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Multicellularity and Signal Transduction. General concepts of signaling Identifying signaling molecules G-protein coupled receptors cAMP Ca++. 1. Multicellularity. Cellular Specialization Separation of functions Different lineages of cells Established in development

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slide1

Multicellularity and Signal Transduction

  • General concepts of signaling
  • Identifying signaling molecules
  • G-protein coupled receptors
  • cAMP
  • Ca++

1

slide2

Multicellularity

Cellular Specialization

Separation of functions

Different lineages of cells

Established in development

Ectoderm- ex. neurons, epithelium

Mesoderm- ex. muscle, blood

Endoderm- ex. gut, liver

Maintenance in adulthood

Tissue-specific stem cells

proliferation and differentiation

2

slide3

epinephrine

Adrenaline/epinephrine in blood stream alters physiology

Cellular components translate signal into action

Sensing Environmental Signals

Ex. “Fight or Flight” Response

3

cell communication

Cellular components translate signal into action

Cell Communication
  • Cells have the ability to communicate with each other
  • Signaling molecules function within an organism to control:
  • Allows cells to respond to many environmental signals
  • Signaling molecules are synthesized and released from certain cells
slide5

Molecular Components that Sense and Respond to External Stimuli

External signal- ligand

Signal receiver- receptor in plasma membrane

Messengers- intracellular components that communicate signal

Effectors- intracellular players that mediate response to signal

5

slide6

Signal Transduction

  • General Steps:
  • Synthesis of signaling molecules by signaling cell
  • Release of signaling molecule
  • Transport of signal molecule to target cell
  • Binding of signal molecule to receptor on target cell
  • Initiation of intracellular signal transduction
  • Specific changes to cellular functions
  • Feedback regulation

6

slide7

Fast vs. Slow Responses

Signaling Cell

Signal sent from cell or environment

Target cell

-expresses receptor that binds to signaling molecule

-contain intracellular proteins that transmit signal

-changes occur in target cell

fast response

slow response

Receiving Cell

7

cellular response to signaling
Cellular Response to Signaling
  • (1) changes in protein activity or function
  • (2) changes in the amounts of specific proteins
  • The first type of response occurs more rapidly than the second
  • Changes in the activity of pre-existing proteins
cellular response to signaling1
Cellular Response to Signaling
  • Several intracellular proteins or small molecules contribute to signaling:
    • Cytosolic enzymes
    • Ligand binding to a receptor
    • G proteins
    • Small molecules
first and second messengers
First and Second Messengers
  • Binding of ligands (the first messengers) to cell-surface receptors leads:
  • Binding of ligands can also cause a decrease in concentration of the second messenger
receptors
Receptors
  • Most signaling molecules are too large and/or hydrophobic to get through the cell membrane
  • Need protein receptors
signaling
Signaling
  • Rapid responses to the environment are primarily mediated by the nervous system and by hormones
  • Cells that generate these signals are found in the pancreas, pituitary glands, neurons, hypothalamus
slide15

Signaling

  • Exocytosis of the stored molecules
  • Released molecules only last for seconds or a few minutes
  • Short term responses
  • Released signaling molecules travel to target cells
signaling1
Signaling
  • Synthesis and
  • Release of the signaling molecule
  • Transport of the signal to the target cell
  • Binding of the signal by a specific receptor 
  • Initiation intracellular signal-transduction pathways
  • Specific changes in cellular function, metabolism, or development
  • Deactivation of the receptor
  • Removal of the signaling molecule to terminate response
signaling2
Signaling
  • Endocrine: signaling molecules are synthesized and secreted by signaling cells (endocrine cells)
  • Autocrine: cells respond to substances that they release themselves
signaling3
Signaling
  • Paracrine: signaling molecule released by a cell affect only nearby cells
slide19

Synthesis, Release and Transport of Signal: Endocrine

Secreted signals

Endocrine:

Long distance signal

Hormone secretion into blood stream

17

slide20

Synthesis, Release and Transport of Signal:

Paracrine and Autocrine

Secreted signals

Paracrine:

Short distance signal

Signaling molecule secreting from cell adjacent to target cell

Autocrine:

Self signal

Target cell secretes its own signal

21

signaling4
Signaling
  • Membrane bound signals on one cell bind to receptors on adjacent cells to trigger differentiation
  • Some can act in short and long ranges
slide22

Signal between adjacent, attached cells

Cell-cell contact

Signaling cell expresses membrane-bound ligand and target cell expresses membrane-bound receptor

23

slide23

Strategies to Identify Signaling Components

  • Biochemical
  • Molecular
  • Genetic

24

slide24

Identification and purification of a receptor

Affinity Chromatography

Couple ligand to bead and load

Column

Run receptor-containing extract over

Column

Receptor binds to ligand on beads

Wash and discard flow through

Add free ligand in excess

Receptor binds to free ligand

Collect ligand-receptor complexes

Sequence peptide

Isolate gene

Express putative receptor gene and

test binding of ligand

Biochemically

25

slide25

Identification and purification of a receptor

Molecular cloning

Identify cells lacking receptor; do not bind ligand and do not respond

Introduce collection of cDNA isolated from cell that do express receptor and respond to signal

