mod 2

Endocrine Signaling

Ex. epinephrine, insulin

Signaling molecules are synthesized and secreted by signaling cells

Transported through circulatory system

Affect distant target cells expressing the receptor

Paracrine Signaling

Ex. neurotransmitters, growth factors

Signaling molecules secreted by a cell - affect only nearby target cells expressing the receptor

Some bind to ECM

Autocrine Signaling

Ex. growth factors

Cells respond to signals they secrete themselves

(the ligand is secreted and then lands on the cell’s own receptors)

ex. tumor cells overproduce and respond to growth factors

Membrane Protein Signaling

signal neighboring cells by direct contact with surface receptors

ex. delta notch

Agonist

binds to receptor to create similar response to original ligand

Antagonist

binds to receptor (or other location) to prevent receptor from producing the response

k on

kinetic rate constant for complex formation

at equillibrium, rate of complex formation is equal to reate of dissociation

koff

kinetic rate constant for complex dissociation

Kd

dissociation constant

quantifies the affinity of a ligand for it’s receptor

(koff/kon) and is equal to l at 50% occupation

lower equals stronger affinity

cAMP

Generated from ATP by adenylyl cyclase

activates PKA

cGMP

generated by guanylyl cyclase

activates PKG and specific cation channels

IP3

Made from PIP 2 by phospholipase C

soluble so it is released into cytoplasm

opens channels to release calcium from ER to increase ca levels intracellularly

DAG

made from PIP 2 by phospholipase C

membrane bound

activates PKC after combining with calcium

GEF

catalyze dissociation of bound GDP and replacement to GTP

(not phosphorylation it takes the whole thing off and places a GTP)

This activates the protein

Turned off by its own gtpase activity on protein

GAP

helps accelerate inactivation of gtp proteins

Does not turn it off itself, gtpase activity on gtp binding proteins do it but this accelerates it

\

Heterotrimeric G proteins

activated by direct interaction with surface receptors (GEF)

Monomeric G proteins

activated by GEFs that are activated by surface receptors or other proteins

Binding Assay

used to study ligand-receptor interaction and determine their affinity (kd)

involves using increasing concentrations of the ligand and measuring ligand bound to receptor

PAM

modulator that facilitates ligand binding and increase of signaling by binding at allosteric site

NAM

modulator that slows or inhibits ligand binding causing weakening of signal

binds at allosteric site

GPCR

Receptor contains 7 membrane alpha helices

heterotrimeric

has membrane bound effector protein

Steps:

Ligand binds to GPCR

It acts as a GEF and alpha unit is swapped to GTP

This causes dissasociation and beta and gamma go target ion channel proteins while alpha carries on signal

alpha then has its own gtpase activity to hydrolyze gtp back to gdp = off

Forster Resonance Energy Transfer (FRET)

Detects protein-protein association or dissociation

Wavelength of emitted fluorescence changes when two dif fluorescent proteins interact

Emission of one protein has to overlap with absorption of second protein

Basically if theyre connected the lower wavelength one lights up and the other protein is close so the light form the first protein causes it to light up and it puts out its light which proves theyre connected

\

G alpha s

Regulate the activity of adenylate cyclase → alters cAMP levels

stimulatory = increase in cAMP

G alpha i

Regulates the activity of adenylate cyclase → alters cAMP

inhibitory = decrease in cAMP

the beta gamma subunit is also inhibitory and will decrease excitability of ion channels (hyperpolarization)

G alpha q

stimulates phospholipase C which leads to mobilization of intracellular calcium

Muscarinic Acetylcholine Receptors

\
* M1 receptors are found in the brain and are involved in learning and memory. (use ip3/dag = gq)
* M2 receptors are found in the heart and regulate heart rate and contractility. (inhibitory = gi)
* M3 receptors are found in smooth muscles and glands and are involved in the regulation of various physiological processes such as digestion, urination, and sweating. (use ip3/dag=gq)

\

Opioid Receptors

Use Gai protein to inhibit adenylene cyclase = decrease cAMP

Gby unit opens k channels and blocks sodium channel so theres a build up of k on outside = hyperpolarization = less excitable

this can lead to pain relief because neurons not firing as frequently

are in both pre and post synaptic terminals

B-arrestins

block g protein activation causing GPCR desensitization

Through steric interference

it does this after receptor is used a lot

Arrestin can act as a docking site to interact with other signaling molecules that normally wouldnt be engaged by g protein

It also endocytoses the entire receptor and degrades it

Adenylyl Cyclase

Takes ATP and makes it cyclic so that it becomes cAMP

Phosphodiesterase (PDE)

takes off the phosphate bond from cAMP so that it’s not longer cyclic and returns to being AMP

Protein Kinase A (PKA)

