D103 Intracellular Signaling Components & Secondary Messengers (ALS 18, Vid 31)

what performs intracellular signal transduction

both proteins and small molecules

• changes in conc of small molecules, such as cyclic nucleotides, Ca2+, and lipids can also convey signals that diffuse from their subcellular site of production

• small molecules that transduce signals via a change in theri conc are referred to as “second messengers”

how are small molecules synthesized

by enzymes using readily available precursors, or can be released from subcellular stores

second messengers

small intracellular signaling molecules that relay the initial signaling response inside the cell (ex - second messengers include Ca2+, IP3, DAG)

• does not include all components (does not include PKC)

• usually small, non-protein diffusable molecules who intracellular conc can be changed rapidly during signaling

5 common intracellular second messengers

1. cAMP: activates PKA (hydrophilic)

2. cGMP: activates PKG and opens cation channels in rod cells (hydrophilic)

3. DAG: activates PKC (hydrophobic)

4. IP3: opens Ca2+ channels in the endoplasmic reticulum (hydrophilic)

5. Ca2+: released from ER or mito; 4x greater mag than in resting cell or cytoplasm (hydrophilic)

rate of conc change of second messengers

intracellular conc can be changed rapidly during signaling

• regulated via synthesis, degradation, or channels and pumps for Ca2+

qualities of second messengers

• chemically diverse (lipids, inorganic ions, cyclic nucleotides)

• function in discrete subcellular components

• encoded by a change in conc of second messenger

how is the net conc of a second messenger determined

the sum of its rate of synthesis and degradation by enzymes, some of which are the direct targets of the heterotrimeric g protein

• enzymes/channels that modulate intracelluar levels can switch on and off rapidly to quickly change conc

4 major target proteins of activated trimeric g proteins

• adenylyl cyclase

• phospholipase c

• cGMP phosphodiesterase

• K+ ion channel

what doesd phospholipase C prduce

IP3 that stimulates the release of calcium ions from the ER

• generates DAG and IP3 from PIP2

how does phospholipase C work

activated by Ga subunit of heterotrimeric GTPase

• cleaves PIP2 to produce 2 independent second messengers IP3 and DAG

DAG

hydrophobic lipid that can diffuse in 2D within the inner leaflet of the lipid bilayer

IP3

a hydrophilic molecule that can diffuse rapidly thr =u the cytosol

• can bind IP3 receptors on surface of ER and mediate rapid release of Ca2+ (another second messenger)

Ca2+ in phospholipase C pathway

once released from ER after IP3 receptor-ligand binding, Ca2+ can bind to and activate PKC

• can also bind to calmodulin

what does PKC do once activated

phosphorylates target proteins on serine and threonine residues

what do IP3 receptors mediate

in response to stim, IP3 receptors in ER mediate rapid release of calcium from ER into cytoplasm

• IP3 receptors are highly dynamic

• Ca2+ is quickly released and reabsorbed

what does phospholipase CB activate

serves as an enzyme that activates an inositol phospholipid signaling pathway

phosphatidyl inositol (PI)

a sugar (inostial) that’s phosphoryalated, which is attached to a glycerol moelcule that has 2 fatty acyl chains

• can be further modified by lipid kinases (PI3 kinase, PI4 kinase, PI5 kinase) or phosphatases

• cleaage of PIP2 by PLCB generates two second messengers: one hydrophilic (IP3), one hydrophobic (DAG)

pleckstrin homology domain

found on phospholipase C to help localize the enzyme to its substrates via binding to PIP2

• PH domains can bind to PI(4,5)P2 and PI(3,4,5)P3

• PH and C2 domains in PLC’s help target the PLC to its membrane-localized substrate

how do phospholipases differ

different phospholipases (C, D,A) can cleave PIP2 at different locations to generate different molecules that affect additional signaling pathways

• C = DAG + IP3

• D = phosphatidic acid

• A = arachidonic acid

arachidonic acid

important intermediate in synthesis of prostaglandins that play important roles in inflammation

how is signaling via PLCB turned off

• many PKC isoforms remain active from extended periods

• IP3 is rapidly dephosphorylated by lipid phosphatases to form IP2

• IP3 can also be phosphorylated by lipid kinase to form IP3 (IP4 can also function as a signaling molecule, but targets are poorly understood)

