---start biochem.lec.12.5.96---

note: know 3rd and 4th questions from 2nd exam for the final, for dr 
jacencko's section.

for 3rd exam:
17 pts atchison, avahdani 9 pts, orsini 17 pts, pehrson 5,  shapiro 52.

for final:
atchison 16, avahdani 16, holzbaur 16, jacencko 4, miller 8, orsini 6, 
pehrson 16, shapiro 18.

lab writeup due...
lab counts as 10% of your grade! (phew! that's a bit of luck!)
--

dr shapiro discussed receptor pathways. we discussed one pathway using 
trimeric G proteins in vision etc. there is also the steroid receptor 
pathway, and the membrane bound receptor pathway, which uses a lot of 
kinases (enzymatic phosphorylation cascade). we'll be talking about the 
latter, and how growth factors elicit them.

GROWTH: increase in cell # or cell size. if you eat a lot, fat cells get 
bigger, don't increase in number. BUt we're talking today about cell 
proliferation, increase in number. all growth factors can cause 
proliferation, but also involved in ECM formation, cell motility, 
inducing cells to secrete stuff, proliferate, or differentiate.

how do these factors induce proliferation?

stages of the cell cycle:

G1, S, G2, M. 

during G1 the cell prepares for DNA synthesis. during G1 growth factors 
have their effects on the cell. during S phase, DNA is replicated, lots 
of histone synthesis, cell doubles DNA content. cell enters G2 period, 
lots of stuff goes on w/cyclins, then cell enters mitotic phase and 
divides into two daughter cells. then it can enter G0 - a dormant phase, 
- from which it could be restimulated to enter G1. vast numbers of cells 
in body are in G0. 

growth factors can enter the cycle in the G1 phase. 

GROWTH FACTORS:

proliferative effects, antiproliferative effects, effects not related to 
proliferation...see chart table one first page of handout.

categories:

small, proteinaceous: egf, tgf a, tgf b, etc

insulinlike: insulin

steroids

GROWTH FACTOR FAMILIES:
epidermal growth factor EGF: includes EGF, TGF a, et al

Platelet derived growth factor: PDGF AA, PDGF AB, et al

transforming growth factor b: tgf b1 tgf b2 et al.

interleukins: tons of them!

fibroblast growth factor
insulin like growth factor

see chart on handout...

So, what do all of these little molecules do? they ALL bind to CELL 
SURFACE RECEPTORS. these are NOT 7 alpha helical receptors of sensory 
systems. these are different, though they do have helical regions. each 
factor will bind to specific receptor on the cell membrane. this is an 
extracellular event. it causes a conformational change in the receptor 
protein which causes an effect INTRACELLULARLY.

we're going to discuss the induction of cellular proliferation by the 
MITOGEN ACTIVATED PATHWAY _ starting with binding of growth factor to the 
receptor.

the receptor undergoes a conformational change. some of these receptors 
have kinase activity on the cytoplasmic tail, which is activated when the 
receptor binds the growth factor. the receptors may dimerize as well.

when the receptor is activated the carboxy terminal portion is 
phosphorylated on specific tyrosine residues (see handout)

the phosphate groups on the tails of the receptor interact with other 
protein: Grb2. Grb2 will not interact with the NONphosphorylated 
receptor. there is a part of Grb2 which does interact with phosphorylated 
tyrosine: that's the SH2 domain of Grb2. (Grb2 is highly conserved in 
organisms, aka Sem2, DRK) SH2=sark homology 2. anyway, SH2 domain 
interacts with phosphorylated tyrosine residue. when the Grb2 interacts 
with the phosphotyrosine, IT undergoes a conformational change, and then 
it interacts much more efficiently with Sos protein. Eg, Sos won't bind 
well w/Grb2 unless the Grb2 is bound to the phosphotyrosin. So Sos 
interacts with the SH3 domains of Grb2. so the growth factor binds to 
receptor, receptor is phosphorylated on cytoplasmic tyrosines, interacts 
with Grb2 SH2 domains,  then Grb2 interact with Sos on the SH3 domains of 
Grb2. Once Sos bound to Grb2, it can then bind to Ras. Sos is a guanine 
nucleotide releasing protein, so it bumps off guanine nucleotides.. and 
it will knock GDP off of Ras. Ras is a G protein, which binds GTP/GDP, 
but it isn't a trimeric one, it's a monomeric protein. So, the inactive 
Ras/GDP is initially attached to the cytosolic side of membrane. After 
receptor is activated etc, Sos binds with Ras, causing release of GDP and 
binding of GTP - activating Ras.  now, all of this is happening near the 
membrane because of course ras is bound to the membrane as is the 
receptor. note that if Ras isn't bound to membrane, it won't work. 
Position is vital to these reactions. So, what does the activated Ras do?