Has regulatory and catalytic subunits

the cAMP binds to the regulatory units which releases the catalyctic subunits making it active

glycogen

stores glucose for when you don’t need it

breakdown occurs in response to epi and glucagon

synthesis involves incorporation of UDP glucose

Glycogen Synthase

incorporates UDP glucose into chain

activated PKA from increased cAMP phosphorylates this protein acting as an INHIBITOR

= no glucose storage

decrease in cAMP leads to release of active form of PP which promotes this protein

Glycogen phosphorylate

Activated by PKA

degrades glucose for when body needs rapid immediate energy supply like epi and glucagon

CREB

transcription factor

phosphorylated in response to PKA = increase in cAMP

forms complex with coactivator cbp/p300 and binds to regulatory elements in promoters of multiple enzymes involved in gluconeogenesies (pyruvate → glucose)

increase of transcription

AKAP

leads to localization of PKA and cAMP phosphodiesterase in same spot

anchors the regulatory subunit of PKA and PDE to the membrane

negative feedback loop = clsoe control of cAMP level and PKA activity

some PKA phosphorylates the PDE causing decrease in cAMP = inactivation of PKA

Found in heart muscle cells

Phospholipase C

Cleaves PIP 2 into two pieces (Dag and Ip3)

Ip3 is the soluble portion and can go into cytoplasm to open calcium channels

Dag is the membrane bound portion

Functions: proliferation, differentiation, apoptosis

Phosphatidylinositol (PI)

membrane lipid

becomes PIP2 which is precursor through Pi4 and pip5 kinase

Phospholipase C hydrolyzes phosphate bond to yield the membrane associated DAG and soluble IP3

\

Glycogen phosphorylase Kinase (GPK)

binds to calcium to increase glycogen degradation in times of immediate sugar need

also activated by PKA

Rods

sensitive to low light intensity

detect shades of grey

provide us with night vision when we can recognize shapes but not colors

found throughout retina besides center of fovea = blind spot

Rhodopsin

GPCR that senses light in rod cells

Activated by absorbing energy from photon of light = conformation change

Activated receptor activates Gt protein (transducin)

within seconds turns back off

Involves cGMP and GMP instead of amp

low cGMP

when light is present

through activation of PDE turns into GMP

causes gated calcium and sodium channels in eye to close

= membreane is hyperpolarized = less neurotransmitter released = no glutamate

glutamate acts inhibitory for light

then arrestin binds and turns it off until it gets removed so rhodopsin can reactivate

high cGMP

no light is present

PDE is off so cGMP remain high and open ion channels causing membrane depolarization = neurotransmitter released = increased glutamate

Transducin

G protein t

involved in rod light pathway

Rhodopsin kinase

phosphorylates the light activated rhodopsin

the more active the rhodopsin is, the more phosphorylation

More phosphorylation = inhibition of Gat

Phosphorylation leads to inactivation by arrestin

Receptor Tyrosine Kinases (RTK)

Has 3 signaling pathways

Ras/Map , Phospholipase C, Pi3 Kinase

cytosolic domain contains INTRINSIC catalytic kinase site

two ligands bind two ___ extracellular domains which homodimerizes the receptors bring together their two poorly active kinase domains. They phosphorylate each other on tyrosine residues which provide docking sites

Fibroblast Growth Factor (FGF)

two of these ligands bind to two receptors to dimerize

heparan sulfate helps enhance binding

Epidermal Growth Factor (EGF)

Uses HER family as receptors

the receptor contains four extracellular domains and ligand induces dimerization

Results in asymmetric kinase domain dimer

donor kinase c terminal binds to acceptor kinase n terminal and activates acceptor kinase

Acceptor kinase then phosphorylates tyrosine residues on both receptors

HER receptors

family of four receptor tyrosine kinases

they bind EFG and other EGF family members like neuregulins

HER2 does not directly bind ligand but facilitates signalling by forming heterodimer with 3 or 4

Her2

Receptor for EGF and others that does not have its own ligand binding site

Thus cannot exist as homodimer

However, can bind to other receptors like 3 or 4

Makes cells more sensitive to signaling and overexpressed in cancers

Therapeutic target - inhibits heterodimerization of this with other members of the family or increased endocytosis of receptor

Lysosomal Degradation

Used for degrading EGFR (HER1) (AN RTK)

After ligand binds, EGFR is recruited to clathrin coated pits and cytosolic side is phosphorlyated allowing binding to cbl which tags the receptor with ubiquitin to be degraded

Proteasomal degradation

Used for degrading HER2 (AN RTK)

Does not have ligand binding so when stability factors unbind, CHIP (ubiquitin ligase) tags with ubiqutin and it’s degraded by _____

