Module 14 - Intracellular Signaling and Transduction

included on exam 4

intracellular receptors

receptors that are recognized by signal molecules

many are transcription reculators

signal molecules can be steroids or amino acid-derived hormones, e.g., which are small and lipid-soluble

response requires expression of hormone-activated genes

cortisol

a steroid hormone

produced by the adrenal cortex (near the kidneys)

targets many organs

effects: adaptation to long-term stress by raising blood glucose levels and mobilizing fat

can bind to an intracellular receptor to bind protein to DNA

steroid hormones

lipid-soluble hormones derived from cholesterol

e.g. cortisol, estradiol, testosterone

contain 3 6-membered rings and 1 5-membered ring

the cortisol receptor, and binding of cortisol

cortisol receptor is a transcription factor

in absence of cortisol, receptor remains inactive in the cytosol

cortisol enters cell and binds to receptor → conformational change occurs → receptor becomes an active transcription regulator

active protein goes to cell nucleus and binds to specific regulatory DNA sequences of target genes

signal transduction

the relay of an outside signal through an intracellular signaling cascade involving a number of intermediate molecules with different activities

leads to final targets

can be in pathways

signal transduction pathways

triggered when an external signal molecule binds to a surface receptor → becomes activated

activated receptor relays signal to cell’s interior → initiate intracellular cascade of molecular events leading to a response

domains of a G protein-coupled receptor

receptor (extracellular domain)

7 transmembrane domains

activator of G protein (cytoplasmic domain)

G protein

a membrane-bound protein complex with three subunits

activated by GTP

when inactive, all three subunits are associated, alpha subunit has a GDP bound to it

when receptor is activated, G protein binds to it:

GDP dissociates from alpha-subunit, replaced with GTP

alpha-subunit dissociates from beta and gamma subunits

beta and gamma subunits remain associated and act as a single unit (beta-gamma complex)

both alpha-subunit and beta-gamma complex are active, can interact with different targets

activated alpha subunit of a G protein

can interact with and regulate activity of membrane-bound target proteins

target proteins are often enzymes

inactivation of G protein

hydrolysis of GTP → re-association of alpha with beta and gamma → inactivation of G protein

activated beta-gamma complex

in heart muscle cells: directly opens a K⁺ channel in response to ACh from parasympathetic neurons (target protein is an ion channel)

result: reduced frequency of cardiac muscle contraction

when alpha subunit reassociates with the complex, K⁺ channel closes

epinephrine (adrenaline)

produced by the adrenal medulla

stimulates glycogen breakdown in anticipation of muscular activity

stops glycogen production

used as a neurotransmitter by sympathetic neurons as part of fight-or-flight response

epinephrine receptor

a G protein-coupled receptor

activated G protein activates adenylyl cyclase (AC) → AC catalyzes conversion of ATP → cAMP

result: elevated cAMP levels → activation of protein kinase A (PKA)

cAMP

cyclic AMP

a second messenger, activates PKA

ATP → cAMP + PP_i (activation, catalyzed by AC)

cAMP → AMP (deactivation)

PKA

protein kinase A

cAMP-dependent

promotes glycogen metabolism

when inactive, is a tetramer with 2 regulatory (R) subunits bound to 2 catalytic (C) subunits

activated by cAMP: cAMP binds to R subunits → C subunits are released

kinase activity of released C subunits phosphorylates multiple effector proteins

glycogen breakdown in skeletal muscle cells

a fast response to PKA activation

PKA phosphorylates/activates phosphorylase kinase

phosphorylase kinase phosphorylates and activates glycolgen phosphorylase

glycogen phosphorylase breaks up glycogen into glucose units (as glucose-1-P)

phosphoglucomutase isomerizes G1P into G6P → G6P enters glycolysis

calmodulin

a small Ca²⁺-binding protein that is involved in modulation of many cellular processes by activating many calmodulin-dependent kinases and phosphatases

activated by 4 Ca²⁺ ions

activated by a pathway

calmodulin activation pathway

begins with activation of a G protein

activated alpha subunit activates phospholipase C (PLC)

PLC catalyzes hydrolysis of phosphatidyl inositol biphosphate (PIP₂) into diacyl glycerol (DAG, membrane bound) and inositol triphosphate (IP₃, released as free cytoplasmic messenger)

DAG and IP₃ both can activate other cellular functions

IP₃ is ligand for IP-gated Ca²⁺ channels on ER

released Ca²⁺ ions bind to calmodulin → activates Ca²⁺/calmodulin-dependent (CaM) kinases

Wnt proteins

a large famimly of secreted signals

name is fusion of wingless gene in Drosophila + integrated, a vertebrate homolog

involves a signaling pathway

the Wnt signaling pathway

involved in cell differentiation, establishment of embryonic axes, and cell adhesion

activates beta-catenin (a transcription factor, important for transcription of cell differentiation genes in neighboring cells)

canonical pathway:

if no Wnt signal present: destruction complex forms in the cytoplasm

a complex containing APC (a tumor suppressor) is active → targets beta-catenin for proteolytic degradation

if Wnt signal is present: Wnt binds Frizzled (a GPCR) → Frizzled activates Disheveled through a G protein

