Exam 4

What is the body's primary anabolic hormone?

Insulin (energy storage)

Insulin

A protein hormone synthesized in the pancreas that regulates blood sugar levels by facilitating the uptake of glucose into tissues
Growth factor
Stimulates sugar/AA uptake
Signals building macromolec,cells,tissues

What is a primary function of RBCs?

Consume a lot of the body's glucose (to run glycolysis)
Makes small amts ATP

What happens when glucose reaches the pancreatic b cells?

High glucose promotes insulin release

Where is glucose primarily taken up?

Muscle and fat (both have insulin stimulated glucose transport)
Stimulates glucose storage and glycogen formation

How does the body get a hunger signal?

Sugar uptake lowers blood glucose lvls

When happens in response to lowered blood sugar at night?

Blood sugar decreased —\> glucagon released—\>glycogen b down to raise bs levels

Pathway of glucagon release

Glucagon bind GPCR—\>
G protein alpha is stimulated—\>
GTP bound alpha activates adenylyl cyclase—\>
ac hydrolyzes atp to convert to cAMP—\>
cAMP activates PKA—\>
PKA phosphorylates phosphorylase kinase—\>
PPkinase phosphorylates glycogen phosphorylase—\>
Phosphorylated glycogen phosphorylase hydrolzyes glycogen to GLUC6Phos—\>
B down

pre-proinsulin

an inactive precursor molecule of insulin that includes a signal peptide
Translated/translocated to RER

How to make mature insulin

Cleave proinsulin with endopeptidase
Exoprotease makes mature insulin monomers
Package & fuse insulin vesicles to raise insulin lvls

Insulin structure stored vs in blood

Stored: hexamer
Blood: monomer

Insulin receptor effects

increase:
glucose uptake
AA uptake
ACoA to FA
glucose —\> glycogen,
protein synth
increase Na/K atpase
decrease:
pyruv to glucose (gluconeogen),

Insulin receptor structure

Already dimer
Two extracellular ⍺ subunits and two transmemb β subunits linked together by disulphide bridges.
Tyr kinase domain on cyto side of β subunit

How is insulin receptor regulated?

Active as long as ligand bound
Downregulated through internalization

Why cant the insulin receptor dimer phosphorylate itself if already a dimer?

Domains are physically to far apart
Need conformational change in the tyr kinase domains to bring together and phosphorylate

IRS1 (insulin receptor substrate 1)

Docking protein that has a PTB domain and binds the insulin receptor

How does PTB domain complex with the insulin receptor?

• brings receptor's kinase domain in close proximity to c term of IRS1 to phos other tyr
• when tyr is phosphorylated on IRS 1, SH2 dock on IRS 1
• SH2 concerned with C side

adaptor proteins

link signal proteins together (grb2)

docking proteins

supply receptors with other tyrosine phosphorylation sites (IRS1)

scaffold proteins

organize groups of intracellular signaling molecules into signaling complexes (ksr)

FRET (fluorescence resonance energy transfer)

measures whether two proteins are in contact with one another
Put proteins close and match fluorophores so emission of A overlaps with excitation of B
Need close or contact

IP3 function

go to ER and stim Ca release
Get IP3 from phospholipase cleavage

PIP3 function

recruits proteins to membrane (serves as bind site) (ring is inside membrane)

PIP3 structure

2 acyl chains in membrane
Glycerol backbone
Phosphotdiester link to hexameric ring

Where is PIP3 phosphorylated?

3 position in ring

PI3K

Lipid kinase that converts PIP2 into PIP

PI3K

P85 has SH2 domain to bind to IRS1
P110 is the catalytic subunit of PI3K

PI3K pathway

Growth factor/ insulin bind insulin receptor—\>
Receptor phosphorylates IRS1—\>
IRS1 (close to receptor) phosphorylates receptor—\>
One of IRS phos sites is PTB for p85—\>
P85 binds IRS with SH2 domain—\>
P85 undergoes conf change and bind to p110 to activate and form PI3K—\>
p110 converts pip2 to pip3

PI3K pathway effects

Promotes cell survival, proliferation and chemoresistance
One of most commonly alter pathways in tumors

What binding domain does PIP3 contain?

PH

Proteins with PH domains and where they come from

Akt & PDK1
Both ser/thr kinases
Recruited from cytoplasm to plasma membrane

How do Akt and PDK1 interact with PIP3?

