BI 256: 15/cell signaling

How do yeast communicate?

yeast secrete peptide mating factor, lets the find a mate in essence by sensing presence of other cells, pheromone type signaling system

What are yeast?

unicellular, divide asexually, close genetic relatives to humans

When yeast detect other cells, what happens?

form a shmoo, try to find a mate to reproduce with, opposite mating types to make a diploid zygote, sensed by secreting a compound neighboring yeast receive

How is cell communication involved in fertilization?

egg communicates w/ sperm for directions, 1/14 million reach fallopian tube, once there sperm gets chemical signals to continue forward

Do humans use pheromones?

humans can perceive to some extent, body odor between M/F differs from bacteria in skin producing odors,

What do pigs use to communicate?

androstenedione, released so male pigs can communicate to female pigs ready to mate, in human creams for males to get females

How is androstenedione seen in humans?

genetic makeup influences how you perceive it, SNIP variation, 1/3 population doesn’t smell it, 1/3 as cat pee, 1/3 as vanilla/floral

What must all cells do?

communicate w/ e/o to coordinate cellular functions & responses

How can cells make decisions?

integrate multiple signals, make most decisions based on info gained from outside world, cells get many 1 time & decide most appropriate response

What kinds of signals are there?

• ABC together = survive

• ABC + DE = survive, grow & divide

• ABC + FG = survive & differentiate

What happens if there is no stimuli from the environment?

apoptosis w/o contact from neighbors/signals

What are first messengers?

extracellular signal molecules, bind to receptor proteins in target cell’s PM

What are signal transducers?

receptor protein in PM, transmembrane protein, transduces signal from outside to inside the cell

What happens inside the cell after first messengers & signal transducers?

secondary messengers/intracellular signaling proteins relay signal from receptor to effector proteins

What are effector proteins?

carry out physiology of the signal

What is an example of an effector protein?

• metabolic enzyme altering metabolism

• transcription factors/regulator proteins altering gene expression

• cytoskeleton → alter cell shape & movement

What happens if a cell doesn’t have a receptor for the signal molecule?

signal can’t be transduced

What is signal transduction?

extracellular signaling pathway leading to change in the cell, taking something happening outside the cell & converting into cellular response inside the cell

What are cell surface receptors?

Proteins on the cell membrane that bind to signaling molecules, initiating a cellular response, receive hydrophilic signaling molecule

What binds to surface receptors?

hydrophilic signal molecules binds, then signal transduced inside the cell, binds outside cell in aqueous environment, receptors help inside otherwise can’t cross PM

What is an example of a cell-surface receptor?

insulin receptor, LDL receptor, out outside because signal molecule is hydrophilic, can’t cross PM into cytoplasm on its own, like Clathrin

What are intracellular receptors?

in the cell, nucleus or cytosol, receive signals from carreir protein

What signals does the intracellular receptors get?

small hydrophobic signal molecule, delivered to cell by carrier protein based on chem, carrier protein diffused to enter, binds & activates

What are examples of intracellular receptors?

T & P receptors, bound by carrier proteins & delivered to cell, diffuse through & activate IC receptor

What is contact-dependent signaling?

cells have to touch for signal to be transmitted, membrane bound signal molecules must physically interact w/ membrane bound receptor on target receptors

What must occur in contact-dependent signaling?

physical interaction between 2 cells to pass signal

ex - contact inhibition

What is the difference between contact dependent & paracrine signaling?

Contact-dependent signaling requires direct cell-to-cell interaction & membrane bound signaling, while paracrine signaling involves the release of signals from one cell that affect nearby target cells without direct contact.

What is paracrine signaling?

A form of cell signaling where signaling molecules are released by a cell and diffuse over short distances to nearby target cells, influencing their behavior, hydrophilic into area

When does signaling count as paracrine?

when diffusion of signal molecules is over a short distance

What cell types use paracrine signaling?

Various cell types, including immune cells, nerve cells, and endocrine cells, utilize paracrine signaling to communicate with neighboring cells.

immune cells → inflammatory signals released by WBC/phagocyte to recruit other immune cells to the site of infection, increased inflammation & immune activity

What is synaptic signaling?

A specialized form of paracrine signaling that occurs at synapses, where neurotransmitters are released by neurons and bind to receptors on adjacent target cells, typically influencing nerve signal transmission

What is endocrine signaling?