Ras stimulates Raf - which is only activated by the GTP bound type of 
Ras, not the inactive GDP bound Ras. Raf is a kinase that phosphorylates 
Mek protein. Mek is also a kinase and Mek phosphorylates MAP Kinase or 
MAPK. MAP = mitogen activated protein kinase. Mitogen is the growth 
factor stimulating cells to divide. So, this is tremendous signal 
amplification which occurs when you link multiple kinases like this. MAPK 
has multiple targets: cell cycle regulators and transcription factors 
that require phosphorylation for activation. the transcription factors 
turn on growth related genes. all of these kinases phosphorylate on 
serine and threonine, whereas the receptor was phosphorylated on tyrosine 
residues as previously discussed.

so this kinase cascade turns on genes which are needed for the cell to 
proliferate. 

what about other pathways? this is just one cell surface receptor 
pathway. other pathways involve diacylglycerol, inositol phosphate, etc. 
they involve protein kinase C which has a whole other set of substrates 
from MAPK. though protein kinase C can interact with Raf as well. 

some pathways involve cAMP
cAMP is associated with activation of protein kinase A which has ITS own 
set of substrates. about protein kinase A...
it can also cross the growth factor pathway at Raf.

integrin pathway: surface proteins sense changes in ECM, and get 
stimulated and send signal to the Ras pathway, telling the cell to 
divide.

additional pathway involves Jak , which phosphorylates STAT pathway of 
kinases. the Jak kinase pathway can also lead into Raf pathway. STAT is a 
primary transcription factor.

what do we need to knwo? Mitogen activated pathway, growth factor 
receptor - the major pathway as outlined above.

so...cell starts proliferating. now what? need to be able to make it 
STOP! how do you do that? 

the terminal kinase in the cascade is MAPK. there is a MAPKphosphatase 
which DEPHOSPHORYLATES MAPK and shuts it off. the regulation is not 
totally clear. but as soon as a cell gets signal to divide, MAPK is 
activated, and within 30 min, MAPKPhosphatase levels increase within the 
cell and start shutting it off. then if you need more proliferation you 
need to resignal the cell.

another regulatory point is Ras, which must be bound to GTP to be active. 
Ras has an inherent GTPase so as soon as it binds GTP it starts cleaving 
it to GDP, so then it inactivates itself and it no longer stimulates Raf. 
so this is a very important regulatory point.

another protein called GAP (GTPase activating protein) stimulates the Ras 
GTPase to make it shut itself off more quickly.

what about the receptor? can you shut it down? these receptors are often 
dimerized, internalized, and degraded in lysosomes. that's a powerful 
mechanism for turning them off :)

so, who cares about all this? why should we care? how can we use this 
info? (besides to pass the test...)

well, it was exciting to discover this in the lab, for many reasons, 
including sheer joy in understanding. but later we found that diseases 
are related to this pathway - cancer is related to defects in this 
pathway, for one thing. overexpression of growth factors - many oncogenes 
are growth factors, overexpressed or which have very high affinity for 
receptor. if receptor is forced to stay on all the time, the cell will 
keep being stimulated to divide...

what about Grb and Sos? if Grb is overexpressed, cells are made 
tumorogenic (they are transformed, he said ..). Ras - 50% of human cancer 
has Ras mutations in it. if Ras GTPase is mutated so that it isn't 
efficient, Ras is overactive. Or, you could lose the GAP protein, and 
then Ras will remain active too long, and this is the case in 
neurofibromatosis - which is due to a defect in the GAP protein. that 
defect has to be homozygous to cause the disease...it's a recessive 
oncogene.

