9. Signal Transduction

Question 1

Steps in cell signaling

Answer 1

Synthesis and release of the signaling molecule by the signaling cell
Transport of the signal to the target cell eg through blood, ECM, simple diffusion, direct contact, signal released by cell acts back on it,
Detection of the signal by a specific receptor protein and transduction (processing) of the signal
A change in cellular metabolism, function, or gene expression
Termination of signaling (if none, can lead to cancer)

Question 2

Types of Receptors

Answer 2

1. G protein-coupled receptor- external ligand L binding to receptor R activates intracellular GTP-binding protein G, which regulates enzyme that generates intracellular second messenger X
2. Receptor tyrosine kinase- extracellularly, ligand binding L activates tyrosine kinase activity by autophosphorylation > kinase cascade > kinase activates transcription factor, altering gene expression inside nuclear envelope
3. Receptor guanylyl cyclase- ligand binding to extracellular domain stimulates formation of 2nd messenger cyclic GMP, cGMP, from GMP
4. Gated ion channel- opens/closes in response to conc of signal ligand of membrane potential
5. Adhesion receptor (integrin)- binds molecs in extracellular matrix, changes conformation, thus altering its interaxn w cytoskeleton
6. Nuclear receptor- hormone binding allows receptor to regulate expression of specific genes

Question 3

Cell Surface Signaling

Answer 3

Signal made by signaling cell
Released by signaling cell
Transported to target cell
Binds to cell-surface receptor
5-7 (signal transduction proteins and second messengers- convey signal intracellularly > conveyed to effector protein (6.) which (7a) produces second messenger, which then carries out signaling process > possibly affect metabolism (modify cellular metabolism, func, movement) OR (7a) taken down to nucleus to affect gene expression, development)
8. Shut down signaling process
9. Get rid of signal at cell surface

Question 4

G Protein Coupled Receptors

GPCR

Answer 4

Seven transmembrane alpha helices
Some extracellular and intracellular domains
Extracellular domains-where ligand of signal/hormone/nt binds @binding site
Ligands eg hormones like glucagon, epinephrine
Intracellular domain-where interaxn w G protein

Other names for GPCR:

• Heptahelical receptors
• Serpentine receptors (wind back and forth across pm)
• Seven transmembrane receptors

Question 5

G Proteins

Answer 5

Trimeric or monomeric proteins that bind GTP or GDP
Have intrinsic GTPase activity (hydrolyze GTP > GDP; not other way) > switch on/off by itself

Helper proteins avail eg GAP, GEF

• GAP inactivates G protein
• GEF activates G protein

G proteins = “switch proteins”

• Active when GTP-bound
• Inactive when GDP-bound

In cell: [GTP] > [GDP] and both compete for G protein binding

Question 6

GPCR Signaling

Answer 6

G-Protein Coupled Receptors (GPCRs) are -helical integral membrane proteins
G-proteins that couple to GPCR are heterotrimeric () (3 diff subunits) membrane-associated proteins that bind GTP
-Gs, Gi, Gq
G-proteins mediate signal transduction from GPCRs (signal from ligand to GPCR to G-protein) to effector proteins
-Adenylyl (=adenylate) cyclase (↑Gs (stimulatory), ↓Gi (inhibitory))
-Phospholipase Cβ (↑Gq- activates phospholipase C, beta subunit)
Effector proteins (adenylyl cyclase, phospholipase) produce second messengers
Hormone=first messenger (but can’t get into cell bc hydrophilic)- delivers signal at cell surface/pm > GPCR > G protein > effector protein > prod second messenger that can travel thru cell

Question 7

Four common intracellular second messengers

Answer 7

cAMP activates protein kinase A

• Adenylyl cyclase -> cAMP
• cAMP adenine (base) produced by adenylyl cyclase from ATP, linked to 5-C sugar ribose, linked to phosphate through diester bond (linked at 3’ and 5’ carbon gps) > transduce G sub s pathway

Phospholipase C -> PIP2 -> DAG, IP3 (can func in G sub q pathway)
>DAG (activates PKC), IP3 (opens Ca2+ channels in ER)

Guanylyl cyclase -> cGMP- won’t go over this lec (cGMP activates PKG, opens cation channels in rod cells)

Question 8

cAMP Activates PKA

Answer 8

cAMP > (hydrolyze diester bond) > AMP (attached to only 5’ carbon, no longer 3’ C)
pKa can’t be activated by AMP
Epinephrine and glucagon both added through GPCR, convey signal to G sub s, which activates adenylyl cyclase > inc in cAMP, which activates PKA
Insulin stim phosphodiesterase, dec cAMP, keeps PKA in inactive state
Epinephrine/glucagon usually does one thing; insulin does opposite