Neurotrophins

NGF BDNF and NT-3

ligands for Neuronal growth and survival

Use high-affinity TRK receptors (RTKs) for signaling (A-C)

Use p75 as low affinity receptor

\

TRKs

receptor tyrosine kinases that have high affinity for neurotrophins

From A-C and can use all three pathways (PKC,Pi3K, MAPRAS)

affect neuronal growth

BDNF

neurotrophin that binds to trkb

psychiatric drus impact it’s activity in the brain

critical role in antidepressants (SSRIs) and ketamine = increase in this ligand in cortex

Cytokine Receptors

some create homodimer complex while others create heteromulimer complex

Do the same pathways as RTK plus an extra Jak/STAT pathway

LACK INTRINSIC KINASE ACTIVITY

have to activate a separate kinase

In absence of ligand, binds to a JAK tyrosine kinase and forms homodimer but poorly active

Ligand binding brings JAK kinase domains together and they phosphorylate each other on tyrosine residue = activation

Epo

Cytokine that triggers red blood cell formation

in response to oxygen drop

JAK

Tyrosine kinase on cytokine receptor cytosolic domain

poorly active until ligand binds causing domains to come close so they can phosphorylate eachother

They activate STAT transcription factors

STAT

transcription factor activated by JAK kinase

Sh2 domain binds to cytokine receptor where JAK then phosphorylates it’s C terminal

It dissociates and homodimerizes which creases a NLS (nuclear localization signal)

Translocates to nucleus and binds to promotor sequences

\

Sh2

domain on STAT transcription factor that binds to cytokine receptor to allow phosphorylation of c terminal by jak

also domain on shp1 for degradation

Western Blotting

detects protein or phosphorylated protein

Use an antibody against the antigen on the protein and seperate using gel electrophoresis to measure bands

found that increasing ligand increases phosphorylation (helps to detect kinase activity)

Cytokine Short-Term Regulation

phosphotyrosine phosphatases (SHP1)

The sh2 domain of SHP1 binds to activated receptor allowing it to dephosphorylate the JAK activation loop = inactivating kinase activity

\

Cytokine Long-Term Regulation

SOCs protein negative feedback

STAT5 induces SOCS protein expression which then binds to residues on JAK2 blocking the binding of other singaling proteins

Then it targets receptor for degradation using ubq tag

SHP1

removes phosphate from JAK kinase on cytokine to inactivate it = short term regulation

SOCS

protein that binds to JAK2 on cytokine and blocks other binding proteins

Then it tags the receptor with ubq for degradation = long term regulation

Ras/MAP kinase pathway

monomeric g protein activates kinase cascade (Raf, MEK,MAPK) which activates transcription factor to induce transcription

The kinase activates the gtp binding protein through an adapter protein (GRB2) via sh2 domains

GRB2

An adaptor protein used to allow a kinase to be able to activate a g protein since g proteins need gtp and kinases only phosphorylate

binds to receptor using sh2 domain

Then it recruits a GEF (SOS) using its sh3 domain

The GEF activates the g protein (Ras)

sh3 domain

On grb2 (adaptor protein) that allows it to bind to a GEF (SOS) so that the sos can activate the ras g protein

Sos

a GEF that is activated by the adaptor protein gbr2 through it’s sh3 domain

It activates Ras

Raf

kinase activated by Ras g protein

has a 14-3-3 on n terminal autoinhibitory domain that is phosphorylated onto the catalytic side to keep inactive

an active ras dephosphorylates one of them so that 14-3-3 is released and Raf can be activate

activates MEK which then phosphorylates Map kinase which is the big boss that phosphorylates a bunch of dif proteins

Mutations in Raf reposible for a lot of skin cancers

MAPK

kinase activated by MEK in Ras pathway

It is dimeric and phosphorylates p90rsk which enters the nucleus and phosphorylates transcription factors (SRF)

Then the dimer itself enters nucleus and phosphorylates transcription factors (TCF)

The phosphorylated factors bind genes with serum response element (SRE) and induce transcription

\

p90rsk

kinase activated by MAPk that goes into nucleus and associates with SRF (serum response factor) to bind to sre region (serum response element) of gene

TCF

transcription factor that mapk dimer phosphorylates in the nucleus which binds to sre (serum response element)

it is already bound to promoter region of gene and mapk comes in and phosphorylates it

Pull Down Assay

used for detecting ras activation

You place raf (ras’s target) onto beads

you also place ras into the tube with the beads

Control: no ligand and you see that the ras does not get pulled down to bottom with beads

inclusion of ligand causes ras to be activated and binds to the raf on the beads and gets pulled down and shows up on paper

