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Basic cell characteristics relevant to this exam- membranes:

- enclose cell, define boundaries- separation of external/internal environment to allow essential chemical reactions to take place- maintains ion gradients across membranes; membrane potential

- how to communicate through semi-permeable membrane?- receptors embedded in cell membrane that interact with specific molecules and transmit signal

to the cell, signalling a response/reaction

Cell signalling

- must communicate w/ environment and one another,whether free living or part of a body

- find food, mates; avoid predators if free living- in muticellular organisms, cells must

communicate with one another b/c havespecialized functions

- various methods of signal transduction- ligands are made and secreted by some cells

- proteins/peptides: insulin, glucagon,epidermal growth factor, TGF-

- chemicals: epinephrine, retinoic acid,estrogen, testosterone

- gases: nitrous oxide- other cells express receptors specific to certain

ligands, generally on surface of cell- some chemicals and gases can diffuse

through membrane unassisted, and havereceptors within the cell

- cell communication regulates fundamental

processes of a cell:- proliferation- gene expression- changes in cytoskeleton

- motility- morphology

- mating- adhesion- cell death (apoptosis)- development/differentiation

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- four types of intercellular communication:- endocrine:

- tissues producing ligands are located agreat distance from responding tissues;ligand travels through the blood totarget cells

- ex: insulin and other hormones- paracrine:

- ligand producing cells are locatednearby responding cells

- ex: neurotransmitters, growth hormones- autocrine:

- cells respond to signals they producethemselves

- a given ligand can be both a paracrineand autocrine signal

- ex: epidermal growth factor EGF- juxtacrine:

- cells must be adjacent; ligand andreceptors are both membrane-bound

- ex: notch, ephrins, semaphorins- most receptors initiate an intracellular cascade

leading to a response- binding of ligand to receptor induces a chain

of events within cell, including conformationalchanges and post translational modifcationsthat activate intracellular enzymes

- mostly de-/phosphorylation by phosphatases/kinases - very tightly regulated

- human genome codes for 600 different

protein kinases and 100 differentphosphatases- exception: hormone receptors, which directly

bind DNA when bound to ligand- ligands bind specific receptors

- mutate one amino acid at a time in ligand or receptor to findresidues essential for interaction btw the two

- binding is mediated via weak interactions- GH and GH receptor:

- eight amino acids in GH defined as responsible for 85%of the energy responsible for tight binding to receptorthrough weak bonds

- two tryptophan residues on receptor are responsible formost of the energy to bind hormone, but others are also involved

- binding of growth hormone to receptor is followed by binding of second receptor tohormone using different amino acids on hormone

- the essential residues for this interaction are held in the correct positions by the rest ofthe proteins structure

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- x ray crystallography and determining 3D protein structure- purify and concentrate protein, hope it crystallizes- x ray through crystal and look at dispersal pattern to find

structure- very high resolution - 0.1nm

- can see which atoms interact with other atoms- but can only be used on proteins that crystallize; no

hydrophobic regions can be identified via x ray crystallography- NMR can determine structure of a protein in solution

- can be used on proteins that do not crystallize- same idea as MRI- replace all H with deuterium, induce vibrations with huge

magnets, then deduce structure from positions of hydrogen- not as high resolution as x ray crystallography, but better than nothing

- a ligand binding to its receptor is a chemical reaction

R + L RL

Kd =[R] [L]

[RL]

KoffKon

Kon

Koff

Kd equals conc. of ligand when half of receptors are bound to ligand. if [R] = [RL], then Kd = L. Lower Kd means lessligand required to occupy 50% of receptors

- sensitivity of a cell for a ligand is determined by two factors:- affinity of ligand for receptor

- high affinity receptor:- at low Kon, Koff is much lower than Kon. RL complexes are very stable and

can therefore act at low concentrations- high affinity ligands can reach effective dose even at very long distances

despite dilution of ligand - endocrine signalling- low affinity receptor:

- at high Kon, Koff is greater than Kon. RL complex is not stable- low affinity ligands only likely to reach effective dose at short distances

(high ligand concentration)- used in auto/juxta/paracrine signalling

- experimental determination of Kd (high affinity ligand)

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- typical cell surface receptor present in1000-50,000 copies/cell (0.1-0.5% totalprotein on plasma membrane)

- radioactively label ligand- incubate with responsive cell type at

different concentrations (curve A) for 1hr at low temp to prevent endocytosisof ligand/receptor complex by more orless shutting down cell functions