Akt docks on PIP3
PDK1 phos Atk in the membrane and activates
Atk releases into cyto and phos many proteins n such

Akt

central regulator for many processes
• stim glucose transp, glycolysis, glycogen synthesis, cell proliferation (anti anti), protein synth
• Inhibits apoptosis

What inhibits Akt?

PTEN (competitive)

PTEN

lipid phosphotase that dephosphorylates the 3' positon back to pip2 (turn off)
Tumor suppressor mutated in cancer
When pip3 formed PTEN and Akt battle for binding

Why do PTEN mutations cause cancer?

because longer/robust signal from pip3 to akt
Less competition for akt

E: what happens when one ph domain has higher/lower binding affnity?

Why do many kinases need 2 phosphorylation events to fully activate?

Each one gets to half speed (partial activation)

What is rapamycin?

drug with strong
immunosuppressant/antiproliferative properties
binds to and inactivates mTOR (discovery)

mTOR

ser/thr kinase - target of rapamycin (targets kinase activity of mTOR and inhibits)

How does Rapamycin inhibit mTOR?

Binds to FKBP-12 and complex associates with mTOR to inhibit

mTOR complex 1 (mTORC1)

With raptor
• Sensitive to rapamycin
• Indirect reg by Akt
• Stim cell growth
◦ ribosome production
◦ Protein synth
◦ Inhibit degradation
◦ Stim nutrient uptake
• inhibit autophagy (ULK1/2)

mTOR complex 2 (mTORC2)

With rictor
• rapa insensitive
• Regulate Akt by ser phosphorylation
• Reg cytoskeleton/cell metab

mTORC1 activation pathway

Activate Akt —\> activate mTOR—\> increase protein synth with phosphor, increase glycolysis, stim glucose uptake

mTORC1 features to remember

centrality, downstream Akt, many diff substrates when activated
Upregulates machinery for daughter cell growth

How is mTOR indirectly regulated by Akt?

Active Akt phos and inactivates Tsc2 (GAP protein& Rheb substate)
Rheb accumulates in GTP bound form which activates mTORC1

What do prokaryotes use for signaling?

2 component sys with phos transfer

What do mTOR and ULK1/2 do?

M: Growth regulation
U: self destruction regulation

Autophagy

cells degrade themselves in response to fast/nutrient deprivation (components of the cell) to make building blocks

Which organelle is autophagy linked to?

Lysosome (primary hydrolytic organelle)

Autophagy mechanism

damaged prot/organelles engulfed by dub membrane and make autophagosome, fuse with lysosome and degrade

Why degrade mitochondria?

Energy sources decline, shut down ox phos, depolarize membrane and signal degrade

What is the key kinase that activates the autophagy cascade?

ULK1/2

ULK1/2

Substrate for mTOR

How do you inhibit autophagy?

Phos ULK12 by mTOR

When is mTOR active/inactive?

Active: day (lots glucose/insulin) (nutrient rich)
mTORC1 complex raptor, phos ser on ULK (inactive)

Inactive: night (sleep/nutrient poor)
ULK complex active
Ampk phos tsc2 activates (ser/thr kinase en debt)

Cytokine signal paths

Image

PIP3 path/inhibition

Image

TGFβ Receptor super Family (common ancestors )

Receptor Serine/Threonine Kinases (growth factors)
• 2 receptors not seq related to each other, both ser/thr kinases
• Cleavage rxn, dimerization released with activation signal

How does TGFB receptor activation occur?

Bind ligand type 2 phosphorylate type 1 receptor (activate kinase activity)
Both receptor homodimer combine physically associate to become tetramer

What is the typical signaling in TGFβ?

Paracrine or autocrine in cancer

R-SMAD

substrate for ser/thr kinase t1 subunit

What does R-Smad associate with?

CoSmad
2RSmad 1CoSmad trimer

SARA

scaffolding proteins anchor Smads and receptors
PIP3P binding protein binds only 3 position

TGFB pathway

TGFB receptor binds T2-\>T2 phosphorylated-\>T1 phosphorylation by T2 and activate kinase activity-\>T1 phosphorylates RSmad-\>RSmad CoSmad trimerize
-\> translocation to nucleus and transcription

Typical role of TGFB

repress proteins in cell cycle control (tumor suppressor)
Mutations in pathway potentiate growth

What does TGFB repress to inhibit proliferation?

Myc, Cyclin D

What happens to TGFB in cancer?

Tumors become TGFB resistant and oncogenic (receptor downreg or mutant)
High TGFB expression changes responsiveness

What are the two major ways tumor suppressor TGFB can be altered in cancer?

