OBJ - Signaling Transduction & Chemical Messengers I & II

Question 1

Explain cell signaling and how it integrates stimuli to body physiology

Answer 1

• internal/external stimuli that tell the body to react; needs to be sensed and transmitted to target organ/tissue/cell
• metabolic cues are interpreted by receptors and transmitters by signaling cascades
• necessary for homeostasis in the body

Question 2

Draw a generalized signal-transduction cascade.

Answer 2

1) release of chemical messenger (by secretory cell)
2) reception of the chemical messenger (by target cell receptors - either on membrane or in cell - cytoplasmic or nuclear)
3) delivery of the message inside the cell (crosses the membrane)
4) signal transduction by:
- signal transducer proteins
- secondary messengers
5) activation of effectors that alter a physiological response
6) termination of the signal
* *like a river; always runs upstream (1) to downstream (6)

-Specificity of response - based on where the receptor is on the cell (membrane/cytoplasm)

• ligands = chemical messengers
• diseases can “strike” any of these 6 steps

Question 3

Explain how signal transduction is amplified

Answer 3

Enzymatic cascade (primarily)
Secondary messengers (part of cascades, but increase speed)

Question 4

Identify the five major types of chemical messengers

Answer 4

1) Neuropeptides (Nervous system)
2) Hormones (Endocrine)
3) Cytokines (Immune System)
4) Eicosanoids (Injury - i.e. inflammation)
5) Growth Factors - Cell proliferation

Question 5

Describe the three modes of action chemical messengers used to signal

Answer 5

1) endocrine - through the blood/hormones bathe whole body and cells with receptors respond
2) paracrine - adjacent cells through interstitial fluid (i.e. nervous system or Antibody/Antigen)
3) autocrine - same cell, vessicle release/exocytosis

**lot of cells can do both Paracrine & Autocrine

Question 6

Intracellular vs Extracellular transcription factors

Answer 6

Intracellular - chemical messenger must be lipophilic to diffuse through membrane

• Acts in Cytoplasm or nucleus = gene specific for hours-days for effect
• Steroid hormone/thyroid hormone
• Cortisol = gene transcription

Extracellular - plasma membrane receptor to pass on signal across plasma membrane

Question 7

Three types of signal transducers and three types of second messengers (3)

Answer 7

Signal transducers:

1) SH2 domain proteins-adapter proteins
2) Monomeric G-protein RAS
3) Heterotrimeric G-proteins

Second Messengers: (nonprotein molecules that amplify the signal, diffuse & have a fast response relative to proteins that turn on genes)

1) Phosphatidylinositol signaling
2) cAMP
3) Ca++

Question 8

Cortisol

Answer 8

Receptor lied in cytoplasm; once activated can:

• enter nucleus & turn on gene transcription for anti-inflammatory proteins
• stay in cytosol & prevent pro-inflammatory proteins from being transcribed

net result: reduces inflammation

Question 9

Explain plasma membrane receptors

Answer 9

Common Features:

1) extracellular domain that binds the chemical messenger
2) membrane spanning region
3) chemical messenger binding induces a CONFORMATIONAL change in the receptor
4) Intracellular domain that initiates signal transduction (proteins/second messengers)

Question 10

Identify the three major classes of plasma membrane receptors (ion channel, kinase or bind kinase, heptahelical); and describe their common feature

Answer 10

1) Ion channel receptor
2) Reeptor kinases/receptors that bind kinases (JAK/STAT)
3) Heptahelical receptors (ETC)

Question 11

Understand Ion channels and their biochemical characteristics and functions

Answer 11

• FAST response
• ions flow down their electrochemical gradient into cell (changing charge of cell)

Example: the nicotinic acetylcholine receptors in nervous system (“reward circuits”) & muscles(neuromuscular junction - cobra venom/Sux paralyzes)

-normally twisted shut & closed, once Acetylcholine binds to it; a conformational change opens the receptor; ion gated channels > release ACh

Question 12

Understand Kinase receptors and signaling cascades

Answer 12

• Either a kinase receptor or acting as a kinase
• posed for action to transduce cell signals

Kinase = a type of enzyme that transfers phosphate groups from high-energy donor molecules, such as ATP, to specific substrates, a process referred to as phosphorylation; which results in signal transducer binding

Kinase -> phosphoralates (adds Pi)
Phosphatases -> dephosphoralate (removes (Pi)

Question 13

Understand Ras/Map kinase signaling

Answer 13

1) Epidermal Growth Factor binds to receptor which dimerizes & autophosphorylates
2) this opens a binding site and SH2 protein binds to receptors/EGF
3) Signal is transduced via small G-protein RAS
RAS is bound to GDP = inactive
4) Signal (with help of GAPs) causes release of GDP & binds to GTP => activing RAS
5) creates a conf change, RAS binds & activates kinase (MAPKKK) & signal is transduced
**RAS’s intrinsically hydrolyses GTP->GDP, RAF dissociates & signal stops
6) MAPKKK gets phosphorylated to MAPKK
7) MAPKK gets phosphorylated to MAPK
8) MAPK gets phosphorylated to become transcription facto
9) genes are turned on