• DAG can be phosphorylated by DAG kinase to produce phosphatidic acid

how is ca2+ store in the er regenerated

ca2+ released to the cytosol is rapidly pumped out of the cytosol by calcium ATPase pumps and anti-porters

1. when Er Ca2+ decreases, STIM1 oligomerizes and activates Orai1, allowing rapid influx of Ca2+ into cytosol, near ER

2. once in the cytosol, ER Ca2+-store pumps rapidly move Ca2+ back into the ER lumen

STIM1

an ER transmembrane protein that has an ER-lumenal EF-hand domain that senses Ca2+ levels

• can also regulate TRP channel opening

Orai1

an example fo a CRAC (Ca2+ release-activated Ca2+) channel

where is PKC found

plasma membrane, cytoskeleton, and nucleus

PI-PLCs in vertebraes

have 13 different kinds

• enables tissue-specific coupling of various receptors to production of IP3 and DAG

PKCs in vertebrates

> 10 different PKCs

• provides a tissue specific and selective response to various lipid second messengers

adenylyl cyclase

an effector enzyme that produces cAMP

• large multiple TM domain protein

• > 8 isoforms in mammals

• catalytic domain is located on cytosolic face of membrane

• most isoforms are regulated by GPCR and Ca2+

G(as)

stimulates AC by mediating a conformational change in catalytic doamins of AC Z

what does activation of adenylyl cyclase result in

increases the cellular concentration of cAMP

• cAMP acts as a small intracellular mediator in both prokaryotes and eukaryotes

• cAMP prodiced from ATP via action of adenylyl cyclase

cAMP synthesis

constantly synthesized at relatively low levels in cells, and is being constantly broken down by constitutively active cAMP phosphodiesterases

• in resting cells, there is usually a high rate of turnover of cAMP (lifetime of a cAMP molecule is relatively short

conc of cAMP in cytosol

around 10^-7 M, but this can change by around 20x within seconds of a receipt of a signal

activation of PKA

via binding of cAMP

• causes a conf change in the regulatory subunits which results in release of catalytic subunits

• CNB: cyclic nucleotide binding site (2 of these per regulator subunit)

activation of target gene expression via PKA/ CREB

CREP cAMP response element

CREB: protein that binds to CRE sequences

• catalytic subunit of PKA phosphorylates CREB, which inducs CREB to recruit CBP

• PKA can have additional effects on the cell, not just via CREB

• targets for PKA differ depending upon specific cell type

second messengers - related cyclic nucleotides cAMP and cGMP

2 cyclic nucleotide monophosphates

• adenosine 3,5-cyclic monophosphate

• guanosine, 3,5-cyclic monophosphate

how are cAMP and cGMP similar

similar mechanisms of synthesis and degradation

• enzymes that produce (cyclases) or degrade (phosphodiesterases) the cyclic nucleotides determine the steady-state conc of these messengers inside the cell

• adenylyl cyclase and guanylyl cyclase are closely related enzymes: changing 2 AAs is sufficient to convert adenylyl cyclase into a guanylyl cyclase

where is ca2+ found

in cytosol

where is DAG found

lipid bilayer

Ca2+ vs cGMP/cAMP rate of diffusion

Ca2+ = lower

cGMP/cAMP and IP3 = higher

which of the following is false?
A. Cleavage of PIP2 produces two independent hydrophobic second messengers.

B. Viagra is an inhibitor of cGMP phosphodiesterase, which can affect vision.

C. IP3 can be phosphorylated to produce IP4.

D. Ca2+ and DAG each bind to and help activate PKC.

E. Diacylglycerol (DAG) can translocate in the inner leaflet of the lipid bilayer.

A. Cleavage of PIP2 produces two independent hydrophobic second messengers.

Which of the following directly helps PKC phosphorylate cytosolic proteins located close to the plasma membrane?

A. IP4

B. Orai channels

C. PIP2

D. DAG

E. GPCR

DAG

Which of the following is a mechanism that helps to end an IP3-mediated Ca2+ response ?

A. Delaying phosphorylation of IP3

B. Adding a phosphate to IP3

C. Converting IP3 to DAG

D. Pumping Ca2+ into the cytoplasm.

E. Increasing synthesis of PIP2.

B. Adding a phosphate to IP3