- separate unbound ligand bycentrifugation and washing; countradioactivity and cell number

- repeat exp in presence of 100xunlabeled ligand. all specific sitesbound by unlabeled ligand,radiolabeled ligand binds to nonspecificsites (curve B)

- so, curve A - curve B = curve C- graph shows data from experiment with Epo receptors (EpoR) in mouse cells

- experimentally determining Kd with low affinity ligand- if Kd is greater than 10-7 M, then Koff is relatively large compared to Kon.- ligand will dissociate from receptor in time required to separate bound from unbound

ligand- previous experiment will consistently underestimate receptor numbers- add constant amount of radiolabeled, high affinity synthetic ligand to cells and increasing

amounts of unlabeled low affinity competitor of same receptor- unlabeled competitor displaces labeled ligand from receptor and is washed away

- note: agonists mimic function of natural hormone; antagonists inhibit function of naturalhormone

- maximum cellular response does not require activation of all receptors- determining sensitivity of a cell to a signal:- affinity of ligand for receptor- receptor concentration

- fewer receptors = less sensitive to ligand- finding receptors for a given ligand

- receptors are rare - 0.1-0.5% of protein in cell- receptors are usually integral membrane proteins; membrane must be solubilized with non-ionic

detergent and separated from other cellular proteins- above are reasons why they are difficult to purify and identify

- can find receptors by expression cloning- ligand must be identified

- express potential receptor in cell type that does not normally respond to ligand- incubate transfected cell with radiolabeled ligand- calculate amount of ligand bound to transfected cells vs untransfected cells to determine if there

is specific binding- separate cells with bound ligand by FACS (fluorescent activated cell sorting)

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- during this process, cells are diluted enough that each droplet only contains one cell at most- sequence cDNA of cells that are identified as being bound to fluorescent ligand

- can use ligand affinity purifcation/chromatography to identify receptors

-- beads with ligand of interest attached- load candidate proteins

Free

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- ligand is buried in plasma membrane instead of being held onto in the extracellular space- ligand binding pocket is made up of 15 residue side chains from residues in four helices (H3,

H5, H6, H7) and the extracellular loop (E2)- + charge on N atom in epinephrine and cyanopindolol interact with carboxylate side chain of

D121 - specificity- Ligand binding induces conformational changes

- receptor is bound to agonist; helices are in ACTIVE configuration- TM5/H5 is pushed into cytosol; TM6 moves outward

- Gs, G, and G constitute the heterotrimeric G protein Gs.- in active configuration, as shown, TM5 interacts with Gs subunit- N helix of Gs subunit has extensive

interactions with G subunit, as Gs does notinteract directly with G subunit.

- Binding of N and 5 helices of Gs with TM5

and 6 of GPCR opens up G subunit.- GDP is evicted from pocket and replaced with

GTP- binding of GTP induces conformational changes

in Switch I and II regions of Gs- G proteins act as molecular switches

- conformational changes in G protieins aremediated by two switch domains

- Switch I and Switch II regions bind terminal g-phosphate of GTP

- GTP hydrolysis causes release of -phosphate,which causes relaxation of Switch I and II

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regions.- Conformational changes control ability of G

protein to interact with different proteins- so, GTP hydrolysis induces off state, and the

time to hydrolyze GTP is variable- note: two types of G proteins - trimeric/large G

proteins directly bound to and are activated byreceptors and monomeric/small G proteinsindirectly linked to receptors by adaptors

- G proteins exist in two states: GTP-bound onand GDP-bound off states.

- There are small monomeric G proteins that actas helpers

- GEFs: guanine nucleotide exchangefactors; help to remove bound GDP andreplace it with GTP to activate Gproteins

- GAPs: GTPase activating protein; stimulatehydrolysis of bound GTP to promoteinactivation of G protein.

- conformational changes control ability of G protein tointeract with different proteins.

- RGS = regulators of G protein signalling

- GDI = guanine nucleotide dissociation inhibitors

- these small proteins also contribute to the length oftime G protein remaines in active state.