RAS is a G protein that acts as a regulator, enzyme itself, & molecular clock (intrinsic hydrolysis activity - converts GTP-> GDP & inactives)

GEFs = Guanine Nucleotide Exchange Factors GDP-> GTP

GAPs = GTPase ACtivating Proteins
- catalyze hydrolysis of RAS GTP -> GDP

Question 14

Describe the general structures of the receptor tyrosine kinases and the process that converts them from inactive proteins to active enzymes

Answer 14

1) Growth factor binds & dimerizes
2) Autophosphorylates
3) Binding of Adaptor proteins (Grb2 & SOS (GEF))
4) Complex is assembled
5) GEFs excahnge GDP for GTP
6) RAS binds to RAF & initiated MAP Kinase pathway

Question 15

Define autophosphorylation and its role in the signal-transduction process

Answer 15

• Homodimer phosphorylates each other

- Epidermal Growth Factor

Question 16

Explain the role of SH2 domains in tyrosine kinase function

Answer 16

SH2 domains contain proteins that are commonly used as signal transducer proteins

subtle differences in SH2 domains -> give specificity to signal

Question 17

Understand that mutations in Ras can cause cancer

Answer 17

Mutations RAS loses ability to hydrolize GTP -> always bent up & active all the time, always bound to RAF and always keeping MAP cascade on -> LOTS of cell division/growth/cancer

Cancers due to signalling cascade:
~25% (general)
30-50% lung/breast carcinoma
>90% pancreatic

HER2 = monomers that can dimerize without a signal

30% Breast Cancers overexpress

Question 18

Activation of the insulin receptor

Answer 18

1) conformational change in alpha induces a conformational change in beta; kinase domain activation & autophosphorylation of Tyrosine beta subunits
2) allows adapter proteins (IRS = insulin receptor substrates) to bind to receptor & then IRS gets phosphorylated

3) allows IRS’s binding sites to open up for SH2s & transduction diverges
- Grb2 -> RAS/MAP kinase cascade
- PI3 kinase -> PDK/Akt kinase cascade

** can also signal through IP3/DAG if Phospholipase C cleaves PIP 2 instead of being phsophorylated

Question 19

Describe the structure of the insulin receptor (IR) and the significance of the insulin-dependent dimerization of the receptor.

Answer 19

always a dimer but subunits on either side of cell membrane (alpha = exterior; beta = interior)
**NOT poised for action - always turned off; need signal to become activated

Insulin = Tyrosine Kinase receptor

Increases transcription of genes that raise blood glucose levels

Question 20

PI3 kinase cascade

Answer 20

Once Insulin Receptor is activated:

• RAS/MAP kinase cascade
• PDK/Akt kinase cascade

1) kinases phosphorylate phosphatidylinositol to PIP2 = PI 4,5 bis-phosphate (2 = free phosphates)
2) PIP 2 gets phosphorylated by PI3 kinase to PIP3 -> activates protein kinase cascade
3) PIP3 binds to PH domain on PDK1 (phosphoinositide-dependent kinase 1)
4) actiated PDK1, conf cahnge & activates kinase activity
5) PDK1 phosphorylates Akt/PKB (Protein kinase B) and causes it to dissociate which then phosphorylates proteins downstream causing an increase in the number of glucose transporters @ plasma membrane

PH = Pleckstrin Homology Domain
* plasma membrane is a reservoir of messenger molecules

Question 21

Understand Transforming growth factor signaling

Answer 21

SERINE/threonine kinase receptors that are heterodimer (2 different receptors - Type I & II)

• TGF-Beta primarily involved in cell growth and differentiation

Cytokine dimer (i.e. TGF-beta) attaches to Type II cause it to phosphorylate & activate/heterodimerize Type I by phosphorylating a Serine

• activated Type I binds & phosphorylates a Smad
• change in Smad’s confirmation makes it pop off & then goes to form a dimer with a Co-Smad
• R-Smad/Co-Smad translocaes to nucleus to bind to DNA & turn on genes

*Pancreatic Cancer deletes chromosomes of Smad4 = CoSmad -> can’t turn on genes to inhibit epithelial cell growth; resulting in overgroth & metastasis

Question 22

JAK/STAT signaling

Answer 22

• important for immune function: IL-2, gamma subunit, JAK3 are responsible for B-, T- & NK cell differentiation - mutations -> SCID
• very direct pathway
• JAK is always associated with the receptor because receptor dimer does NOT have intrinsic kinase activity (needs kinase activity of cytosolic protein Janus Kinase - JAK)