Endocrine signaling is a type of long-distance cell communication in which hormones are secreted into the bloodstream by endocrine glands, affecting target cells far away from the site of release.

When does signaling count as endocrine?

diffusion signal molecule needs help of the circulatory system

How can signals act over short periods of time?

signaling molecule binds to cell surface receptor, activates fast signaling cascade, only alters protein function by de/phosphorylating or rearranging cytoskeleton for motility

How can signals act over long periods of time?

slow, altering gene expression & protein synthesis, change to express different proteins, alter cell behavior

What do fast & slow signals have in common?

ultimately alter cytoplasmic machinery & cell behavior

How can signals be bidirectional?

pass through gap junctions, narrow water-filled channels allowing direct contact between 2 adjacent cells, epithelial, allow exchange of ions & small molecules, direct pore, connect cytoplasms

Where is gap junction transport seen?

epithelial cells, necrosis & cell death

easy to pass intracellular signaling molecules to neighboring cells, apoptosis/necrosis spread through gap junctions

How can signal molecules have different effects on different cells?

extracellular signal carries little info/context, only induces cell to respond according to predetermined state, depends on cell & genes expressed, 1 signal can activate multiple receptors or vice versa

How is acetylcholine used across cell types?

• heart pacemaker cell → GPCR interacts, decreased rate of firing

• salivary gland cell → GPCR interacts, secrete saliva

• skeletal muscle cell → acetylcholine gated ion channel, leads to muscle cell contraction

How do cell-surface receptors relay signals?

relay signals via intracellular second messengers, inside cell transducing signal,

How can second messengers be controlled?

proteins, activity can be controlled by phosphorylation

turn on w/ protein kinase transferring P on, turn off w/ protein phosphatase taking P off

How else can second messengers be controlled?

GTP binding proteins, use GAFs & GEFs, GTP = on, GDP = off,

How doe cell surface receptors use other second messengers/not proteins?

cyclic AMP & Ca2+

How is Ca used in cell signaling?

Ca is kept in ER, pumped out to extracellular space w/ Ca ATPase pump, Ca binding proteins keep inactive in cytosol

What are the types of cell surface receptors?

• ion channel-coupled receptors

• G-protein coupled receptors

• enzyme-coupled receptors

What are enzyme-coupled receptors?

most common is receptor-tyrosine kinases/RTK, single pass transmembrane proteins on PM, cytosol has conserved tyrosine kinase domain that binds to signal molecules

How are RTKs activated?

dimerization & trans-autophosphorylation, when each subunit binds a signal protein, phosphorylates other on tyrosine residue & activates receptor

• signal protein binds, bringing 2 RTKs together/dimerize, trans-auto phosphorylate, activate kinase domains, make binding sites for signaling proteins

signaling protein shape induces dimerization of RTKs → brings close to trans-autophoshprylate e/o, recruits signaling proteins & helps pass signal down stream

How does trans-autophosphorylation work?

swap several P across e/o, make landing pads/recruitment sites for additional proteins

What can RTKs activate?

Ras, small GTPase anchored to PM, activate RTK = activate Ras, downstream effects, always on independent of upstream, overactive mutant removes need of RasGEF to activate

How does RTK activate Ras?

1. RTK activated by trans-autophosphorylation

2. Recruits Grb2/adaptor protein & SOS/Ras-GEF which facilitates GDP-GTP exchange on Ras, activating it.

Ras-GTP = active

Ras-GDP = inactive

Once Ras is activated, what happens?

MAPK cascade activated, short live activation, MAP kinase cascade carries signal into nucleus to change gene expression

Ras → MAP kinase ³(Raf) → phosphorylate & activate→ MAP kinase ² (Mek) → phosphorylate & activate → MAP kinase (Erk)

What is Erk?

MAP kinase, 1 of most important signal molecules, master of cell proliferation, phosphorylate proteins for initiation quick changes in protein activity & gene expression

If you were making a drug to target a cancer, what part of the Ras pathway would be blocked?

Inhibiting Ras or its downstream effectors such as Raf or MEK could prevent cell proliferation and survival signals in cancer treatment, want to go as low as possible = Erk, upstream = more negative side effects, only want cancer gone not healthy

What is the clinical relevance of Ras?

