Unit 8

Glucocorticoid action

ligand → hormone (glucocorticoid), receptor → receives signal (transcription factor), transducer → transmits signal to effector (HAT), effector → activated in response to signal (genes)

G-protein

a protein that binds GTP and GDP to cycle between active and inactive states (ras, ran, arf, rabs)

GEF

guanine exchange factor, removed GDP, allowing GTP to bind

GAP

activates GTPase activity of G-protein, converting bound GTP to GDP

G-Protein coupled receptors (GPCRs)

largest family of cell-surface receptors (1000); 7 transmembrane alpha helices, cytoplasmic c-terminal domain bound to heterotrimeric G proteins, extracellular ligand-binding domain, and IC domain with GEF activity; ligand binding → conformational change → receptor’s cytosolic domain activates a G protein at inner plasma membrane; activated G protein dissociates and modulates enzymes or ion channels, initiating signal transduction to intracellular targets

Heterotrimeric

composed of alpha (binds GTP/GDP), beta, and y subunits (bind a)

≈

Resting state

Ga-GDP bound to GBy as a trimer

Activation

ligand-bound GPCR acting as a GEF exchanges GDP for GTP on Ga

Dissociation

Ga-GTP separates from GBy; both species can regulate targets

Termination

Ga intrinsic GTPase hydrolyzes GTP → GDP; Ga-GDP associates with GBy

G-protein coupled receptors are regulated Ga GEFs

activated GPCRs serve as GEFs for Ga, promoting GDP release and GTP binding to switch G proteins on

Adenylyl Cyclase

membrane enzyme that converts ATP → cAMP; stimulated by Ga, discovery stemmed from finding GTP is required for hormonal AC activation

Adenlyl Cyclase (cAMP from ATP) Hormonal Activation

receptor with bound ligand stimulates the exchange of GTP for GDP; activated Ga dissociates from receptor and B/y; activated GTP-bound Ga binds to and activates adenylyl cyclase; binding of hormone promotes the interaction of the receptor with a heterotrimeric (inactive) G protein, activating Ga

cAMP

second messenger mediates many hormonal responses; made by AC; degraded to AMP by cAMP phosphodiesterases; primary action is activating PKA; in some cells it directly opens the Na+ channels independent of PKA

PKA or Protein Kinase A

tetramer; phosphorylates Ser residues on targets; active C subunit can enter the nucleus and phosphorylate CREB to induce CRE-containing genes; protein phosphatase 1 reverses PKA-dependent Ser/Thr phosphorylation, settling response magnitude/duration

Turning off the signals

phosphodiesterase degrades cAMP → AMP, lowering PKA activity; GAPs regulate G-protein signaling for heterotrimeric G proteins, and accelerate Ga GTP hydrolysis returning Ga to GDP-bound off state

Type 1 protein phosphatases (PP1)

dephosphorylate PKA substrates, terminating PKA outputs

Phosphodiesterase

degrades cAMP to AMP

RGS or Regulator of G-protein Signaling

to turn off the signal RGS stimulates Ga-GTPase activity

Regulation of Glycogen Breakdown by phosphorylation

1) cAMP diffuses through the cytosol and activates PKA, 2) PKA phosphorylates and activates phosphorylase kinase, 3) phosphorylase kinase phosphorylates and activates glycogen phosphorylase, 4) glycogen phosphorylase breaks down glycogen (each step amp)

• PKA activation of glycogen breakdown/glucose release pathway

epinephrine receptor → activates G protein → activates AC → increases cAMP; activates PKA → phosphorylase kinase activated → glycogen phosphorylase activated → glycogen → glucose-1-phosphate; demonstrates signal amplification at many steps

Regulation of cAMP dependent protein kinase

PKA held in “off” by regulatory subunits; cAMP binds to regulatory subunits of PKA; cAMP binding to R releases the active catalytic subunit of PKA; allosteric activation of kinase