- how do trimeric G proteins mediate signals from GPCRs?- each has three peptide subunits: , , - subunit binds GDP (and GTP) and exhibits GTPase

activity- , subunits are attached to plasma membrane- binding of ligand to GPCR stimulates exchange of GDP for GTP; when bound to GTP, G

proteins are activated- GTPase activity of subunit hydrolyzes GTP to GDP, inactivating G protein- rate of GTP hydrolysis determines how long G protein remains active- note: signal is amplified downstream of activated receptor

- receptor remains in active state as long as ligand is bound. activated G proteinsdissociate from an active receptor, therefore one active receptor activates multiple Gproteins

- subunit dissociates from subunit; each complex can activate multiple effectormolecules

- primary signal is amplified considerably

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- what are effector molecules?- enzymes that produce small molecules that act as

second messengers- molecules that act as second messengers include:

cAMP, cGMP Ca2+, diacyl glycerol and inositoltriphosphate (IP3)

- subunit determines which effector molecules arecontrolled by the G protein

- Gs activates adenylate cyclase- the number of effector molecules activated depends

on how long G protein remains in on state- this depends on how quickly GTP is hydrolyzed; if G

protein binds a synthetic, non-hydrolyzable analog ofGTP (like GTPS), it is permanently in the on state.

- How does Gs-ATP interact with adenylate cyclase?- adenylate cyclase is a multipass transmembrane

protein; catalytic units are on the cytoplasmic side- X ray crystal structure: ultimate test of protein

interaction (highest resolution, but can beproblematic)

- does not work on intermembrane proteins- work around: crystallize only soluble catalytic

domains with Gs- A3-b5 loop of Gs subunit and helix of switch II region

interact with enzyme- forskolin activates adenylate cyclase

constituitively- produced by indian coleus plant

(Coleus forskohlii)

--- different types of G subunits have different effects

- Gs G proteins: subunit activates adenylatecyclase

- cholera toxin attaches ADP ribose fromNAD+ to subunit of Gs; inhibitsGTPase activity of Gs, causing it toremain active. elevated cAMPin gut causes large efflux ofwater and Cl-, resulting insevere diarrhea

- Gi G proteins: subunit inhibitsadenylate cyclase

- pertussis toxin attaches ADPribose to the subunit of Gi,preventing it from interactingwith receptors. as a result, Giprotein remains bound to GDPand inactive (cannot inhibitadenylate cyclase), resulting inincreased cAMP levels

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- remember: s = stimulates adenylate cyclase; i = inhibits adenylate cyclase- cAMP synthesis occurs very rapidly

- make a cell express protein that fluoresces when cAMP is bound- cAMP accumulates very rapidly- cAMP is synthesized by adenylate cyclase, degraded by cAMP phosphodiesterase (PDE)- second messengers amplify signals (1 adenylate cyclase molecule makes ~1000 cAMP

molecules/second)- other second messengers include cGMP, Ca2+, and IP3

- cAMP activates PKA (cAMP dependentprotein kinase)

- PKA is composed of two regulatorysubunits and two catalytic subunits

- regulatory subunits inhibit catalyticactivity of enzyme

- each regulatory subunit binds twomolecules of cAMP

- binding of cAMP to CNB regionsinduces conformational changefrom purple strand to green (img onnext page)

- each subunit has a flexible linker connecting to AKAP binding site and dimerization domain- so cAMP must bind and inhibit regulatory domain to allow catalytic domain to become active

- second messengers provide second amplification of signal, as seen in the diagram below- as mentioned previously, maximal cellular response does not require activation of all receptors

due to this amplification

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- how do we know which part of GPCR interacts with G protein?- epinephrine bindes two types of GPCRs, each expressed in different cell types and elicit

different effects- -adrenergic receptors are expressed in smooth

muscle cells lining blood vessels, intestinal tract,skin, and kidneys

- binding of epinephrine triggers constrictionof smooth muscle

- active -adernergic receptors stimulateGi

- -adrenergic receptors are expressed in liver, fatcells, and heart cells

- binding of epinephrine triggers release ofglucose and fatty acids from liver and fatcells

- makes heart beat faster- active -adrenergic receptors stimulate

Gs- experiment:

- inject WT or chimeric receptors intoXenopus oocytes

- treat oocytes with epinephrine agonist

- measure cAMP levels directly- why Xenopus:

- large oocytes, easily manipulated- experiment shows that c terminal portion of

receptor is responsible for binding Gs; loop of receptor is responsible for binding Gi

- how do we prove two proteins associate within a livingcell?