Add ligand X and look for clones of cells that do respond to ligand

Isolate cDNA that confers response

Sequence

Confirm by reintroducing putative receptor cDNA into cells and retesting

26

slide26

X

Genetic Isolation of signaling molecules

B

cone

B

cone

B

R7

B

R7

A

A

A

Define a cellular output for activation of receptor

B cell becomes R7 neuron when signal detected

Signal Target

Selective killing of A cell results in B cell becoming a cone cell instead of R7 neuron

A cell sends signal to B cell to become R7 neuron

Perform genetic screen for loss of cellular output that resembles loss of signaling cell

B cell becomes cone cell instead of R7 neuron

Loss of signal can be from mutation in ligand, receptor, or intracellular effectors

27

gpcrs
GPCRs
  • Most common class of receptors is called G Protein-Coupled Receptors (GPCRs)
  • About 900
  • Activation of these receptors alters gene expression
  • Many different receptors, one ligand
slide28

GPCR signaling in different tissues can elicit different responses

epinephrine

Increase in cAMP levels

Glycogen breakdown

Glucose release

Increase in intracellular Ca++

Muscle Contraction

30

gpcrs1
GPCRs
  • G protein coupled receptor ligand binding triggers the G protein to exchange GDP for GTP

29

cellular response to signaling2
Cellular Response to Signaling
  • Two large classes of GTPase switch proteins are used in signaling.
    • Trimeric
    • Monomeric
slide32

G-protein coupled receptors

7-pass transmembrane receptors

N-term extracellular

C-term cytoplasmic

Associate with trimeric G-proteins

Act as GEF for G-protein

Lead to increases in second messengers

cAMP

Ca++

Effector molecules found in membrane

32

gpcrs2
GPCRs
  • All GPCRs have the same orientation in the membrane

33

gpcrs3
GPCRs
  • Exterior surface consists mainly of hydrophobic amino acids
gpcrs4
GPCRs
  • TrimericG proteins have three subunits, α, β and γ
  • During signaling, the β and γ subunits remain together
  • Without ligand, the α subunit is bound to GDP
slide36

Trimeric G-protein

Composed of 3 subunits: α, β, and γ

α contains GTPase activity

α and γ tethered to plasma membrane via lipid tails

When α bound to GDP, also interacts with β and γ−−> This is the off position

When GPCR activated, binds to G-protein and acts as a GEF

Stimulates exchange of GDP for GTP in the α subunit

36

gpcr signaling1
GPCR signaling
  • Ligand binding causes a conformational change,
  • When GTP binds, another conformation change
  • Usually, Gα remains anchored to the membrane
  • Gα-GTP can inhibit the effector
  • Gβγ is sometimes freed from α to signal through an effector protein
  • GTP is hydrolyzed by α, which has GTPase activity
  • This switches α back to the GDP state and prevents any further activity
  • Binding of α to the effector protein can also cause hydrolysis
  • It then quickly reassociates with βγ, ready to repeat the process
gpcrs that regulate ion channels
GPCRs that regulate ion channels
  • Acetylcholine receptors in the heart muscle activate a G protein that opens K+ channels
  • Ligand binding leads to the opening of the associated K+ channels
gpcrs that regulate adenylate cyclase
GPCRs that regulate Adenylate Cyclase
  • Adenylatecyclase is a very common effector protein of GPCRs
  • When blood sugar levels are low, cells have a demand for glucose
  • In the liver, these two bind different GPCRs, but the response is the same
  • Activation and inhibition of cAMP occurs though many different ways and in different cell types
gpcrs that regulate adenylate cyclase1
GPCRs that regulate Adenylate Cyclase
  • Binding of prostaglandin PGE1 or adenosine to their GPCRs inhibits adenylatecyclase
slide47