Epinephrine

ligand

7TM G-protein binder

receptor

Ga

transducer

cAMP

transducer

PKA

transducer (serine/ threonine kinase that adds phosphates to hydroxyl group)

Phosphorylase Kinase

transducer

Glycogen Phosphorylase

effector

CRE

cAMP response element; enhancer

CREB

cAMP response element binding protein; when phosphorylated, binds to CRE and recruits co-activator

Effectors

the free catalytic subunit of PKA translocates to the nucleus and phosphorylates CREB which activates genes

Type 1 protein phosphatase

dephosphorylates proteins in serine/threonine

• Counter-regulatory hormones

epinephrine, norepinephrine, and glucagon

• Insulin

is secreted in response to high blood glucose. Signals glucose is high, store energy. Stimulates glycogen synthesis, inactivates phosphorylase by activating type 1 phosphatase.

• There are many types of Ga subunits- Gas Gai Gaq Gaolf

• Receptor tyrosine kinases

extracellular ligand-binding domain, single transmembrane helix, cytosolic Tyr kinase domain; ligands include EGF, NGF, insulin, and many growth factors

• Ligand binding (EGF, PDGF, insulin)

causes receptor dimerization.

• Autotransphosphorylation

increases kinase activity and creates phosphotyrosine docking sites for downstream signaling proteins

• pTyr sites on linker regions

are binding sites for proteins with SH2 domains; leads to membrane localization, complex assembly, phosphorylation, and activation of downstream effectors

SH2 domains-

protein domains that bind phosphotyrosine-containing peptides.

Ras

little squiggle is C-terminal farnesylation (ras is inserted into the cytoplasmic leaflet of the PM); turned on by Ras GEF; off by Ras GAP

• Two main signaling pathways downstream of RTK- Ras/raf/MAPK (Erk) and PI3K/mTOR/PDK1/Akt (PKB)

• Ras/raf/MAPK (Erk) pathway

cascade of kinases; activated by RTK autophosphorylation recruiting GEFs, which load GTP on Ras; Ras-GTP → Raf dimer activation → MEK → ERK activation; outputs= ERK phosphorylates cytosolic and nuclear transcription factors; control cell proliferation, differentiation, and survival

Activated ERK

translocates to the nucleus where it phosphorylates and activates TF Elk-1

Activation of Ras, Raf, and ERK

1) autophosphorylation creates binding site for SOS (Ras GEF), 2) Ras-GTP recruits Raf (MAP kinase x3); 3) Raf phosphorylates MEK (Map kinase x2); 4) MEK phosphorylates ERK (Map kinase), 5) ERK (Map kinase)

KSR

organizes Raf-MEK-ERK into a scaffolded signaling cassette, ensuring specific and efficient signal flow; ERK dissociates after activation to translocate to the nucleus

• KSR scaffolding protein- Ras/Raf/MEK/ERK mitogen-activated protein kinase (MAPK) pathway.

• Erk

(Extracellular Signal-Regulated Kinase) is a vital signaling protein that acts as a molecular switch to relay external messages—like growth factors—into the cell nucleus to control essential processes such as cell division, growth, and survival.

• Erk => Elk1 => immediate early genes.

IP3

binds to irs receptor on ER/SR and stimulates calcium release; activation of Gaq leads to the activation of PLC which cleaves off the inositol tri-phosphate (IP3) from phosphatidyl inositol

Olfactory signal transduction

Ga-GTP binds to and activates AC; cAMP activates channels that depolarize the cells

JNK and p38 MAPK

distinct MAPK cascades preferentially activated by inflammatory cytokines and cellular stress; often associated with inflammation and cell death; like ERK, can enter nucleus to regulate transcription

PI3K/Akt pathway

Autophosphorylation creates binding site for P13 kinase (SH2); P13k phosphorylates PIP2 → PIP3 at the membrane; PIP3 recruits Akt, PDK1, MTORC2 via their PH domains, PDK1 and MTORC2 phosphorylate/activate Akt; Akt/PKB phosphorylate many downstream targets; lipid kinase and protein localization