Disheveled inactivates the APC-containing complex

stable beta-catenin enters nucleus, joins and activates a transcription factor (TCF) complex, promotes transcription of cell differentiation genes

RTKs

receptor tyrosine kinases

enzymic receptors with single transmembrane domains that form dimers upon binding of a signal

3 steps in activation

steps in activating an RTK

1) binding of signal as a dimer causes formation of receptor subunit dimers

2) cytoplasmic TK domains of the receptor subunits phosphorylate each other

3) phosphorylated tyrosines become binding sites for signaling proteins that contain an SH2 domain

the Ras-MAP pathway

a pathway that controls cell proliferation and differentiation in response to growth factors

signal molecule: mitogen (promotes mitosis)

activated RTK is recognized by adapter protein (an SH2 protein)

adapter protein recruits Ras-activating protein (a GEF)

GEF activates Ras (a monomeric GTP-binding protein) by exchanging its GDP for a GTP

Ras-GTP activates a MAP kinase-kinase-kinase → triggers MAP kinase cascade of activation of protein kinases by phosphorylation

Ras can hydrolyze its GTP → GDP to deactivate itself (Ras has GTPase activity)

importance of involving many intermediates before a cellular response

1) signal can be amplified

2) each step ban be a target for regulation/modulation of the response

2 is especially important in controlling cell proliferation

MAP kinase and Ras: importance of many intermediates

MAP kinases and Ras are coded by proto-oncogenes

some mutations turn them into oncogenes because activity of gene products becomes independent of GTP or phosphorylation → independent of growth factors → result: abnormal cell proliferation

(mutated lack of independence on intermediates → abnormal proliferation → cancer)

Drosophila compound eye

eye has 800 ommatidia

each ommatidia consists of 8 photoreceptor cells and 12 lens cells

all 20 cells differentiate from eye epithelial cells

R8 differentiates into a photoreceptor cell first → then induces 7 neighboring cells to become photoreceptor cells → end with cell R7

R7 differentiation involves Boss, Sevenless, Drk, and Sos

remaining 12 cells of the ommatidia (1 eye unit) become lens cells by default

Boss

bride of sevenless

a signal protein on the surface of cell R8

involved in R7 differentiation in Drosophila eye

blue in the picture

Sevenless

an RTK

Boss receptor on the surface of R7 in Drosophila

activates a Ras pathway

green in the picture

can be activated by phosphorylation to connect to Drk

Drk

downstream of receptor kinase

an SH2 protein

connects to phosphorylated Sevenless to activate Ras

red/orange in the picture

Sos

son of sevenless

a Ras-GEF

binds to Drk to activate Ras by swapping Ras’s GDP for a GTP

teal in the picture

immunoprecipitation

one way to test whether protein-protein interactions are occurring

combine a mixture of purified molecules (e.g. cell extract) with antibodies specific to one molecule (molecule A, in the picture)

antibody and molecule A will aggregate and precipitate, collected by centrifugation

anything bound to A will also precipitate

elucidating signal transduction pathways

can identify receptor binding sites, or order of signaling proteins and Ras

different methods to elucidate both

elucidating the identification of receptor binding sites in a signal transduction pathway

mutate specific sites, and use co-immunoprecipitation of receptors and SH2 proteins to see what protein did not bind

elucidating the order of signaling proteins in a signal transduction pathway

a protein upstream from Ras (e.g. X) regulates Ras

a protein downstream from Ras (e.g. Y) is regulated by Ras

if X is mutated and Ras is normal, no signaling will occur

if X is normal and Ras is mutated (oncogenic active), signaling will occur (Ras rescues the pathway)

if Y is mutated and Ras is normal, no signaling will occur

is Y is normal and Ras is mutated (oncogenic active), no signaling will occur (Ras cannot rescue the pathway)

cytokines

soluble signals secreted by the lymphoid system (part of the immune/defense system)

receptors activate transcription factors, but do not have intrinsic enzymatic activity → receptors depend on associated protein kinases (they are non-receptor tyrosine kinases (NRTKs))

activated by dimerization of receptor subunits

activation of cytokine receptors

binding of signal causes receptor subunit homodimers to form

JAKs (non-RTKs) into close proximity so they can phosphorylate and activate each other

JAKs

janus kinases

non-receptor tyrosine kinases

phosphorylate each other, then the cytokine receptor subunits they are associated with

phosphorylated receptors are then recognized by STATs

part of the cytokine receptor and JAK-STAT pathway

STATs

signal transducers and activators of transcription

DNA-binding proteins

phosphorylated and activated by JAKs

part of the cytokine receptor and JAK-STAT pathway

2 STATs form a dimer → go to nucleus → activate cytokine-responsive genes

the interferon-gamma response pathway

natural killer cells can distinguish malignant from health cells, kill malignant cells by cell-mediated immunity

NK cell has different activating receptors, healthy vs malignant cells have different activating ligands

when NK receptors bind to malignant ligands, cytokines (IFN-gamma) is released

IFN-gamma binds to a receptor → triggers a JAK-STAT pathway in target cell → activates expression of the IDO gene → causes cell death

IDO

idoleamine dioxygenase

an enzyme that catalyzes the degradation of tryptophan → causes a target cell to die of tryptophan starvation

released by NK cells when a malignant cell binds