RTK
• proteins dock by modular interaction domains (remember SH2, SH3, etc etc), sites of protein protein binding that don't affect function
• examples of docked proteins are PI3-kinase and PLCgamma (PLCbeta associates with GPCRs)
• adaptor proteins with lots of modular domains connect docked proteins to other proteins that DON'T have these awesome domains
• example being RAS
• Ras and Rho families are the only monomeric GTPase families to relay signals from surface receptors
• as a family, they can coordinate one signal to many pathways
• Ras has lipid groups to keep it anchored to the cytoplasmic leaflet of the membrane
• RTKs activate Ras through adaptors and Ras-GEF
• case study: fly eye is composed of ommatidia, which are sets of 8 photoreceptor cells and 12 accessory cells
• the Sevenless (Sev) mutant gene causes flys to be blind in UV light, because mutant Sev prevents normal development of the R7 photoreceptor
• BUT it turns out for some blind flies, the Sev gene is kept normal: it's the protein Bride of Sevenless (BOSS) who's mutant
• Boss is a 7pass transmembrane ligand on the R8 cell, and binds to the R7 Sev RTK (see, Sevenless is an RTK, a receptor.  BOSS is the ligand.  That's why it's "bride of Sevenless," because it binds to the Sevenless receptor)
• when they bind, the R7 precursor cell is induced to develop into R7
• even if other cells express Sev (which they do), only R7 touches R8, so only the Sev on R7 receives the ligand
• Steps to signal transduction
1. Boss binds Sev-RTK
2. Sev-RTK is phosphorylated and an adaptor DRK with an SH2 domain docks
3. a special Ras-GEF called Son of Sevenless (SOS) is also bound by DRK's 2 SH3 domains
4. SOS makes membrane-bound Ras get a new GTP and the activated Ras continues the signal with cytoplasmic free proteins
• see, SOS is Son of Sevenless because it's activated when Bride of Sevenless binds to Sevenless receptor = love child (haha, biologists are both bored and lewd)
• Now that Ras is active, where do we go next?
• Ras-MAP-kinase signaling pathway: Ras activates MAPKKK (Raf), who activates MAPKK (Mek), who activates MAPK (Erk), who activates gene regulatory proteins in the nucleus
• these gene regulatory proteins quickly turn on "early genes," who are part of the primary response
• early genes produce more gene regulatory proteins that go to activate later genes for the secondary response
• in this way, there is a rapid response and a more delayed response
• this pathway is how the signal goes from surface (where RTKs and Ras await) to the nucleus (where DNA and genes hide)
• lots of positive and negative feedback: MAP kinases activate both downstream targets and its own activators, BUT they also increases transcription of phosphatases and deactivate its own activators (Erk blocks Raf)
• different sets of 3 MAP kinases form MAP modules in different pathways
• however, many modules share one or more of the same kinases
• scaffolds prevent cross-talk by associating with the sensor, so that activated kinases that bind to the scaffold are automatically next to its downstream targets also docked to the scaffold protein
• this makes sure the activated kinase doesn't wander off and find other targets of other pathways
• Rho GTPases regulate the cytoskeleton (actin and microtubules) in response to special guidance receptors
• inactive Rho is bound to GDI, which prevents association with the GEF
• Eph RTKs are the receptors that activate surface-bound GEFs that activate Rho
• ephrins are the guidance ligands that bind Eph RTK
• e.g. most commonly used for directing axonal growth
• target cells express the appropriate ephrin on its surface
• the axon's growth cone has Eph RTK that binds ephrin
• Rho changes the cytoskeleton to grow in the direction of the bound ephrin to shift the axon thataway
• PI3-kinase is another protein that docks to RTK
• PI3 phosphorylates PIs, usually to make PIP3
• PIP3 floats around until the PTEN phosphatase turns it into PIP2
• PIP2 is nabbed by PLCbeta or PLCgamma and cleaved into IP3 and DAG for signaling
• RTKs activate PI3-kinase to make PIP3 available to make PIP2 available for signaling
• PIP3 have special PH modular domains (pleckstrin homology) that are super specific (because PIP3 and PI3 tend to be involved in growth and related pathways that are vulnerable to become cancerous)
• PI3-kinase-Akt pathway receives signals from IGF, the "SURVIVE AND GROW" signal
• RTK activates PI3 kinase, which makes PIP3, which recruits Akt and PDK1 (phosphoinositide-dependent protein kinase 1) to the plasma membrane
• Akt is activated there and phosphorylates many targets, producing a growth response
• e.g. one target is called Bad, which causes apoptosis, but phosphorylation of Bad makes it dock onto a scaffold protein, preventing its function
• a special TOR protein promotes ribosome production, protein synthesis, and nutrient uptake/metabolism, so it is activated by the Akt pathway for growth purposes

pg. 916 - 935