Cell bio exam 3: cell signaling

What is the fundamental problem of cell signaling?

 How does a cell “transduce” the binding of an extracellular ligand to elicit short-term and/or long-term intracellular physiological responses?

group of hormones that are cell signal that actually enter cell:

• Steroids, retinoids, thryoxine

• They are more hydrophobic and diffuse through the pm. Do not need an integral membrane protein receptor .

• Their receptors are in the cytosol and are inactive until they are bound.

• Goes straight to nuc .activate transcription of target genes 

classes of signaling receptors

1: G protein coupled receptors

• very rapid change

• changes cellular physiology / metabolism

• reversible (physiology goes back to what it was before signal once the signal ends)

• short term immediate effects

• not synthesizing any proteins , not going to nuc, not changing transcription

2: rtk signaling

• receptor tyrosine kinases

• proliferation

• slower, long term effects

• genetic reprogrammnig, going to nuc, changing transcription , making proteins

• end goal is go make G0 cell enter the cell cycle and initiate dna synthesis

signal amplification

• One signal binding to one receptor gets amplified. Turn on multiple g proteins, multiple kinases. So the signal is increasing dramatically. 

• The signal is in the namomolar change. The change in response isin the millimolar range. 

4 schemes for intracellular signaling

endocrine signaling: most common in multicellular organisms. The source of siganling is physical distance away from target. Example glucagon / insulin which are secreted into blood stream and their target is way down. Requires small diffsuable molecules that are transported over large distance 

paracrine signaling: signal and target are adjacent. No cell - cell contact, but they are next to each other , have a synapse . for growth factors, nerve cells

autocrine signaling:  target sites on same cell/ cell produces the signal and also receives it . proliferation

Plasma memabrne attached proteins. Cell that are close enough that their integral membrane proteins hook up. Cell - cell contact. 

hormone

• any diffusable molecule that participates in endocrine signaling. 

agonist

 natural derivative of hormone. Have lower or higher affinity based on want, and can mimic effect of hormone. Do the same thing as hormone

• acts as cell signal , like hormones

GCPR pathway

signal activates receptor

receptor is activated . next target is g protein

• Called heterotrimeric g protein complexes. Alpha,beta, gamma sub parts. beta is peripheral. Alpha and gamma are lipid anchored proteins. 

  • We are about alpha. Is the gtp binding protein, is the gtpase. Is the one that activates effector enzymes

  • alpha starts gdp bound (inactive), and also bound to beta and gamma 

  • signal-receptor coplex comes over and is the gef 

  • gdp dissociates and exchanged with gtp . alpha is not gtp bound , active state 

  • alpha subunit dissociates from beta and gamma, and goes to the effector enzynme. turns it on 

Effector enzymes produce second messengers (small diffusable molecules like camp) that transduce the signal to the inside of the cell. 

at some point, G alpha will hydrolyze its gtp. determines rate / burst of effector protein making camp .. g alpha hydrolyzes and dissociates from effector, which turns it back off. Goes back to beta/gamma

• Hormone is still bound to receptor = cycle continues 

• Hormone is not bound = cycle stops 

• Has a bursting pattern of making camp. 

10000 fold amplification of the signal to the second signal. 

effector enzymes

produced second messengers (small diffusable molecules, like camp) that transduce the signal to the inside of the cell.

is turned on by g alpha

otherwise off

heterotrimeric g protein complexes

• Alpha,beta, gamma sub parts. beta is peripheral. Alpha and gamma are lipid anchored proteins. 

  • We are about alpha. Is the gtp binding protein, is the gtpase. Is the one that activates effector enzymes

the receptor goes here after being turned on by signal 

GPCRs are coupled to heterotrimeric G protein complexes (e.g., Gs, Gi, Go, Gq Families)

• Three families : alpha subunit functions: . there are 16, here are four

  • G s alpha = activates adenyly cyclase. Increases camp 

  • Go / gq = activiates this integral membrane protein (see above). Get two second messages. One is polar head (IP3) and opens Ca channels (more in). DAG are two fatty acids and stay in PM 

  • G i = inhibitory g protein. Decreases camp. Actviates a gi that interacts with adenyl cyclase and drives down camp production

• Cells have high diversity and felixibily on physiology bc of their diversity of gpcrs. 

gcpr structure

The human genome encodes ~850 GPCRs: All have 7 membrane spanning a-helices

The ligands for many GPCRs are unidentified so these GPCRs are called “orphan receptors

The modular structure of GPCRs means you can make chimeric receptors by fusing the ligand binding module from 1 receptor (e.g., bAR) with the activation module from a different receptor (e.g., AchR)

• Has ligand binding domain and activation module. Can be separated. 