Ip3/Dag signalling

Used by g proteins AND Rtk/cytokine receptors

Phospholipase C hydrolyzes PIP2 into ip3 and dag

PI3K

lipid kinase that adds phosphates to phospholipids in position 3 of ring

Used by RTKs and Cytokine Receptors

this creates PIP3

Pip3 then binds to PKB and PDK2

\

PKB (AKT)

phospho kinase B

partially activated by PIP3 by binding to its PH domain

fully activated once pdk1/2 joins

Used to synthesize glycogen from influx of glucose / insulin by inhibiting inhibitor of glycogen synthase

Pip3

Created by Pi3k

partially activates PKB by binding to ph domain

also activates PDk2

Pdk2 helps fully activate the PKB

PDK2

Activated by PIP3

It helps finish activating PKB (AKT)

PTEN phosphatase

dephosphorylates PIP3 back into Pip2 which ends the pi3k pathway

this is deleted in many advanced cancers causing uncontrolled cell growth

Tuberous Sclerosis Complex (TSC)

inhibits the pi3k signalling pathway along with pten

its a gap protein, inhibits mtor

can be inhibed by pKb (akt)

TGF-b receptor

limits growth not promote

display SERINE kinase activity

activate SMAD transcription factors

RI,RII,RIII complex

RII always active without ligand and once ligand comes it brings together RII with RI which is phophorylated by RII

RI can then phosphorylate smad

RIII is an optional helper that will bind the ligand and bring it to the RII

Complex of RI and RII together make up the receptor

\

integrin

helps release tgf b from it’s strait jacket ltbp/garp segments so that it can bind to the cell-surface receptor

Garp/ltbp

straight jacket / arm domain on tgf-b that prevents it from binding to receptor and keeps it stuck in ECM

tgf beta

LTBP/Garp keep it inactive by covering it up but integrin releases from cover

ligand forms dimer

transformign growth factor, inhibits growth

importin

brings smad to nucleus

RAN

monomeric g protein that brings the importin back to the cytoplasm after it dropped off smad

Ski/histone

This represses smad function by binding to it while its on promoter region

It blocks the mh2 domain which is necessary for transcriptional activation

Then it activates histone deacetylation which represses gene expression

This is negative feedback because it is created by the protein being transcribed

Notch

receptor that interacts with delta on adjacent cell

undergoes proteolytic cleavage that releases the cytosolic segment that can go and modulate transcription

cleaved by ADAM 10 cell surface protease

mechanism of lateral inhibition cuz it stops surrounding cells of delta from matching it

Wnt Signaling Pathway

Binds to frizzled receptor (Fz)

In absence of this, transcription factor is bound to promoters but has a groucho that inhibits gene activation

b catenin becomes phosphorylated on serine and threonine residues and ubq tagged and degraded

With this present, LRP co receptor binds to the complex that initially phosphorylated the b cateninin = no phosphorylation = increase in b catenin in cell

the b catenin goes inot the nucleus and binds to Tcf which displaces the groucho = gene transcription

b catenin

binds to tcf to promote transcription by displacing groucho

Only available in high quantities when wnt ligand is present so that the lrp can prevent complex from tagging and phosphorylating it for degradation

LRP

co-receptor that binds to a complex when wnt is present to prevent b catenin from being phosphorylated and degraded

Adipocyte differentiation

The pi3k pathway with insulin promotes induction of fat

wnt and tgf b inhibits induction

NF-kB

the master transcriptional regular of the mammalian immune system

made up of p50 and p65 subunits

bound to inhibitor i-kba at rest

agents like infection, uv etc activate Ikb kinase which phosphorylates the inhibitor so that a ubq ligase can tag it

inhibitor gets degraded and free ___ can go into nuclues and induce specific transcription

Cerebral Cortex

Has 6 layers

small neurons for processing complex info at top

big pyramidal cells with descending nerve fibers to different areas at bottom

Neural Stem Cell formation

The cells bubble up and go to top

Newer cells float all the way to the top which pushes older cells lower

Thus, newest cells at top of cortex and oldest cells at bottom layer of cortex

Reeler mouse

spontaneous autosomal recessive neurological mutant mouse that walks funky

has cerebellar hypoplasia (no gyration = did not grow enough)

purkinje cell plate did not form , its a cluster cell mass instead of a nice layer

organize disruption, the cells are just all over the place in different directions

Reelin

extracellular ligand that reeler mouse lacks

glycoprotein

knew that it was extracellular because of pattern in sequence that was common in others

Controls radial neuronal migration and layer formation

Basically, surface neuron secretes this to tell the other neurons how to line up correctly

\

Dab1

adaptor protein in signaling pathway for reelin

leads to phosphorylation and degradation

Src-family Kinases (SFKs)

phosphorylates Dab after reelin signal

is inhibited by pp2