-PKA=tetrametric form: 2 catalytic and 2 regulatory subunits > inactive
-cAMP converts inactive pka to active pka
>cAMP binds to regulatory subunits, they will disassoc from catalytic sites and 2 catalytic subunits separate and are now active

Question 9

Glucagon and Epinephrine

Answer 9

Glucagon is produced by the α cells of pancreas in response to low blood glucose
Glucagon receptors are present in liver and adipose tissue; absent in skeletal muscle tissue
Epinephrine is a “fight or flight” hormone produced by adrenal medulla
Epinephrine binds to adrenergic receptors (α1, α2 and β) in many tissues (mainly beta)
Antagonists of β-adrenergic receptors (β-blockers) are widely used as anti-hypertensive drugs

Question 10

Epinephrine Signaling via Gs

Answer 10

1. Epinephrine binds to (beta-adrenergic receptor) GPCR, which undergoes conformation change that allows it to interact w trimeric G protein (G sub s in this case)
2. Allows GDP to be removed from G protein so that alpha subunit can pick up GTP
3. Weakens interaxn btwn alpha and beta subunits (alpha released from Gs (beta, gamma) and becomes mobile in membrane, interact w adenylyl cyclase, which then becomes active and converts ATP into cAMP), step 5.
4. cAMP activates pKA > phosphorylation of cellular proteins by PKA causes cellular response to epinephrine
5. PKA also to nucleus to modify transcription factor of phosphorylation and affect gene expression
6. ## Signal terminated by hydrolysis to AMP (process promoted by insulin)Glucagon signaling is similar, but instead of binding to beta-adrenergic receptor, it binds to own receptor

Question 11

Termination of GPCR Signaling

Answer 11

Kill second messenger
Inactivate G protein (self-inactivation although GAPs can affect/help this process)
-Since all proteins are GTPases, activated GTP protein hydrolyzing to GDP bound form (can no longer interact w effector protein), thus released and affinity for alpha subunit (1st molecule) increases so it comes back to form initial trimeric complex

Clicker-low PKA > which mut?
Inactivating mut in adenylyl cyclase

1. Gs w GDP turned off; can’t activate adenylyl cyclase
2. Contact of Gs w hormone-receptor complex causes displacement of bound GDP by GTP
3. Gs w GTP bound dissociates into alpha and beta-gamma subunits. Gs, alpha-GTP turned on; can activate adenylyl cyclase
4. Gs, alpha-GTP hydrolyzed by protein’s intrinsic GTPase; Gs, alpha turns itself off. Inactive alpha subunit reassociates w beta-gamma subunit

Question 12

Some bacterial toxins covalently modify Gα-subunits

Answer 12

• Cholera toxin ADP-ribosylates (transfers ADP and ribose from NAD molec) Gsα on an Arg to freeze it in the active, GTP-bound state (↑cAMP→ ↑Cl-,H2O,HCO3- (opens channels, thus high efflux and accompanying Na2+ ions) →Diarrhea)- inhibits GTPase; activates alpha sub s
• Pertussis toxin ADP-ribosylates Giα on a Cys to freeze it in the inactive, GDP-bound state (↑cAMP→Whooping cough) (unclear mechanism)- inhibits subunit dissociation; inactivates alpha sub i
• The net result of action of BOTH toxins is an overproduction of cAMP
• The bacterial toxins that cause cholera and whooping cough (pertussis) are enzymes that catalyze transfer of the ADP-ribose moiety of NAD+ to an Arg residue of Gs (in the case of cholera toxin) or a Cys residue of Gi (pertussis toxin). The G proteins thus modified fail to respond to normal hormonal stimuli. The pathology of both diseases results from defective regulation of adenylyl cyclase and overproduction of cAMP.

Question 13

Signaling via Gq

Answer 13

1. Hormone binds to specific receptor in extracellular space (e.g., Norepinephrine) (e.g., α1 receptor)
2. Receptor causes interaxn w G sub q trimeric G protein (and sim to G sub i, G sub s), active of alpha subunit, allowing it to release GDP and pick up GTP, be separated from beta/gamma subunits and travel across membrane to interact w effector proteins (which is phospholipase C (PLC) in this case, not adenylyl cyclase)
3. PLC- hydrolysis of PIP2 > 2 prods (DAG and IP3)
4. IP3 travels to ER and interacts w IP3 receptor (ion channel in ER membrane), inc cytosolic [Ca] from 0.1 mM to 1mM (10x increase)
   - ER=storage for Ca (cytosolic lvls usu low vs high lvls in ER
5. Ca + DAG activates PKC (assoc w membrane) > phosphorylates target proteins > cellular responses to hormone
6. PKC not only target for Ca, other proteins are targets eg calmodulin (nxt slide)

Question 14

Ca2+ - Another Second Messenger

Answer 14

Ca2+ binds to calmodulin
Ca2+-bound calmodulin activates CaM-kinases (protein kinases)

