Toolbox/Lecture 7

adenylate cyclase
An enzyme that takes AMP as substrate and synthesizes cyclic AMP (cAMP). cAMP is secondary messenger in many signal transduction cascades. cAMP, for example, binds to the regulatory subunits of protein kinase A to free the catalytic subunits. There are nine different isoforms of adenylate cyclase that respond differently to the various kinases, elevated calcium levels, and to G protein subunits other than the stimulatory alpha subunit (by which all adenylate cylcases are activated.)

cAMP Phosphodiestrase
An enzyme that takes the second messenger cyclic AMP (cAMP) as a substrate and hydrolyses the phosphodiester bond, creating AMP. Important enzyme in shutting off signal transduction cascades.

Isoproternol
An agonist of the beta andrenergic receptor, a receptor for the catelchomines epinepherine (adrenaline), and norepinepherine (noradrenaline).

Beta adrenergic receptor
A receptor for the catelchomines epinepherine (adrenaline), and norepinepherine (noradrenaline). It is one type of G-protein coupled receptor or GPCR, in which ligand receptor binding activates a G protein which in turn activates an adenylate cyclase. The beta adrenergic receptor resembles rhodopsin. All GPCRs have 7-transmembrane domains. There is an extracellular domain and an intracellular signaling motif. For the beta-adrenergic receptor, the 3rd cytosolic loops binds the heterotrimeric stimulatory G protein.

G-proteins
G protein is a heterotrimeric protein that exists in two forms, a GTP bound form, and a GDP bond form, and acts as an on/off switch in signal transduction cascades. The trimer is activated when it binds GTP. When activated the trimer separates into two subunits, one being the alpha subunit, the other being a heterodimer of the beta and gamma subunits. The alpha subunit activates its target adenylate cyclase, causing synthesis of the cAMP second messenger. Recent research suggests that the beta/gamma subunit also has downstream targets. G protein to GTP binding is mediated by an enzyme called the Guanine nucleotide Exchange Factor or GEF which exchanges GDP for GTP in the G-protein binding site. The alpha subunit has intrinsic GTPase action, and therefore can turn the entire trimer into the inactivated GDP-bound form, however hydrolysis of GTP to GDP by the alpha subunit is accelerated by the enzyme GAP or GTPase activating protein. When in the inactivated GDP bound form, G protein reforms the heterotrimer.

Sodium Fluoride and G protein activation
Sodium fluoride and aluminum fluoride are G protein activators.

Narcoleptic dogs
Narcolepsy is a primary sleep disorder, whose prominent symptom is excessive sleepiness. It was first identified by Jean-Babtiste in 1880. In the 1950s the narcoleptic syndrome was defines as consisting of four symptoms: (1) daytime sleepiness, (2) cataplexy,thbe reversible loss of muscle tone (3) sleep paralysis, and (4) hypnagogic hallucinations. After the discovery of REM sleep, it was discovered that patients with narcolepsy begin sleep with REM sleep, whereas normal sleep begins with non-REM sleep. Narcolepsy has been associated with a class II antigen of the major histocompatibility complex on chromosome 6 at the HLA-DR2 or HLA-DQW1 locus. HLA-DR2 is also associated with autoimmune diseases such as multiple sclerosis and rheumatoid arthiritis, raising the possibility that narcolepsyt has an immunological basis. Some dogs are narcoleptic, and their narcolepsies are similar in most respects to human narcolepsy, except for the mode of genetic transmission. In narcoleptic dogs, abnormalities have been found in cholinergic and monoaminergic synaptic transmission, important components of REM sleep regulation. Dogs with narcolepsy have more muscaniric M2 receptors in the pons, suggesting a defect in cholinergic sensitivity. Consistent with this, cholinergic antagonists inhibit and agonists exacerbate canine cataplexy. Norepinephrine function also seems abnormal in that the number of α-2 receptors in the locus ceruleus is larger than normal. Moreover, the density of dopamine D2 receptors is greater both in dogs and in humans with narcolepsy. Some of the selective serotonin reuptake inhibitors reduce cataplexy in dogs and humans, implicating serotonergic systems at least in cataplexy. A group of researchers at Stanford University led by Emmanuel Mignot, MD, PhD associate professor of psychiatry at Stanford University School of Medicine, used a technique called positional cloning to pinpoint the “narcolepsy gene” in dogs. In the August 6 issue of Cell (*Mignot, E., et al. "The Sleep Disorder Canine Narcolepsy Is Caused by a Mutation in the Hypocretin (Orexin) Receptor 2 Gene." Cell, August 6, 1999.), Mignot and his colleagues report locating two defective versions of the narcolepsy gene, one in Doberman pinschers, the other in Labrador retrievers. The gene, known as hypocretin receptor 2, codes for a protein that juts out from the surface of brain cells and that functions as an antenna, allowing the cell to receive messages - transmitted via small molecules called hypocretins - from other cells. The defective versions of the gene encode proteins that cannot recognize these messages, in effect cutting the cell off from essential directives, including perhaps messages that promote wakefulness. Mignot predicts the finding will not only help the roughly 135,000 Americans who suffer from narcolepsy, but in time it will shed light on two of the biggest mysteries in sleep research: how and why we sleep.