- genetics identifies genes in a molecular pathway (mutants give results in similar phenotypes)

- cAMP

+ cAMP

+ cAMP

- cAMP

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- this does not prove physical interactions- method 1: use bait/prey model (yeast

two hybrid)- fuse gene encoding ligand to a

DNA binding domain (usuallyGAL 4 TF)

- fuse DNA encoding potentialbinding partner to transcriptionalactivation domain of GAL4

- express two constructs in yeaststrain that has GAL4 DNAbinding site upstream of anartificial reporting gene (like GFPor w/e)

- both chimeric proteins mustinteract to activate reporting gene

- bait cannot activate, butcan bind

- prey cannot bind, but canactivate

- method 2: fluorescence resonance emission transfer (FRET)- somewhat less artificial than yeast 2 hybrid system

- can be more reliable results, but the proteins are still artificial- basic idea:

- attach yellow fluorescent protein (yfp) to subunit and cyan fluorescent protein(cfp) to the subunit

- shine 440nm lighton cfp, it emits490nm light

- shine 490nm light

on yfp, emits 527nmlight- so, if the and

subunits areassociated, only527nm light can bedetected, but oncedisassociated,490nm light will bedetected

- method 3: co-immunoprecipitation (pull down assay)- two cells, one with Rac inactive (no PDGF), one with Rac active (PDGF)

- lyse, mix w/ agarose beads with p21 binding domain; then pull down assay- anything bound to Pac1 will be pulled out- when Rac inactive, does not associate with agarose beads- when Rac active, will show up when probing for RacGTP

- what happens after activation of PKA?- PKA regulatory subunits bind AKAP (a kinase associated/activating protein)- AKAPs control subcellular localization of PKA and may also bind other regulatory proteins

(diagram on pg 10)

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- PKA phosphorylates target proteins- GPCR binds ligand, activates Gas- Gas activates adenylate cyclase, leading

to a rise in intracellular cAMP- cAMP binds regulatory subunit of PKA,

releasing catalytic subunits frominhibition

- catalytic PKA subunits translocate tonucleus and phosphorylate targetproteins, including CREB transcriptionfactor

- CREB = cAMP response bindingprotein

- phosphorylates to pCREB andforms dimer that can bind to CRE

- pCREB binds to CRE, recruits CBP/P300to DNA and activates transcription oftarget genes

- CBP/P300 interact withtranscription machinery andactivate transcription

- CRE = DNA binding site- PKA controls a lot of transcription factors

- strategies for downregulation of response to GPCR activation- mAKAP (membrane associated A kinase Associated protein) anchors PDE and PKA to outer

nuclear membrane in heart muscle- PDE breaks down cAMP- PKA phosphorylates PDE,

stimulating activity; providesnegative feedback control as

cAMP activates PKA- close proximity of PDE andPKA provides tight local controlof cAMP concentration and, byextension, PKA activity

- separates cAMPconcentration from restof cell

- AKAP15 anchors PKA to cytosolic face of PM in heart muscle cells, near Ca2+ gated channel- basically: GPCR starts turning itself off once it turns on to prevent pathways remaining active for

too long or staying too highly active- very tight regulation

- GPCR modulate many physiological events- one of the most important is glucose metabolism

- pancreas detects high blood sugar and releases insulin from cells- insulin moves through body and promotes rapid takeup of blood glucose (GLUT

recruitment to cell membranes)- also stimulates glycogen synthesis in muscles and liver

- glucose binding to cells prevents release of glucagon- when glucose is not bound to cells (low blood sugar), glucagon is released

- glucagon receptors are GPCRs in liver - signals breakdown of glucose to maintainblood sugar levels

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- glucose metabolism is controlled by cAMP levels- muscle and liver: glucose-1-phosphate from glycogen is converted to glucose-6-

phosphate and enters glycolytic pathway to generate ATP- liver contains a phosphatase that converts glucose-6-P to glucose, which is then

exported by GLU2 glucose transporter- so glycogen in liver can be degraded to glucose and transported elsewhere in the body

- GPCRs also regulateglycogen storage

- epinephrine orglucagon-stimulatedactivation ofadenylatecyclaseenhancesproduction ofglucose-1-P

- directlyinhibitsglycogen

synthesis- indirectly activates glycogen degradation

- when epinephrine is removed, cAMP drops and process is reversed- reversal is mediated by phosphoprotein phosphatase- PP normally removes phosphates from glycogen synthase, thereby activating it