A lot of oncogenes are serine threonine kinases. 50% of known oncogenes 
are transcription factors. why? because they are at the end of the 
cascade, and there is hardly any regulation at that point, so it is very 
hard to compensate for a mutation at that point in the pathway. 

cancer is a multimutation disease. a single mutation isn't likely to 
cause cancer on its own. but many cancers have mutations of the pathway 
present.

in handout is list of oncogenes of known location in the proliferation 
cascade.these were mostly isolated from retroviruses.

in response to question: receptors sometimes dimerize and become 
internalized, not all of them.

FYI table of retroviral oncogenes, don't need to memorize them.

each step in this pathway can be regulated, so in a perfect world you 
should be able to INTERVENE. how can points in this pathway be targeted 
for regulation by new drugs? hmmmmm......

drug companies currently screen for compounds by doing bioassay of 
10000-50000 compounds til they find one that inhibits biological 
function, then they try to modify the one that works. this is all random, 
there's no real intelligent mechanism happening ....

----break-----

see list of diseases in handout for non-cancer related defects in 
receptor pathways. also some defects in g proteins unrelated to cancer; 
these are mostly in trimeric g proteins.

so, we've looked at growth factor pathway, and mentioned that there are 
specific points that are really important and one of those points had to 
do with Ras: it MUST be associated with the membrane. but is not normally 
associated with membrane: must be MODIFIED to attach to membrane. it 
attaches to membrane via a farnesyl group, which is put onto it by a 
farnesyltransferase. the farnesyl group is added to a CAAX sequence in 
the Ras (that's a cysteine, two aliphatic AAs, and then anything). once 
the farnesyl is added, ras can associate with the membrane. Now, what if 
you flooded the cell with the CAAX sequence? the farnesyl transferase 
wouldn't know what to do! there would be so much extra substrate floating 
around, effectively competing with Ras for the farnesyl transferease, so 
to speak. So less Ras would get farnesyl transferase to attach the 
farnesyl to it. so ras wouldn't associate with the membrane.

so suppose you have cancer with mutant Ras. what if you put a lot of CAAX 
into those cancer cells? You'd prevent the Ras from associating with the 
cell membranes! this does in fact work in culture. tumorogenic cells will 
revert to normal growth properties under these circumstances....

seediagram in handout: receptors with pathways ABCD around them. suppose 
there were a defect in pathway D. what if you could target the 
phosphotyrosine interaction with that specific SH2 domain? you could shut 
down that pathway.

any specific interaction in any of these pathways could potentially be 
interfered with. a new class of drugs called moleculomimetics are being 
designed to target specific interactions in these pathways. it is hoped 
that a new generation of clinical compounds will be developed in the not 
too distant future....

now if you target a pathway is it going to affect just the mutant cells, 
or all cells? This would affect ALL the cells. hopefully, it would be 
less toxic than current chemo, though - instead of KILLING all 
proliferative cells, you could just turn off some pathways. maybe you 
could combine these drugs with monoclonal antibodies to target specific 
cells.....

now if you stop the pathway, the cell may go through apoptosis and die 
anyway, and this could end up killing tumor cells....

how else might you be able to use growth factors clinically? stimulate 
damaged tissue cells to regenerate? 

TREATMENTS using GROWTH FACTORS:

four are being used in treating wounds: PDGF (platelet derived), TGF b, 
FGF (fibroblast gf), EFG (epidermal gf).

PDGF: made in platelets, which are made by megakaryocytes. PDGF is 
dimeric growth factor which binds to specific receptor. that receptor is 
found mostly on fibroblasts, monouclear phagocytes, endothelial cells, 
smooth muscle cells. can stimulate proliferation, chemotaxis, production 
of proteins needed for ECM. 

TGF b (transforming) made in most cells, binds to specific receptor. very 
confusing factor, has many different effects, positive and negative. will 
inhibit differentiation of fibroblasts into fat cells. will stimulate 
other cells to differentiate: stimulates muscle and cartilage 
differentiation, bone, as well as epithelial cells. very pleotrophic 
growth factor. 

