BCOR 011 Lecture 19 Oct 12, 2005 Lecture Outline

I. Cell Communication – Signal Transduction 1. Types of intercellular communication Chapter 11 2. The primary receiver – Receptors

3. - the concept of AMPLIFICATION 4. Types of receptors 5. Ion Channels – Membrane depolarization 6. Trimeric G-Protein coupled receptors - the cAMP signal pathway - the phophatidyl inositol pathway, Ca++ release 7. Tyrosine Kinase – MAP Kinase Cascade 8. Internal cytosolic receptor systems

External signal is received and converted to another form to elicit a response 1 2

External signals are converted to Internal Responses General principles:

• Cells sense and respond to the environment 1. Signals act over different ranges. Prokaryotes: chemicals Humans: 2. Signals have different chemical natures. light - rods & cones of the eye 3. The same signal can induce a sound – hair cells of inner ear chemicals in food – nose & tongue different response in different cells.

• Cells communicate with each other 4. Cells respond to sets of signals. Direct contact 5. Receptors relay signals via Chemical signals intracellular signaling cascades.

3 4 Signals act over different ranges 1º messenger Endocrine Paracrine Cells local long distance ex. nitric oxide, detect ex. estrogen, histamines, signal & epinephrine Effector prostaglandins Like Fig 11.4 respond Enzymes Target Enzymes 2º messengers

Signal transduction: ability of cell to translate Neuronal/Synaptic direct contact receptor-ligand interaction into a change in behavior ex. neurotransmitters or gene expression Cell-cell recognition5 6 ex. delta/notch

Each protein in a signaling pathway Primary – Amplifies the signal by activating multiple Messenger Secondary Target Messengers Enzymes copies of the next component in the pathway

EXTRACELLULAR CYTOPLASM FLUID Plasma membrane 1 primary signal- activates an enzyme activity, processes 100 substrates per second

1 Reception 2 Transduction 3 Response Primary enzyme activates 100 target enzymes

Receptor Each of the 100 enzymes activates an Activation of cellular response additional 100 dowstream target enzymes Relay molecules in a signal transduction pathway Each of the 10,000 downstream targets Signal molecule activates 100 control factors Figure 11.5 Cascade Effect so rapidly have 7 1,000,000 active control fac8 Receptors relay signals via intracellular Cell-surface receptors -large &/or hydrophilic SIGNALING CASCADES ligands Push doorbell ion-channel-linked

Ring bell

Enzymatic activation Trimeric of G-protein-linked more ENZYMES enzyme-linked (tyrosine kinase)

9 10

Signal Gate closed Ions Ion channel receptors molecule Review: (ligand) Remember the Na+/K+ ATPase (Na+/K+ pump)? [Na+] inside ~10mM; outside ~150mM [K+] inside ~100mM; outside ~5mM Ligand-gated Plasma ion channel receptor Membrane cell has membrane potential ~ -60mV Examples: Gate open Na+ Muscle Contraction Cl- -60mV Nerve Cell communication + Cellular K response - A- + - - + - - Gate close - + + + + 11 12 Figure 11.7 Gated ion channels specifically let ions through membrane “keys”: small molecules (ligand-gated) Acetylcholine: or change in membrane potential (voltage-gated) common neurotransmitter + +++ + + +++ + +++ ++

------60 mV inside

opens ligand-gated Na+ channels on muscle cell and some nerve cells 13 14

Gated ion channels Gated ion channels specifically let ions through membrane specifically let ions through membrane “keys”: small molecules (ligand-gated) “keys”: small molecules (ligand-gated)

+ + +++ + + + + + + + + ++ + +++ + + +++ ++ + + + ++ + + + - - - - - + ------+ + + --- + + - -60 mV inside + + + +10 mV inside

Influx of Na+ ions causes local, transient depolarization of membrane potential

15 nerve impulse (action potential) 16 Gated ion channels Gated ion channels specifically let ions through membrane specifically let ions through membrane “keys”: small molecules (ligand-gated) “keys”: small molecules (ligand-gated) or change in membrane potential (voltage-gated) or change in membrane potential (voltage-gated)