- insulin stimulates a receptor tyrosine kinase (discussed later)- in summary:

- cAMP up; lots of glucagon and low blood sugar- glucagon binding activates adenylate cyclase- cAMP activates PKA- glycogen synthase deactivated by phosphorylation- glycogen phosphatase kinase is activated by phosphorylation- glycogen phosphorylase activated by phosphorylation - degrades glycogen- IP binds to phosphoprotein phosphatase - inhibits activity- active PKA = deactivation of glycogen synthase pathway, activation of

glycogen degradation pathway- cAMP down, blood sugar up

- IP does not get phosphorylated, PP is not bound to IP and is active- PP is relatively indiscriminate

- phosphate removal from GP or GPK leads to inactivation- Phosphate removal from GS leads to activation- blood sugar is maintained but really this is more complicated but main pathway

goes like this.------

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- see here are some other pathways:

-Perception of Light in the Eye- photoreceptors are in the back of the eye; transmit signal forward to interneurons that connect to

retinal ganglion cells, which transmit thesignal to the visual cortex

- two types of photoreceptors:- cone cells, three types; one each for

red, green, and blue.- if one or two are lacking ->

colorblindness- if all are lacking -> blindness

- rod cells; responsible for detecting lowlevels of light- if lacking -> night blindness

- cone and rod responses to light are similar,but cones are less sensitive to light

- photons trigger a molecular cascade inphotoreceptors (transmitted by transducin;inhibited by pigment kinases and arrestin)before transmitting signal forward tointerneurons

- rod cell mechanism- rod cells are stimulated by weak light (like moonlight) over a range of wavelengths

- rod cells are filled with rhodopsin localized to discs in the outer segment (towards back of eye)- these discs are constantly being turned over and must be re-synthesized

- rhodopsin links to trimeric G protein called transducin (Gt); these proteins are only expressed inrod cells

- rhodopsin in humans is similar to bacteriodopsin; but in bacteria acts as light activatedproton pump - general reaction is similar

- if a specific human eye gene is expressed in a fly, it will grow an ectopic fly eye- genes conserved all the way down to jellyfish- very similar in all metazoans

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- photoconversion of retinal- 11-cis-retinal is covalently linked to opsin module of rhodopsin.- when impacted by a photon, retinal shifts to all-trans configuration, causing a

conformational shift and activating opsin- all trans retinal is highly unstable and detaches from opsin, inactivating it- in the dark, all trans retinal converts back to 11-cis-retinal and can rebind opsin - resets

whole system- rhodopsin is a GPCR

- trimeric G protein is called transducin (Gt)- and subunits have lipid attachment to membrane, like other trimeric G proteins- in GDP bound state, and subunits interact, and and subunits also interact;

however, and subunits do not interact directly- when rhodopsin is activated (when 11-cis-retinal converts to all trans retinal), subunit

exchanges GDP for GTP- how we see

- in the dark (rhodopsin is inactive):- PDE is bound to inhibitory subunits and is inactive; cannot hydrolyze cGMP- high levels of cGMP allow sodium/calcium gated channels to remain open,

changing the membrane potential to around -30mV- this low depolarization causes the inhibitory neurotransmitter glutamate to be

releasedat the synapse; so generation of AP in interneurons is inhibited- in the light:

- rhodopsin is activated and in turn activates G t. the subunit of transducin thenbinds to the inhibitory subunits of PDE, pulling them away from the / subunitsand activating PDE

- cGMP is converted to 5-GMP by PDE; lowered concentration of cGMP causessodium/calcium ion channels to close, resulting in a transient hyperpolarization ofmembranes and less glutamate released at the synapse

- when the interneurons no longer sense glutamate being released, an actionpotential is generated and eventually gets to your brain, where it is interpreted as

light- one rhodopsin molecule absorbing one photon is amplified along this pathway and altersmembrane potential by approx 1mV

- rod cells are able to adapt to different light conditions- activated rhodopsin is a substrate for rhodopsin kinase, which will phosphorylate the

cytosolic C terminal tail of rhodopsin- extent of phosphorylation is directly proportional to amount of time rhodopsin is

active- once three sites on the tail are phosphorylated, rhodopsin can be bound by arrestin,

completely blocking activation of Gt.- so: bright light = high activation of rhodopsin = high phosphorylation of rhodopsin = high

recruitment of arrestin = low activation of Gt = cell is desensitized to light

- compartmentalization of transducin and arrestin in different conditions also contributes tothis activity