Akt (PKB)

Ser/Thr kinase promote cell survival and proliferation via phosphorylation of multiple targets; regulates protein synthesis through eIF2B; required for initiation of all protein translation; inhibits GSK3 (stress or low GF activate inhibitory kinases)

Initiation of Translation in euk. cells

initiation met tRNA and GTP bind to eIF2; eIF1, eIF1A, + eIF3 bind to 40S; two complexes bind + eIF5; eIF4 binds the 5’ m7G cap; PABP binds the polyA tail + eIF4; 40S+++ binds eIF4 displacing PABP

FOXO

Akt phosphorylation creates a 14-3-3 binding site, sequestering FOXO in cytosol and reducing transcription of anti-proliferative/pro-death genes; in the absence of insulin or growth factors, FOXO is located in the nucleus where it specifies target gene expression

GSK-3

inhibited Akt; links to metabolism and translation via eIF2B

• PI3K/Akt/mTOR pathway

low AA inhibit mTOR; mTORC2 acts upstream to help activate Akt; Akt inhibits TSC, thereby activating Rheb regulates → mTORC1; AMPK (high AMP/ ATP) activates TSC to inhibit mTORC1 under energy stress; amino acids are additionally required for mTORC1 activity

TOR

target of rapamyacin (immunosuppressant drug)

TOR 1

activates protein translation through activation of S6kinase and inhibition of eIF4EBP1; inhibits protein breakdown via autophagy

• mTORC1

stimulates protein synthesis by phosphorylating s6 kinase and 4E-BP1 (relieves eIF4E inhibition), and inhibits autophagy; when nutrients/energy are low, reduced mTORC1 activity frees autophagy to recycle amino acids; regulates protein synthesis through 4EBP; non-phosphorylates 4E-BP1 binds eIF4E and blocks initiation; phosphorylation releases eIF4E to promote cap-dependent translation

Regulation of eIF4E

low GF- 4E-BP binds and inhibits eIF4E; MTOR phosphorylates 4E-BP and initiation occurs

Receptor Tyrosine Kinases

ligand binding → receptor dimerization; dimerization → autophosphorylation (activates tyrosine kinase and creates binding sites for proteins with SH2 domains); each receptor has an N-terminal extracellular ligand-binding domain, a signal transmembrane a helix, and a cytosolic C-terminal domain with tyrosine kinase activity and binding sites

Non-Receptor Tyrosine Kinases (NRTKs)

ligand binding induces receptor dimerization, leading to the activation of NRTKs as a result of cross-phosphorylation

Cytosine receptors

lack intrinsic kinase activity and associate with NRTKs

JAK family

Janus Kinase; ligand-induced receptor dimerization → JAK activation → receptor phosphorylation → recruitment and tyrosine phosphorylation of STATs (signal transduction activator of transcription) → STAT dimerization and nuclear translocation to drive transcription

JAK/STAT pathway

JAK phosphorylates receptor; STAT binds via SH2 domains; Jak phosphorylates bound STAT which dimerizes via its SH2 domains

Src family

function downstream of cytokine receptors, RTKs, antigen receptors, and adhesion receptors; activates cell migration through attachment to ECM

Insulin => Akt => AS160 a Rab GAP.

Cross talk

can either be positive or negative

Non-receptor tyrosine kinases-

Integrins- and non-receptor TKs.

NFkB/IkB pathway

activation- adaptor proteins → lkB kinase (IKK) → IKB phosphorylation → recognition by B-TrCP ubiquitin ligase → proteasomal degradation of IKB; outcome- freed NF-KB enters nucleus → target gene expression; negative feedback- NF-KB induces IKB gene, restoring inhibition and shaping signal duration

What does the integrin binding to the ECM lead to?

clustering and activation of NRTK FAK (focal adhesion kinase)