Biol-K 324 Lec 15: Signal Transduction #1

Front 1

general signaling

Back 1

1. release of signal

2. binding signal to receptor

3. activation of receptor and signal transduction cascade

4. effector mechanism

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schemes of intracellular signaling

Back 2

1. endocrine

2. paracrine

3. autocrine

4. juxtacrine - plasma membrane attached proteins

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protein kinase

Back 3

1. add phosphate moiety from ATP to specific amino acids within proteins

2. either target serine/threonine residues or tyrosine residues

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protein phosphatase enzymes

Back 4

remove phosphates from substrate proteins

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protein kinase/phosphatase switch

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Front 6

GTPase switch proteins

Back 6

1. GTP-bound active binds effectors that transduce the signal

2. cycling between on and off states is controlled by GEF and GAP activities

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Switch I and II

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1. binding the gamma phosphate of GTP alters the conformation of the GTP switch molecules

2. when GTP is hydrolyzed to GDP, then the switch I and II domains fall back

3. conformation changes are recognized by downstream effector molecules

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secondary messengers

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1. low molecular weight

2. high diffusion molecules that change concentration in response to ligand-receptor (first messenger) activation

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signal amplification

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1. receptor activation produces a series of events that often amplify the signal

2. small amounts of ligand produce profound responses in cell

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receptor binding

Back 10

can determine affinity of ligands and numbers of receptors on cell surface

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physiological response

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1. can be amplified by signal transduction pathways

2. levels change within a narrow range of concentrations

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general G-protein coupled receptor systems

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1. receptor, seven membrane spanning domains

2. receptor coupled to trimeric G-protein

3. membrane bound effector protein

4. feedback regulation and desensitization

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Back 13

G protein-coupled receptor

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G-protein signaling

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1. regulates ion channels

2. regulates adenylyl cyclase

3. regulate cytosolic calcium

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muscarinic acetylcholine receptor

Back 15

1. acetylcholine receptor slows rate of heart contraction

2. G-protein coupled receptor dissociates trimeric G-protein

3. beta-gamma subunit binds and opens a potassium channel, hyperpolarizing the heart muscle cell

4. GPCR

5. effector protein is a K+ channel

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light activated rhodopsin pathway

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1. light indirectly inhibits cGMP gated channel

2. hyperpolarizing the photoreceptor membrane

Front 17

GPCR activation

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activation of transcription

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G-protein coupling with adenylate cyclase

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1. precise control of cAMP levels is produced by balance of different signals

2. in adipose cells, there are competing responses that stimulate or inhibit fatty acid catabolism

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PKA activation

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1. affects cytoplasmic mechanisms like glycogen metabolism

2. also activates transcription by phosphorylating CREB factor

3. with CBP/300, stimulates transcription

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phosphatidylinositol signaling

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Front 21

IP3/DAG pathway

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divergent signal transduction pathway producing calcium and protein kinase C signaling

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smooth muscle relaxation of arteries

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1. endothelial ACh signal activates Ca/CaM

2. Ca/CaM activates NO syntase

3. NO gas diffuses to nearby smooth muscle cell

4. relaxes in response to NO

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ACh signal activation

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Ach --> ACh GPCR --> Phospholipase C --> IP3 --> Ca2+ calmodulin --> NO synthase --> NO --> NO receptor --> GTP hydrolysis (cGMP and PPi) --> protein kinase G --> muscle cell relaxation

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external signals

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1. membrane anchored and secreted proteins or peptides

2. small hydrophobic molecules

3. small hydrophilic molecules

4. gases

5. physical stimuli

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endocrine signaling

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signals from one cell act on distant cells

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paracrine signaling

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signals from one cell on nearby cells

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autocrine signaling

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signals from one cell to itself

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Kd

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1. concentration of ligand at which half the ligand's receptors are occupied

2. determined experimentally and is a measure of the affinity of the receptor for the ligand