JAK Pathway:

• Cytokine binds 2 subunits of the receptor that have JAK on them = forming a dimer
• JAK’s phosphorylate each other
• Phosphorylated JAKS -> phosphorylate the receptors on one of their TYROSINES
• Phosphorylated Tyrosine creates a binding site allowing STAT with specific SH2 domain to bind
• JAK phosphorylates STAT -> conf change & pops off/dimerizes
• Free STAT translocated to nucleus to bind to DNA & turn on genes

*Tyrosine kinase: most common (exception is Smad serine kinase)

Question 23

STAT

Answer 23

Signal Transducer and Activator of Transcriptions

• actually carries out cell signal/net effect

Question 24

Understand Heptahelical receptors

Answer 24

7TM = 7 Transmembrane Receptors (alpha helices with chemical messenger bound inside)
GPCR = G protein coupling receptors

most common plasma membrane receptor

Question 25

Draw and describe how heterotrimeric G-proteins function, and how they play a role in how. Include the point at which cholera toxin acts

Answer 25

Heterotrimeric G-proteins(alpha, beta and gamma subunits) act as signal transducers for:

• cAMP or DAG/IP3 to be secondary messengers and amplify the signal
• act as molecular switches

Question 26

Understand there is a large class of Gα subunits that stimulate different pathways, thus affecting a variety of physiologic functions

Answer 26

Gα subunits resemble RAS is appearance & function (Internally can hydrolyze GTP -> GDP)

1) All 3 subunits are associated with receptor & inactive; Alpha has GDP bound to it
2) Ligand binds to receptor; receptor becomes a GEF letting alpha exchanges GDP for GTP
3) confirmation change & alpha can no longer stay attached to dissociates & activates signaling

** Gα subunit can hydrolyze GTP-> GDP and terminate signal

Use two secondary messengers:

• cAMP
• DAG & IP3

Question 27

Describe the roles of G proteins in coupling a hormone-receptor complex to adenylyl cyclase and in amplifying the stimulus

Answer 27

ß-Adrenergic receptor = GPCR

1) NorEpi binds to ß-Adrenergic receptor causing GEF to exchanges GDP for GTP
2) Dissociates from subunits & moves across membrane to associate with Adenylyl cyclase
that association catalyzes conversion of ATP -> cAMP (G alpha subunit can stay bound, not kicked off)
3) cAMP activates protein kinase A by binding to a regulatory subunit inducing conf change -> Active protein kinase A
4) continuing downstream for ß-Adrenergic physiological responses

Medicine:
Beta blcokers - compete for site and don’t allow HR to increase; treat cardiac arrhythmias

Cholera - Adenylyl Cyclase always one -> diarrhea because osmotic efflux of NaCL from gut
Cystic Fibrosis = keeps Cl channel open

Question 28

Angiotensin Cascade

Answer 28

1) Angiotensin II binds to receptor & conf change -> GEF causes Ga subunit exchanges GDP -> GTP then dissociates & moves along to stimulate Phospholipase C

2) PIP 2 cleaved by Phospholipase C produces the second messengers:
IP3 = inositol 1,4,5,-triphosphate
DAG = diacylglycerol

3) IP3 opens Ca++ channels in ER to increase [Ca++] in the cell
4) DAG + Ca++ allows protein kinase C to bind & turn on cascade & phosphorylate many proteins

**Angiotensin II regulates BP;
AGI -> AGII by ACE
ACE inhibitors block enzyme - net result = lower BP

Question 29

Draw a generic signal transduction pathway and identify the mechanisms responsible for termination

Answer 29

• Stimulus “Terminators” released to stop synthesis
• Messengers diffuse out of cell or degrade
• Desensitizaiton/down regulation - ligand receptor leaves or receptors becomes endocytosed
• GTPases: GTP is hydrolyzed to GDP
• Phosphateases activated to turn off cAMP reducing cAMP to AMP

*done at multiple places in the pathways

Question 30

Appreciate how crosstalk relates to signal transduction

Answer 30

• one or more components of one signal transduction pathway affect another
• there are often shared components that can interact with either pathway
• i.e.: cAMP’s role in regulating cell proliferation by interacting with the MAP kinase pathway; stimulation of adenylyl cyclase on the inner membrane surface to catalyze the conversion of ATP to cAMP

Question 31

Understand the two major regulators of the pathway, SOCS and PIAS of STATs

Answer 31

SOCS (Suppressors of Cytokine signaling)
- inhibit STAT phosphorylation by binding and inhibiting JAKs or competing with STATs for phosphotyrosine binding sites on cytokine receptors

PIAS ( protein inhibitors of activated STAT):
negatively regulated STAT by acting on the nucleus