• G protein binding/activation module determines its role (has no knowledge of what its supposed to do). This part interacts with g alpha s. The gef region 
  

How did phosphorylation evolve as a mechanism to conditionally regulate enzymatic activities?

• Why did cell pick phosphorylation in evolution ? 

• Easy to put on and take off 

• Using atp: atp very abundant 

• Lab found: 

  • Looking at enzymes where phosphorylation turns off. Ex Glycogen synthase: converts glucose to glycogen. Wants to inhibit this to have higher blood sugar. Have a serine or threonine near catalytic site of enzyme. Phosphorlate = large, negative phosphate group inactivates their catalytic activity , turns em off 

  • Looking at enzymes there phosphorylation turns on. Comes from enzymes that were always on. They all required electrostatic interaction between + and - charged AA for their structure. Over evolutionary time: gene duplication. Copy can acquire new mutations. Mutation: - charged AA now S or T. lost the charge = is going to be inducable  bc will not work unless restore - charge through phosphorylation. Phospharlyated = active bc right structure 

    • Ex glycogen phosphorylase. Active = breaks down glycogen to glucose. Higher blood sugar

  • Both of these enzymes work in repsonse to epinephrine 

  • Through evolution : - charged AAs replaced with S or T 

  • Ex Topo2 and enolase. 

  • Further back in evolutionary time = - charged AAs 

  • Newer = T or S

second messengers

• made by effector proteins

• The first two: camp activates pka. Cgmp activiates pkg 

• Third: DAG. actiavtes PKC 

  • Phosphatelipase cleaves and creates DAG and the fourth one. Said this around 20 mins into the recording bro talks too fast 

• IP3: opens Ca channels. More Ca in 

• NO and cgmp are involved in acetlycholine signaling.

How are they generated? (e.g., Adenylyl cyclase)

 What are their targets? (e.g., Protein kinase A)

 How are they degraded or inactivated? (e.g., cAMP phosphodiesterase)

What provides the specificity for S/T kinases vs Y kinases?

kinase structure:

All Protein Kinases Share a Common Mechanism & Generic “epsilon” Structure

• Bc they have a cleft. The atp and substrate binding site. Have to be together bc transferring the P onto the hydroxyl group of the STY . 

• Makes it difficult to design specific kinase inhibitors 

• Substrate specificity: ST vs Y kinase. 

  • Size of the cleft (next slide)

  • Y is pretty big = cna reach atp . Y kinases have deep cleft. S and T cannot get close enough to atp 

  • S T kinases have more shallow cleft . apt is much closer to exterior. S or T are able to com ein contact with atp gamma phosphate. Y doesnt fit 

  • Y kinases have deep cleft. ST kinases have shallow cleft. 

Kinase-substrate complexes are relatively long-lived. This means they can be biochemically isolated, and you can identify the substrates for a given kinase by coimmunoprecipitating complexes using an antibody specific for the kinase.

kinases

• Main players in cell signaling are protien kinases

• Kinases phosphorylate amino acids with OH groups

• classe: 400 S/T kinases in the genome . Y kinases are far fewer. 

  • Serine is preferred over T

• Have tiny group (18) called dual specificity kinases and they can phosphorylate all three of them. 

  • Do not do anything in this pathway 

  • Netierh does Y i think 

  • T phosphorylation going wrong  = underlying cause of cancer 

The 3 classes of protein kinases are based on the amino acids they phosphorylate on target proteins

Target protein activity is regulated by changing [phospho-protein]:[non-phosphorylated protein]

• Use gamma phosphate from atp during phosphorylatin, add it to the protein/amino acid via kinase. 

• Increase kinase = makes more substrate for phosphatase and would reverse it. SO kinase activates the inhibition of the phosphatase that would reverse it . 
  

phosphorlyation:

kinase Bind atp.

Substrate bind kinase.

Transfer phosphate from atp to S/T/Y.

Substrate release.

Adp release

phosphatases

the opposite of kinases

dephosphorlyate 

Note: Phosphorylation activates most proteins but can inactivate others!

have way more of them for Y than s/T bc more concerning when they go wrong.

Phosphatases are inactivated when kinases are activated

• Cells also keep some uninhibited phosphatase

• Resting state cell = phosphate has the upper hand. Default active, need to be shut off by kinase

• Phosphatases activate most proteins. But not all of them. Cannot assume 

what are the α, β, and γ subunits of heterotrimeric G protein complexes?

The α and γ subunits are lipid-anchored proteins, β is a peripheral protein.

function of the Gα subunit of G_s

Gαs increases cAMP levels,

function of Ga subunit of Go/Gq

Gαo increases IP3 and DAG levels,

function of Ga subunit of Go

, Gαi reduces cAMP levels.

What is a "dual specificity" kinase?

A kinase that phosphorylates serines, threonines and tyrosines