Ras is overactivated in 30% of human tumors, upregulation & overactivity of ERK drives cell proliferation

What are GPCRs?

G-protein coupled receptors, made of 7 transmembrane helices, always associated w/ G protein on cytosolic face of membrane, N term outside the cell, protein/ligand binds → C term inside the cell, C3 loop associate w/ G protein/recruits

What are G proteins?

heterotrimeric = alpha,beta, gamma units, alpha & gamma subunits lipid-linked to the plasma membrane, alpha subunit is GTPase, a-GDP = inactive, a-GTP =active, beta sandwiched between a/y

How are G proteins activated?

1. signal molecule binding activates GPCR/binds to internal domain

2. GPCR becomes a GEF, a-GDP/inactive → a-GTP/active

3. a-GTP makes conformational change, dissociates from B/y, stuck to membrane

4. a-GTP binds to target effector activation

   1. enhances GTPase activity of a subunit to inactivate, hydrolyze & turn off, next activation = want to turn off immediately

   2. alt → a-GTP binds to regulator of G protein signaling/RGS = GAP forcing hydrolysis, secon

Which subunit can turn on y/B?

only the G protein alpha subunit can, turns on heterotrimeric G protein

When would the alternative binding of a-GTP to RGS be needed?

secondary level of control, important signaling activation

What happens after By are activated?

activate downstream targets, additional to what alpha activates

How does GCPR adapt to signaling?

• receptor sequestration

• receptor down-regulation

• receptor inactivation

• inactivation of signaling protein

• production of inhibitory protein

What is receptor sequestration?

cells have evolved ways to ignore/dampen down response to signal, respond for too long = tone down sensitivity to it

RS pulls receptor into a vesicle w/ endocytosis, keep in endosome until response again

Can a signal go for a very long time?

No, signals are eventually terminated through various mechanisms, like receptor sequestration or inactivation, exposure to high concentration of signal for prolonged period can lead to adaptation or sensitization

What is receptor down-regulation?

sustained for long period of time, reduce number of receptors on surface, route receptor to lysosome for degradation, downregulation to really ignore signal/decreased sensitivity

What is receptor inactivation?

produce intermediates to block GEF exchange & activation of receptor, receptor is less responsive/non-functional (phosphorylation, inhibitory proteins)

What is inactivation of signaling protein?

inactivate another protein down the pathway to prevent cascade, possibly alpha subunit,

What is production of inhibitory protein?

blocks activity of the pathway or receptor, lead to decreased responsiveness

What causes GPCR desensitization?

Activation of kinases and phosphorylation of receptor, arrestin

What is the difference between GPCR kinase & arrestin?

GPCR kinase phosphorylates active receptors, arrestin proteins bind only phosphorylated GPCR

Is a phosphorylated GPCR on or off?

A phosphorylated GPCR is typically considered "off," as it undergoes conformational changes that inhibit further signaling.

What is arrestin?

inhibits GPCR from interacting with G protein, adaptor protein for clathrin, induces receptor mediated endocytosis to recycle it

What is the process for arrestin?

1. overactivated GPCR starts to be ignored by cell

2. GPCR stimulates GRK/GPCR kinase to phosphorylate GPCR on multiple sites, ATP → ADP

3. arrestin binds to phosphorylated GPCR

4. GPCR is desensitized

How does GPCR kinase/GRK phosphorylate active receptors?

GPCR kinases (GRKs) phosphorylate the active forms of GPCRs on specific serine and threonine residues, which leads to receptor desensitization and the subsequent binding of arrestin.

binding domain on protein, C3 loop on C terminal end, prevents G protein from interacting w/ GPCR, phosphorylate on cytoplasmic tails, recruit arrestin

How do second messengers work in the cellular response?

carry activated signal away from membrane, serve to amplify cellular response, intracellular signaling proteins

What are the 2 main second messengers of GPCRs?