Adenylyl (Adenylate) Cyclase

Enzyme that converts ATP to cAMP

Activated by Gαs

Intergral membrane protein

45

gpcrs that regulate adenylate cyclase3
GPCRs that regulate Adenylate Cyclase
  • cAMP activates Protein Kinases A by releasing catalytic subunits
    • Inactive PKA is a tetramer that has two regulatory (R) subunits and two catalytic (C) subunits
    • Inactive PKA is turned on by cAMP, as each R subunit has a cAMP binding site (CNB-A and CNB-B)
epinephrine
Epinephrine
  • There are different receptors for epinephrine in many different types of cells
  • Mediates the body’s response to stress
epinephrine signaling
Epinephrine Signaling
  • Activation of Adenylatecyclase by epinephrine results in:
    • An increase in cAMP and activation of PKA
    • conversion of glycogen to glucose 1-phosphate
    • PKA phosphorylates and inactivates glycogen synthase
    • PKA promotes glycogen degradation
    • When epinephrine is lost, this whole process is reversed
epinephrine signaling1
Epinephrine Signaling
  • cAMPlevels drop which inactivates PKA
  • Phosphoproteinphosphatase removes phosphate residues from glycogen synthase
  • Phosphoprotein phosphatase itself is regulated by PKA as it has an inhibitor that is activated by PKA
epinephrine signaling2
Epinephrine Signaling
  • At low cAMP levels, when PKA is inactive, the inhibitor is not phosphorylated
  • In the absence of cAMP the synthesis of glycogen by glycogen synthase is enhanced
  • Epinephrine-induced glycogen lysisthus exhibits dual regulation:  
diverse response to camp mediated pka activation
Diverse response to cAMP mediated PKA activation
  • Receptors are in low abundance, but trigger large scale responses
  • Signal amplification
  • Receptors and G proteins can diffuse though the plasma membrane
  • Epinephrine stimulation can increase cAMP levels by 104
slide57

Second Messengers

Small intracellular molecules

cAMP

Ca++

Easily diffusable--> reach locations distant from PM

Amplify signal 100-1000 fold

54

gpcr signaling2
GPCR signaling
  • Animation 15.1
slide59

Signal can be downregulated at several points

  • Receptor
  • decrease ligand affinity when bound to G-protein
  • Phosphorylation of receptor induces internalization
  • Can be target of PKA
  • G-protein
  • -innate GTPase activity turns off signal
  • -Regulators of G-protein Signaling (RGS) proteins act as GAP
  • cAMP levels
  • -phosphodiesterase breaks down cAMP to AMP
  • -regulated by PKA phosphorylation

56

downregulation of signaling from gpcrs
Downregulation of Signaling from GPCRs
  • Termination of signaling is needed to truncate the large amount of signaling
  • Affinity of the receptor for the ligand decreases
  • Intrinsic GTPase activity of α converts GTP to GDP
  • Adenylatecyclase can also act as a GAP and increases GTPase activity of α
downregulation of signaling from gpcrs1
Downregulation of Signaling from GPCRs
  • cAMPphosphodiesterase hydrolyzes cAMP to 5’AMP
  • Receptors themselves can be downregulated by a feedback repression mechanism
downregulation of signaling from gpcrs2
Downregulation of Signaling from GPCRs
  • Receptor can be phosphorylated by a protein call Β-adrenergic receptor kinase (BARK)
arrestin
β-arrestin
  • β-arrestin can desensitize GPCRs
camp localization
cAMP localization
  • Anchoring proteins localize the effects of cAMP to specific regions of the cell
    • PKA is localized by anchoring proteins to particular locations in the cell
    • AKAP15 is tethered to the cytosolic side of the plasma membrane

66

gpcr also activates phospholipase c plc
GPCR also activates Phospholipase C (PLC)
  • The plasma membrane contains phospholipases that are activated by extracellular signals
gpcr also activates phospholipase c plc1
GPCR also activates Phospholipase C (PLC)
  • GPCRs activate phospholipase C
  • Ca2+ ion levels play a key role in regulating the cell’s response to external signals
gpcr also activates phospholipase c plc2
GPCR also activates Phospholipase C (PLC)
  • Acetylcholine stimulates GPCRs in secretory cells of the pancreas and salivary glands
slide70

GPCR also activates Phospholipase C (PLC)

Ligand binds to GPCR

Induces conformational change--> binds to G-protein

Ga exchanges GDP for GTP

Ga-GTP binds to and activates phospholipase

PLC cleaves PI to make IP3 and DAG

66

slide71

GPCR also activates Phospholipase C (PLC)

  • Many second messengers are derived from phosphatidylinositiol
  • Phosphatidylinositol 4,5-bisphosphate is cleaved by into two important second messengers
  • IP3/DAG pathway
phospholipase c activation
Phospholipase C activation
  • GPCRs that activate phospholipase C cause an elevation in cytosolic Ca2+
phospholipase c activation1
Phospholipase C activation
  • GPCRs that activate phospholipase C cause an elevation in cytosolic Ca2+
slide75

IP3 and DAG trigger intracellular Ca++ release

IP3 released into cytoplasm binds to IP3-gated Ca++ channel in ER which releases Ca++ into cytoplasm

DAG stays in membrane but diffuses away where it binds to Protein Kinase C

PKC requires binding to DAG and Ca++ for activation

Active PKC binds and phosphorylates substrates

71

ca2 calmodulin mediates cellular response
Ca2+/Calmodulin mediates cellular response
  • Ca2+/Calmodulin mediates cellular responses
  • cAMPphosphodiesterase degrades cAMP
  • Calmodulincan activate protein kinases
ca2 calmodulin mediates cellular response1
Ca2+/Calmodulin mediates cellular response
  • Calmodulinalso activates a phosphatase that targets transcription factors
ca2 calmodulin mediates cellular response2
Ca2+/Calmodulin mediates cellular response
  • DAG is a secondary messenger formed by phospholipase C-catalyzed hydrolysis of PIP2

79