Calmodulin= flexible molecule, 4 binding sites for Ca
Ca2+-bound calmodulin activates calmodulin-dpt kinases or CaM-kinases > acts on enzymes > metabolic pathway activity changes in cell

Question 15

Adrenergic Receptors

Answer 15

3 diff adrenergic receptors
Binding/effects of E/NE varies based on receptors (and assoc tissues)
Note beta receptors- same hormone, same receptor, but diff effects in diff tissues

-Alpha 1
Assoc w G sub q
Activates phospholipase C (PIP2 -> IP3 -> Ca2+; PIP2 -> DAG); Ca2+ -> smooth muscle contraction

-Alpha 2
Assoc w G sub i- inhibitory G protein which shuts down adenylyl cyclase (ATP -> cAMP), thus dec cAMP (thus smooth musc contraction)
-dec Ca2+ (thus inhibits transmitter release)

-Beta receptors (Epinephrine=preferred ligand)
Assoc w G sub s- stimulatory G protein for adenylyl cyclase (ATP -> cAMP), inc cAMP -> heart musc contraction, smooth musc relaxation, glycogenolysis

–
Clicker- adenylyl cyclase stim by G sub alpha,s (stim), GTP

Question 16

Downstream Signaling

Answer 16

What downstream products affected, result in different actions resulting from THE SAME HORMONE (E) in diff tissues:
-Epinephrine in Heart (Gs)
> Contraction of cardiac muscle
-Epinephrine in Lungs, Intestine (Gs)
> Relaxation of smooth muscle
β-Adrenergic receptor- activated PKA, inc cAMP (which activates PKA)
-BUT downstream targets of PKA diff in 2 tissues (so can give epi shots for diff CC scenarios)

Question 17

Summary of GPCR Signaling

Answer 17

Gs- glucagon, adrenaline, histamine
G, abg -> G,a -> AC, inc cAMP, inc PKA

Gi- opiates, prostaglandins
Opp of Gs- G, abg -> G,a -> AC, dec cAMP, dec PKA

Gq- noradrenaline, vasopressin
G, abg -> G,a -> PLC -> PIP2 -> DAG -> inc PKC
…-> PIP2 -> IP3 -> Ca2+ -> CaM -> inc CaM kinase

Question 18

Receptor Tyrosine Kinases

RTK

Answer 18

Single pass membrane proteins
Long extracellular and intracellular domains
-Extracellular domains of tyrosine kinases=RTK’s and where ligands bind eg hormones, growth factors
-Intracellular domains=light blue=active enz domains=tyrosine kinase domain
>Capable of putting phosphate gps on tyr residues to target proteins (1st target protein is itself; once activated, TK auto-phosphorylates self on tyr residues)
Many RTKs- imp in cell growth, division
Receptor tyrosine kinases. Growth factor receptors that signal through Tyr kinase activity include those for insulin (INSR), vascular epidermal growth factor (VEGFR), platelet-derived growth factor (PDGFR), epidermal growth factor (EGFR), high-affinity nerve growth factor (TrkA), and fibroblast growth factor (FGFR). All these receptors have a Tyr kinase domain on the cytoplasmic side of the plasma membrane (blue). The extracellular domain is unique to each type of receptor, reflecting the different growth-factor specificities. These extracellular domains are typically combinations of structural motifs such as cysteine- or leucine-rich segments and segments containing one of several motifs common to immunoglobulins (Ig-like domains; see Fig. 4–22). Many other receptors of this type are encoded in the human genome, each with a different extracellular domain and ligand specificity eg insulin subunits already together, vs others=mono

Question 19

Signal Transduction with an RTK

Answer 19

1. When growth factor or hormone binds to (extracellular portions of receptor chains of) RTK, 2 subunits of RTK brought together (refer to prev slide), dimer binds to ligand while for monomer, binding of ligand brings 2 subunits together to form dimer
2. Tyr kinase domains (intracellular domains) activated and they autophosphorylate aka cross phosphorylation (one TK phos other TK > gen phos tyr)
3. Intracellular proteins (contain SH2 adaptor proteins which recognize and bind to phos tyr) bind to phos tyr
   Initial steps summary- hormone binding, dimerization, cross phosphorylation, binding of SH2 adaptor proteins

Question 20

RTK Signaling

Answer 20

Ligand binding causes dimerization and auto (cross) phosphorylation of RTK
Adaptor proteins with SH2 domains bind to phospho-tyrosines
Docking proteins (have their own enzymatic activity) bind to adaptor proteins via SH3 domains (on either adaptor or docking proteins)
Concl- intracellularly, phospho-tyr being bound by adaptor protein, which has dock protein bounded