FGF small peptide, 140 AA, binds to receptors on mesodermal type cells: 
fibroblasts, endothelial cells, sm muscle, etc. 

so, you have a wound. there's an injury causing the wound, there's 
hemorrhage, inflamation, granulation, scarring...

so first, hemorrhage. then clotting. during clotting alpha granules from 
platelets spill out their contents. these alpha granules are full of 
growth factors, esp TGF b, PDGF, and FGF. all these factors spill into 
the wound and start inducing things. they stimulate cell proliferation, 
to replace lost/hurt cells. they cause chemotaxis, recruiting helper 
cells. they stimulate production of ECM. the integrins on cell surfaces 
link up to the signalling cascades as noted before...they cause cells to 
migrate toward the wound site. also stimulates release of gf's from the 
matrix as well. FGF and TFG b are released from matrix at this time. so 
matrix is laid down, you get new cells, you start seeing granulation 
tissue forming. collagen, fibronectin, other ECM components: the genes 
coding for them are also turned on by these factors. macrophages come in 
to chew up debris. you see loose cellular association, neovascular, 
differentiation of fibroblastoid cells into myofibrils, which contract, 
to close the wound edges. ultimately the granulation tissue is replaced 
w/new matrix and scar is formed. (" a scar is born" :))

note: fetuses don't scar...don't get infected easily.

also need to heal epithelium- stimulated by EGF epidermal growth factor. 
small, but comes from huge precursor. will stimulate any cell with EGF 
receptor to proliferate. there is a gland which make lots of EGF in most 
animals. this is the salivary gland- the submaxillary gland. so when an 
animal licks wounds, it's adding EGF.

can you use these factors clinically to speed healing? yes. can speed it 
about twofold. so a wound could heal in 3 days instead of a week. can 
also speed healing in conditions when healing is slow, eg in diabetic or 
patient on chemotherapy. 

you can add EGF to burns and wounds and increase healing twofold. need to 
apply topically in an ointment. 

can also apply to surgical incisions. use PDGF and TGFb to surgical 
wounds.

now, what about impaired healing, like in chemo patients? use EGF, PDGF, 
and TGFb. this combo will essentially reverse the inhibition caused by 
chemotherapy. so these factors can be used to heal wounds.

what is the danger of using these growth factors clinically?

TGFb - transforming growth factor. this can cause cancer. that's what 
"transforming" means. so it's not to be used lightly. TGFb has many 
diverse effects. can stimulate or inhibit cell division depending on the 
dose. a low dose will stimulate synthesis of PGDF and proliferation. high 
dose of TGFb will inhibit expression of PGDF receptor, and inhibits 
proliferation.  so dose is really quite crucial. TGFb also induces ECM 
formation.sounds good, right? but if you have too much you can get scar 
formation in the kidney. that is a Bad Thing. 

DECORIN inhibits TGFb. if you give decorin it will inhibit TGFb. so you 
can have animals overexpressing TGFb, you treat them with decorin, animal 
stops getting scar formation in organs.

but if you get rid of ALL TGFb (eg in knockout mice you can delete the 
gene for it or whatever), mice born normal, die about 20 days later of 
massive inflammation. so lack of TGFb is a lethal defect, TGFb somehow 
regulates immune function. so you have to be really carefull with it.

INHIBIN and ACTIVIN are two other growth factors in the TGFb family. they 
control gonadal cells and are antagonistic to eachother. if you lose 
inhibin you see gonadal stromal tumors. so the balance is very important.


see handout for chart of potential uses for growth factors.


some clinically used growth factors include TGFb for retinal wounds
erythropoietin for anemia, granulocyte colony stimulating factor already 
approved.

realize these factors all have diverse biological effects.

-------

next two weeks:

tomorrow (12/6), 1pm - Q and A review session in B101. not sure if Dr 
Orsini will be there. Dr Miller is available Dec 19 10 am in rm 13.

Exam Tuesday the 10th hopefully will be back ASAP, will try hard to do it 
w/in a few days.

FINAL on 12/20. the good news is that we'll have recently studied for 1/3 
of it. try to study in small chunks so it is a bit more manageable. at 
the end of the final we will get an evaluation form. we can evaluate the 
course and the instructors. profanity is discouraged and will be ignored. 
please be thoughtful. you can't turn it in that day, though. wait til the 
anger has subsided, then turn it in. we'll get the finals back probably a 
week into the third quarter.










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