Fluorescent Proteins (e.g. GFP)
Green fluorescent protein, isolated from the jellyfish Aequorea victoria, fluoresces upon exposure to blue light. The structure was solved by James Remington and colleagues - the fluorescent group is contained within the B-barrel. Fluorescent proteins are available in many colors and are most often used as reporters of protein expression to better understand cellular signaling.

FRET Imaging
Fluorescence Resonance Energy Transfer (FRET) is the radiationless transfer of energy between two fluorescent proteins. The fluorescent donor is excited at a specific wavelength. This energy can then be transferred to the fluorescent acceptor through a dipole-dipole coupling mechanism. FRET imaging can be used to determine protein-protein interactions, protein-DNA interactions, and protein conformational changes.

For example, one part of a protein is tagged with CFP and another part is tagged with YFP. When the protein is in a certain conformation in which the two fluorophores are far apart, the CFP will be excited but will not transfer its energy to the YFP. The assay would result in the visualization of the CFP wavelength. If a conformational change takes place and allows the CFP and YFP to come close together, the energy transfer will take place. In this case, the assay would result in the visualization of YFP wavelength.

One limitation of FRET is the inherent background noise that results from the direct excitation of the acceptor fluorescent protein. To avoid this problem Bioluminescence Resonance Energy Transfer (BRET) is used in which a bioluminescent luciferase is used instead of CFP as the fluorescent donor. Another solution to determine protein-protein interactions is BiFC. This technique attaches one half of the YFP molecule to one protein and the other half to another protein. When the two halves come together, the complete YFP is now functional.

CaM Kinase II
CaM Kinase II is a calmodulin-dependent protein kinase that shows history-dependent activity, remembering previous calcium pulses through autophosphorylation. CaM kinase II forms a complex with 12 subunits, arranged in two hexamer rings. It is activated by calcium-bound calmodulin, which relieves the autoinhibitory interaction between CaM kinase subunits. Once activated, CaM kinase II proceeds to phosphorylate itself and remains partially active even after the lowering of calcium levels, thereby prolonging the duration of its kinase activity. Consequently, activated CaM kinase II responds non-linearly to calcium oscillations, as (in the absence of calcium) its activity falls more slowly the more it is phosphorylated.

See BIO254:CaMKII

Calcium imaging
Calcium imaging is a scientific technique designed to reflect the calcium status of a particular tissue or medium. In calcium imaging a substance called Fura is used to bind to calcium. When Fura binds to calcium after being exposed to fluorescent light, it fluoresces. The Fura-Ca complex affects the wavelength typically associated with unbound Fura and the resulting fluorescence can be detected by a camera adapted (usually through a microscope) for fluorescent light imaging. A computer-generated image is thus created which can be analyzed according to intensity, which reflects calcium status in the given medium or tissue.