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affinity chromatography

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use to purify receptors even when they are present in low abundance

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immunoprecipitation assasy

Back 30

use antibodies specific for protein kinases to measure kinase activity

Front 31

pull down assays

Back 31

1. use protein-binding domain of a target protein

2. used to quantify activation of GTP-binding protein within a wall

Front 32

GPCRs

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1. large and diverse family

2. seven membrane spanning alpha helices

3. internal ligand-binding pocket that is specific for ligands

4. range of cellular effects

5. coupled to trimeric G proteins (contain alpha, beta and gamma subunits)

6. can function as GEF from conformational change

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fluorescence energy transfer

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demonstrate receptor-mediated dissociation of coupled Galpha and Gbeta-gamma subunits in living cells

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Galpha subunit

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determines the function of the G protein and affords it specificity

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effector proteins

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1. activated or inactivated by trimeric G proteins

2. form second messengers or ion channels

Front 36

rhodopsin pathway

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1. effector protein: cGMP phosphodiesterase

2. activated by Galpha-GTP mediated release of inhibitory subunits

3. reduction of cGMP leads to closign of cGMP-gated Na/Ca channels --> hyperpolarization --> decreased NT release

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terminate visual signaling

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1. GAP proteins inactivate G.GTP

2. Ca sensing proteins activate guanylate cylcase

3. rhodopsin phosphorylation

4. binding of arrestin

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cAMP

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1. ligand binding of GPCR --> adenlyl cyclase --> ATP to cAMP

2. binds cooperatively to a regulatory subunit of protein kinase A --> releases the active kinase catalytic subunit

3. levels measured by hormonal stimulation

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PKA

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1. mediates the diverse effects of cAMP

2. localization restricts cAMP to particular subcellular locations

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GPCR/adenylyl cyclase/cAMP/PKA signaling pathway

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amplified tremendously by second messengers and kinase cascades

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feedback repression

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1. downregulated by GPCRs

2. end product of a pathway blocks an early step in the pathway

3. activation of coupled G proteins is inactiviated

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BARK

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1. phosphorylates cytosolic residues of receptor in its active conformation

2. leads to binding of b-arrestin and endocytosis of receptors

3. deactivate b-adrenergic receptors

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GPCR-arrestin complex

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1. functions as a scaffold that activates several cytosolic kinases

2. initiates cascades that lead to transcriptional activation of genes controlling cell growth

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down-regulation of Gs-coupled receptors

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1. affinity of the receptor for its ligand decreases when GDP bound to Galpha is replaced with GTP

2. intrinsic GTPase activity fo G that converts the bound GTP to GDP is enhanced when G binds to adenylyl cyclase

3. cAMP acts to hydrolyze cAMP to 5'-AMP, terminating the cellular response

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small rise in Ca

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1. induces a variety of responses in different cells

2. hormone secretion, contraction of muscle, platelet aggregation

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phospholipase C enzyme

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1. effector protein activate by GTP bound G

2. cleaves a phospholilpid known as PIP2

3. makes two secondary messangers

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secondary messengers made by PIP2 cleavage

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1. diffusable IP3

2. membrane bound DAG

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IP3

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1. triggers opening of IP3-gated Ca channels in ER

2. elevation of cytosolic free Ca

3. protein kinase C recruited into plasma membrane

4. activated by DAG

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depletion of ER Ca

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1. leads to opening of plasma membrane store-operated Ca channels

2. influx of Ca from extracellular medium

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Ca-calmodulin complex

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regulates activity of cAMP phosphodiesterase and protein kinases

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ACh GCPR stimulation

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1. induces increase in cytosolic Ca

2. synthesis of NO

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NO

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activates intracellular guanylate cyclase to synthesize cGMP

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increase in cGMP

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1. activation of protein kinase G

2. triggers a pathway resulting in muscle relaxation and vasodilation

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glycogen breakdown and synthesis

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1. coordinately regulated by secondary messengers Ca and cAMP