+ + +++ + + +++ ++ + + + + + + +++ + + +++ +++ + + + + + + + + - - + ------+ + ++ ------+ ++ --- - - + + + + + + + + + + + + + + + + + + + + + +10 mV inside + +10 mV inside

Influx of Na+ ions causes local, transient depolarization of Influx of Na+ ions causes local, transient depolarization of membrane potential membrane potential nerve impulse (action potential) 17 nerve impulse (action potential) 18

Gated ion channels specifically let ions through membrane a. polarized Transmission of action potential “keys”: small molecules (ligand-gated) or change in membrane potential (voltage-gated) b. Action potential Initiated by Ligand-gated Na+ + +++ + + +++ +++ + + + + + + + channels opening + Local depolarization Na+ + Depolarization opens - - + ------+ + ++ + + - - - Voltage-gated Na+ - + + + + + + Na+ + channels K+ +10 mV inside re-polarization + + Na channels close K Na+ Influx of Na+ ions causes local, transient depolarization of channels open K+ membrane potential Action potential nerve impulse (action potential) 19 Propagates to as more 20 Voltage-gated channels open Action potential: Signal transmitted to muscle Muscle cell across a synapse nerve impulse; rapid, cell a. self-propagating electrical signal a. Depolarization opens voltage-gated Ca+2 channels Muscle b. Ca+2 rushes in; Vesicles cell fuse with membrane b.

c. Neurotransmitter Muscle released; opens ligand-gated cell Na+ channels on muscle cell Depolarizes muscle cell Signal: electrical to c. 21 chemical to electrical 22

Depolarization of Muscle Cell Turning off the synapse…….. ++ Results in release of [Ca ]: Acetylcholine degraded by acetylcholinesterase or removed Typically in cytosol Ca++ is 10-7 M, by re-uptake & endocytosis maintained ultra-low by active transport a. if not removed…… “pumps” Ca++/ATPases “vacuums” a,b Pesticides Ca++ Stored in c Nerve gases Smooth Endoplasmic b. reticulum Potent enzyme inhibitors Post-synaptic membrane can’t Ca++ from SER c. repolarize In Cytosol Paralysis, Tetany Triggers Activation of Myosin ATPase To “walk along” actin filaments – causing contraction 23 24 Cell-surface receptors -large &/or hydrophilic Trimeric G protein-linked receptors: ligands largest family of cell-surface receptors ion-channel-linked 7-pass membrane receptor ligand

Trimeric Ligand binding G-protein-linked

activates G-protein G-protein GTP enzyme-linked by GTP exchange (tyrosine kinase)

25 26

Signal-binding site G-protein activation Trimeric G-protein-linked receptors “molecular switch” Segment that inactive interacts with G proteins (b) Ligand binds G-protein associates G-protein-linked Activated Inctivate Plasma Membrane Signal molecule Receptor Receptor enzyme (c) GDP-GTP exchange •α-Subunit dissociates GDP G-protein GDP GTP CYTOPLASM (inactive) Enzyme

Activated Effector enzyme Active G-Protein-GTP -> allosteric modulator active of target effector enzyme GTP GDP

P i 27 28 Cellular response Figure 11.7 An activated G -protein-GTP •All G-proteins – similar structure/activation α – Can trigger the formation of cAMP, which then acts as a second messenger in cellular pathways

•There are TWO broad subclasses of First messenger (signal molecule trimeric G-protein-activated signal such as epinephrine) Adenylyl cyclase transduction pathways: G protein

GTP depends on their target effector enzymes G-protein-linked receptor A. adenylyl cyclase ATP B. phospholipase C cAMP Protein kinase A

29 Figure 11.10 30 Cellular responses

G-protein-GTP activation of Effector Enzyme adenylyl cyclase cAMP Inactive produces the 2nd messenger cAMP PKA Activated activates G-protein target enzyme