Cyclic AMP and calcium ions

What is cAMP?

cyclic AMP, uses adenyl cyclase enzyme to take ATP & make cAMP, turn off cAMP w/ cAMP phosphodiesterase into 5’ AMP,

What is the process

1. a-GTP activates adenylyl cyclase (membrane protein)

2. adenylyl cyclase converts ATP →cAMP

3. cAMP diffuses throughout cell, activates protein kinase A (PKA)

4. activated PKA enters nucleus nucleus & regulates transcription of genes, activates genes

What cell responses can be regulated by cAMP?

almost all, ex → adrenaline extracellular signal molecule, targets skeletal muscle, glycogen breakdown for energy as response

What does GPCR do in response to adrenaline?

activate adenylyl cyclase for cAMP, break down glycogen in response to adrenaline, breakdown in muscle cells

phosphoryalte & activate phosphorylase kinase → (P & activate) → glycogen phosphorylase → breaks down glycogen

How is calcium a second messenger?

GPCR second messenger, involved in various signaling pathways that lead to cellular responses, such as muscle contraction and neurotransmitter release, not a protein so quick response

How is calcium managed in the cell?

Cytosolic Ca concentration usually kept low, use time & energy to keep Ca low

• calcium sequestered outside cell, through antiport coupled transport, Na in & Ca out, also P type ATPase pump

• calcium sequestered inside organelles, pump in ER membrane

• Ca binding molecules in cytoplasm

• active Ca import in mitochondrion, coupled transport & symport

Why is calcium kept at low levels in the cell?

low level, any little amount can activate signaling, already want Na in so use that to get Ca out

How is Ca activated?

1. alpha goes, causing B & y to go

2. activated phospholipase c-B cleaves PI(4,5)P2 into diacylglycerol & IP3

3. diacylglycerol has tail regions in PM, IP3 goes to ER

4. IP3 used in open gated Ca release channel, gets Ca out from ER lumen, Ca activates protein kinase C bound to diacylglycerol

What does PI(4,5)P2 break into?

done through activated phospholipase c-B, into diacylglycerol & IP3, diglycerol stays stuck to PM, IP3 freely diffuses/water soluble once free

What can phospholipases do?

modify phospholipids to regulate signaling, breakdown causes:

• IP3 diffuses to ER, opens IP3 receptor letting Ca out

• protein kinase C/PKC translocates from cytosol to PM from increased Ca, Ca & PKC bind

• diacylglycerol/DAG recruits PKC to PM, Ca diffuses to the PM

• Ca + DAG + phosphatidylserine activate PKC, PKC has several downstream targets

What happens to PKC in the live stain?

PKC goes from diffused cytosolic distribution to PM association in response to Ca release from the ER

What is Calmodulin?

abundant protein in all eukaryotes, seen in metabolism & muscle contraction, activated by binding to Ca, activated Ca-Calmodulin bind & activate enzymes

What can signaling networks make?

feedback loops, ±

+ → product promotes previous, self activation of previous

- → product inhibits production of previous/inactivation

What is the pathway of MAPK signaling?

1. PDGF in extracellular space

2. Raf

3. Mek

4. Erk (cytoplasmic)

5. Erk (nuclus

What is heparin?

naturally occuring complex carb, anticoagulant, attenuates inflammation in vascular endothelial cells, regulates vascular smooth muscle proliferation

How does heparin regulate vascular smooth muscle proliferation?

regulates MAPK cascade intermediates, put G1 phase cell cycle block

Heparin receptor at PM triggers cGMP → PKG →MKP-1 in nucleus

How is vascular tone regulated?

determined by balance between vasoconstrictor & vasodilator influences on a blood vessel

What increases BP?

angiotension, major blood vessel contraction signal, increases BP

What decreases BP?

hypertension treatments: diuretics, ace inhibitors, beta blockers, Ca channel blockers

What relaxes blood vessels?

cyclic cGMP, released/caused by NO (nitric oxide) & atrial NAF (natriuretic factor), medically = nitroglycerine (converted to NO)

How do muscle cells relax?

acetylcholine binds at endothelial cell, NO synthase/NOS converts R to NO, NO diffuses across membranes, NO binds to guanylyl cyclase & GTP → cyclic GMP in contracted muscle cell to smooth muscle cell

How do muscle cells contract?

PDE5/phosphodiesterasse converts cGMP to GMP

How is blood vessel relaxation regulated with viagra?

viagra = sildenafil citrate/SC

SC inhibits PDE5, so NO → guanylyl cyclase & GTP → cGMP → SMC relax → vasodilation