Loss of function in core path components
- Receptor mutation
- Mutated SMAD4

Downstream alterations
- Because TGFβ Affects so Many Functions, can Tumor Promote Cancer Progresses

What pathways can TGFB affect?

Tumor promotion: Growth stim, angiogenesis , immune suppression , cell speciation

Tumor suppression: growth inhibition, apoptosis

Atrial Natriuretic Peptide ( ANP) Receptors Structure

Extracellular ligand binding domain
1 Transmembrane domain
Kinase homology domain (KHD)
Guanylyl cyclase domain on cytosol side

Atrial natriuretic peptide (ANP)

hormones secreted by heart in response to high blood pressure
Relax smooth muscle cells in blood vessel/stim excretion of Na with H2O (natriuresis)
Loop with disulf bridge
Also BNP &CNP

Guanylyl cyclase function

GTP to cGMP

Kinase homology domain (KHD)

Becomes phosphorylated (not sure how)
Isn't catalytically active
Dephosphorylation desensitizes and lose ligand (inactive GC)

cGMP

Actions mediated by PKG binding

PKG structure

Ser/thr kinase
Single polypep 2 domains
1 regulatory domain
1 catalytic domain
Domains fused

How is PKG under basal conditions?

PKG is auto inhibited
Bind cGMP to activate catalytic function PKG

How do you turn the cGMP pathway off?

Hydrolyze to to 5' GMP with PDE

ANP receptor pathway

Receptor binds ANP—\>KHD phosphorylated —\>activates GC domain—\> convert GTP to cGMP—\> cGMP binds R site on PKG—\>conf change active PKG —\> targets phosphorylated

What are the targets of PKG?

IP3 Ca channel in ER (independent of IP3)
PKG phosphorylates channel and inhibits

What happens when PKG phosphorylates IP3 Ca channel?

Less Ca release from ER—\>less smooth muscle contraction
More SM relaxation as long as cGMP around

Sildenafil purpose

Treats angina (reduced blood flow to heart)
Relax\= increase blood flow

What controls smooth muscle contraction and allows for greater lvls of blood flow?

Nitrates (nitric oxide NO)
Through rxn of cGMP

PDE5

phosphodiesterase that cleaves cGMP & stops SM relaxation
More blood vessel contraction (smaller bv)
Want to combat

Nitric Oxide (NO)

Highly reactive free radical gas short 1/2 life
Ligand binds to SGC

Which enzymes synthesizes NO witch which amino acid?.

nitric oxide synthases (NOS)
Use Arg as N donor and oxidize
Arg—\> citruline

How was NO discovered as a signal molecule?

Acetylcholine and SM cells

In what form does NO regulate cGMP synth?

Radical

Types of NOS

nNOS: nervous sys signaling(reg enz)
eNOS: vascular signaling (reg enz)
iNOS: killing microbes, virus (reg gene transc)
INOS oxides aas on protein on bug surface and kills

What is the site of NO production?

Endothelial cells through eNOS
Gas diffuses to regulate constriction

SM cell relaxation pathway (soluble GC)

Activate eNOS in endothelial—\> convert arg to cit to make NO—\>NO diffuse between cells—\>NO activate GC—\>GTP to cGMP conversion—\>PKG active—\>Ca channel phos (relaxation)

Soluble Guanylyl Cyclase (SGC)

Cytoplasmic enzyme (not receptor)
GTP to cGMP

How does NO bind to SGC?

Cov bind to the Fe of heme group in GC
Is 6th axial Fe ligand to induce conf change (same place where o binds)

How does SGC convert GTP to cGMP?

GTP binds a b subunit interface
Basic Asp in cat subunit extract proton from 3' OH
3' O attack alpha phosphate

Channels

membrane proteins that form pores that let passive movement of ions (ion channels), water (aquaporins), or other solutes to passively across the membrane along their electrochemical gradient.

Which two important properties distinguish ion channels from aqueous pores?

Ion selectivity
Not continuously open but are gated

Ion channels

Always passive (conc grad)
Small and strip water off ions that go across

Which signals regulate ion channels?

Voltage
Ligand gated (extra/intercell)
Mechanically gated

IP3 is voltage gated

How is synaptic junction signaling regulated?

Ligand gated
Respond to action potential & neurotransmitter fuse with membrane into synapse
released neurotransmitter binds to and opens the transmitter- gated channels on the postsynaptic targ et cell.

What type of bond does acetylcholine have?

ester bond —\> can be hydrolyzed by esterase