Protein Kinase A (PKA)

phosphorylates Active target proteins PKA

Like Fig 11-9 31 32 Reception Binding of epinephrine A Signal to G-protein-linked receptor 1 Protein Kinase A (1 molecule) Phosphorylates downstream target enzymes Cascade Transduction Inactive G protein Active G protein (102 molecules) amplification 102 Phosphorylase kinase Inactive adenylyl cyclase Active adenylyl cyclase (102)

inactive ATP Cyclic AMP (104) 4 Inactive protein kinase A 10 + P Active protein kinase A (104)

active Inactive phosphorylase kinase 5 Active phosphorylase kinase (105) 10

Breaks down Inactive glycogen phosphorylase 6 Active glycogen phosphorylase (106) 10

Starch Response Glycogen Into Glucose 33 Figure 11.13 Glucose-1-phosphate 1034 8 (108 molecules)

What are targets for Protein Kinase A?? How to shut it off? G-protein α-subunit is on a timer

No ligand cAMP regulated pathways

Function target tissue signal

Glycogen breakdown muscle,liver epinephrine Heart rate cardiovascular epinephrine Inherent Water reabsorption kidney antidiuretic Auto GTPase activity hormone Shut-off

35 36 How to shut it off? How do you turn it off?

kinases – phosphatases

Diametrically Opposed…

cAMP- Remember: whether you active phosphodiesterase or inactivate by adding P rapidly cleaves depends on the specific protein cAMP (so short lived) 37 38

What if you can’t turn off cascade? TWO subclasses of trimeric G-protein-activated Vibrio cholera - causes cholera signal transduction pathways: 7 great pandemics, Ganges Valley, Bangladesh

Normal gut: H20, NaCl, NaHCO3 secretion A. target protein adenylate cyclase controlled by hormones via Gs/cAMP signal cAMP-> PKA pathways B. target protein phospholipase C V. cholera – secretes enterotoxin, chemically modifies αGs – no GTPase activity - stays ON

Severe watery diarrhea – dehydration, death

39 40 target effector enzyme PIP is Phospholipase C 2

PLC cleaves a membrane DAG phospholipid (Phoshatidyl inositol) to Lipid Soluble two 2nd Messengers:

InsP3 Inositol-1,4,5-Trisphosphate Water (InsP3) & Soluble

Diacylglycerol (DAG)41 42

DAG Activates Response: Protein Protein Kinase C phosphorylates Kinase C target proteins (ser & thr) (Starts cell growth Cascade) regulation of ion channels InsP cytoskeleton 3 increases cell pH Ligand Protein secretion for ER ligand- gated Ca++ Ca++ Binds & activates calmodulin channels Calmodulin-binding proteins activated (kinases & phosphatases) ↑ Ca++ levels43 44 1 2 A signal molecule binds Phospholipase C cleaves a 3 DAG functions as to a receptor, leading to plasma membrane phospholipid a second messenger activation of phospholipase C. called PIP into DAG and IP . in other pathways. 2 3 Summary - signaling is endocrine, paracrine, synaptic, or direct cell contact EXTRA- Signal molecule CELLULAR - signal transduction is mediated by receptor (first messenger) FLUID - Receptors bind primary signal (ligand) G protein proteins - Some amplification event occurs DAG - Example: ligand gated ion channel opens GTP influx of ions triggers change in activity PIP G-protein-linked 2 (vesicle fusion in nerve end, contraction in muscle) receptor Phospholipase C IP 3 - Example: ligand binds to 7-pass membrane receptor (second messenger) catalyzes GTP exchange

IP3-gated to Ga-subunit of trimeric G-protein calcium channel active Ga-subunit-GTP is allosteric activator of effector enzymes:

Endoplasmic Various - ADENYLATE CYCLASE: makes cyclic AMP Cellular reticulum (ER) 2+ proteins Ca response - PHOSPHOLIPASE C: makes DAG and IP3 activated these second messengers activate target enzymes Figure 11.12 Ca2+ (second Trigger cascades messenger) - Must shut off cascade: removal of ligand, hydrolysis of GTP, ++ 4 5 6 phosphodiesterase, protein phosphatases, Ca ion pumps IP3 quickly diffuses through Calcium ions flow out of The calcium ions the cytosol and binds to an IP3– the ER (down their con- activate the next 45 46 gated calcium channel in the ER centration gradient), raising protein in one or more membrane, causing it to open. the Ca2+ level in the cytosol. signaling pathways.