Question 21

MAP Kinase Pathway Involves Ras – A G Protein

Answer 21

-MAP Kinase pathway for growth factors, incl insulin
-Req monomeric G protein called RAS
-(rev: GPCR=trimeric G protein; RTK=monomeric G protein)
1. binding of ligand to EGF (growth factor)
2. dimerization, kinase activation, auto cross phosphorylation of cytosolic receptor phospho-tyr, which are recog by adaptor proteins (GRB2 –pronounced greb2- here)
3. GRB2 causes docking protein, Sos –pronounced sauce- to bind to GRB2; Sos also interacts w RAS (mem bound), causing RAS to become active (lets go of GDP, picks up GTP [active=GTP bound]; active Ras dissociates from Sos
(Sos acts like GEF, promoting activation of RAS)

4. Active RAS recruits (inc affinity to Raf), binds protein Raf (usu inactive in cytoplasm) (protein kinase- can phosphorylate self; activated when bound to RAS)
5. Activated Raf finds protein MEK (protein kinase, activated thru phosphorylation); GTP hydrolysis > dissociate Ras from Raf
6. Activated MEK can phosphorylate target proteins either on tyr or threonines—does so- phosphorylates MAPK, actives MAPK
   - MAP kinase=mitogen activated protein kinase (aka ERK)
   - Mitogen=growth factor from outside
   - Mitogen activated=RTK
   - RTK activ. > Ras activ. > Raf activ. > MEK activ. > MAPK activ. > activ many transcription factors (which inc gene expression > cell growth, division)

Question 22

Ras Mutations Are Oncogenic

Answer 22

Ras mutations affect its GTPase activity
60% of pancreatic cancers and 33% of colorectal cancers involve Ras mutations

Single AA mutation- 12th position- Gly mutated to Val (bulkier hydrophobic AA)
Result- intrinsic GTPase activity of Ras is lost—locked in active form longer > activ more Ref > active more MEK > activ more MAPK > activ more tf’s (cascading effect of mutation)

Normally
GEF: converts Ras from inactive to active (also, Sos interaxn stimulates GDP-GTP exchange: GTP -> GDP)
Intrinsic GTPase activity converts Ras from active to inactive (mutation cuts short here)- can be stim by GAP but cannot be stim in mutant Ras (thus, signal stays on)
Mutation in GAP also can result in this

Question 23

Ras and Neurofibromatosis

Answer 23

Neurofibromatosis (condition that causes tumors to form in the brain, spinal cord, and nerves) is due to a mutation in NF-1 gene
NF-1 encodes a Ras-GTPase activating protein (GAP)
Loses GAP activity, cannot promote Ras inactivation so Ras active longer, result in neurofibromose (tumours on, under, or hanging off theskin)
Neurofibromose formed all over body

Question 24

Insulin Signaling

Answer 24

1. Insulin binds to receptor (already dimer), activates tyr kinase domain, causes cross phosphorylation (autophosphorylation) on carboxyl-terminal Tyr residues, which activates adaptor protein called IRS-1 (which gets phosphorylated on tyrosines (phospho-tyrosines))
2. Phospho-tyro on IRS-1 recog by SH2 domain of Grb2, which recog Sos, which recog Ras (causes GDP release and GTP binding to Ras), which activates Raf, which activ MEK on 2 Ser residues, which activ MAPK (ERK=extracellular regulated kinase here [same as mitogen activ kinase] on Thr and Tyr residue)
3. Activ ERK phos and activate tf’s eg Elk1, inc gene expression in nucleus (eg phos Elk1 joins SRF to stim transcription, translation of genes for cell div)
   * so insulin=growth factor for lots of cells (anabolic hormone)*

Question 25

Insulin Also Signals via PI 3-Kinase Pathway

Answer 25

Another adaptor protein binding to IRS-1 is PI3K
Activ PI3K turns PIP2 into (uncleaved) PIP3 in membrane bound state
PIP3=docking molec for cytosolic proteins (PKB, PDK1 (phosphor-inositide dependent protein kinase 1)), which have PH domains that allow for binding to PIP3
Results in them coming in close proximity to e.o
PDK1 phosphorylates PKB, activating PKB (after dissociation), which can then phosphorylate many target proteins
ins signaling causes activ of PKB

Question 26

PKB Mediates Insulin Signaling

Answer 26

Active PKB helps in the translocation of GLUT4 (pick up glc from circ) glucose transporters to cell surface in myocytes and adipocytes; thus ins helps clear glc from blood supply
PKB inactivates glycogen synthase kinase-3 (GSK-3) to stimulate glycogen synthesis
PKB activates transcription factors that promote the expression of genes involved in adipocyte differentiation
PKB promotes cell survival by inactivating proapoptotic proteins
PKB may activate protein phosphatase 1 (PP-1) (phosphatase=remove phosphate from target proteins) by phosphorylating its G subunit > explain effects of ins and carb metabolism (ins promotes dephosphorylation of protein that are phosphorylated by PKA)