- in low light, transducin is moved into the outer segment with the rhodopsin-richdiscs, while arrestin is moved out towards the synapses

- in bright light, transducin is moved out of the outer segment, while arrestin ismoved in

- both biochemical and physical regulation of light sensitivity

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- homologous desensitization of GPCR- regulation of rhodopsin in bright light is

an example of homologousdesensitization - the activation ofGPCR also activates enzymes thatblock the activity of GPCR

- happens to all GPCRs- another example: -adrenergic

receptor- activation of -adrenergic

receptor (coupled to Gs) turnson -adrenergic receptor kinase(BARK)

- BARK phosphorylatescytoplasmic C terminal tail,permitting the binding of -arrestin (related to arrestin inrhodopsin pathway)

- arrestin then recruits otherproteins (ex: clathrin; AP2) thatpromote endocytosis of receptor

- after endocytosis, some receptors are recycled back to membrane, while othersare broken down in lysosomes

- other signalling proteins are also recruited, activating MAP kinase cascade (c-Src)- MKK-4, ASK1, and JNK-3 (Jun N-terminal kinase) phosphorylate the c-Jun transcription

factor- so, arrestin serves both to turn off GPCR as well as activate other pathways; serves as

scaffold for proteins recruited for these functions- second messengers

- IP3 (inositol 1,4,5-triphosphate)

- synthesis:- phosphatidyl inositol (PI) is converted to PI 4-phosphate (PIP) through thehydrolysis of ATP by PI-4 kinase

- PIP is converted to PI 4,5-bisphosphate (PIP2) through the hydrolysis of ATP byPIP-5 kinase

- PIP2 is converted to IP3 by phospholipase C- (PLC) and diacylglycerol. bothIP3 and DAG act as second messengers; IP3 in the cytosol and DAG in themembrane

- activation of PLC:- acetylcholine activates GPCR, triggering activation of Gq or Go, which activate

the effector protein, PLC- IP3 is not very stable; within a second, the phosphate group on C5 will be removed by

hydrolysis, forming inositol 1,4-bisphosphate which is not able to act as secondmessenger

- so, needs something to stabilize it and prevent hydrolysis

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- CaM kinase has catalytic and inhibitory domains- when inactive, catalytic domain is bound to inhibitory domain, blocking the active

site- CaM binds to inhibitory domain, displacing the catalytic domain, and opens active

site- CaM kinase autophosphorylates

- only partially activated by CaM binding, must autophosphorylate on inhibitorydomain in order to be fully active

- this prevents CaM kinase from inactivating immediately when Ca2+ levels drop andCaM no longer binds to the inhibitory domain

- only inactivated when another enzyme removes phosphoryl group from inhibitorydomain, allowing it to once again bind to the catalytic domain

- CaM kinase plays a role in learning and memory- use aplasia (sea slug) as model organism

- big neurons that are easily manipulated- simple neurological circuitry, easy to dissect- simple behaviors, but display learning behaviors- so can figure out molecular/cellular basis of learning and memory

- create mini-brain from one sensory and one motor neuron in order to see whathappens when learning and memory formation occur

- also use aversive stimuli to teach aplasia that the environment is dangerous- after enough time, the slug will begin to react defensively for longer periods

of time if exposed to aversive stimuli previously- mechanism:

- synapse gets stronger with more exposure to stimulus- short term: more neurotransmitter is released, so more receptors

recruited to post synaptic membrane = increased signal strength- long term: eventually neurons physically change, creating new

synaptic connections, ensuring continued increased signalling- post synaptic cells have two kinds of receptors: AMPA and NMDA

- AMPA opens in response to glutamate binding; influx of sodium ions- sodium ions change membrane potential, removing Mg2+ ionblocking channel and allowing the NMDA channel to open whenglutamate/glycine are bound

- Ca2+ enters cell, activating CaM kinase; triggering exocytosis ofvesicles with more AMPA receptors, further sensitizing the synapticconnection

- in summary: AMPA open --> NMDA opens --> CaM kinase active -->more AMPA exocytosed to membrane --> increased sensitivity

- AMPA receptors will be continually added until CaM is inactivated byprotein phosphatase

- mechanism is more or less the same in humans, as well as Drosophila

- Ca2+

levels are triggered by several stimuli, including fertilization of oocyte and fusion ofpronuclei within egg

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