Chapter 16Cell Communication

Extracellular signal molecules can act overshort or long distances

Figure 16-3

Overview of signal initiation and responses

1. Receptor recognizesstimulus (3 typesshown)

2. Signal transferred tocytoplasmic surface ofreceptor

3. Signal transmitted toeffector molecule

4. Cessation of response

Signal transduction - signal conversion

Figure 16-13

First messenger

Second messengerEffector

Figure 16-5

Same signal molecule can induce differentresponses in different target cells

Although heart and salivary gland have samereceptor, different intracellular effector proteinsare activated

Extracellular signals bind to cell-surface or intracellularreceptors

Carrier protein

Figure 16-8

Animal cells get multiple signals

Figure 16-6

Extracellular signals can act rapidly or slowly

Fig. 16-7

signal transduction -- stimulusreceived by cell-surfacereceptor is different thansignal released in cell interior

signal pathways -- series ofdistinct proteins that alter theconformation of thedownstream protein

Figure 16-13

Signaling proteins can relay, amplify, integrateand distribute incoming signal

First messenger

Second messenger

Second messengers

small, nonprotein intermediaries acting in signaltransduction

e.g., cAMP, calcium, lipid-derived

Figure 16-17a

3 classes of cell surface receptors

1. Ion channel coupled receptors

Figure 16-17b

3 classes of cell surface receptors

2. G-protein-coupled receptors

3 classes of cell surface receptors

3. Enzyme-coupled receptors

Figure 16-17c

Basic characteristics ofcellsignaling systems

Many intracellular signaling molecules actas molecular switches

shape change by phosphorylation

Figure 16-15

may cause a conformationalchange in the protein

may be part of a proteinbinding site

may be added to serine,tyrosine, or threonine

may inc. or dec. proteinsactivity

Protein phosphorylation

most substrates are otherenzymes

Protein kinases/phosphataseschange the shape/activities ofthe proteins they modify

continual balance of kinaseand phosphatase activities

human genome ~500different kinases and ~100different phosphatases

Many intracellular signaling molecules actas molecular switches

shape change by GDP/GTP exchangeG-proteins are NOT kinases

Figure 16-15

The activity of monomeric GTP-binding proteins iscontrolled by two types of regulatory proteins

Figure 16-16

GAP - GTPase-activating proteinsGEF - Guanine nucleotide-exchange factors

G-proteins areNOT kinases

2 alternate types ofsignal transductionpathways

1. G protein-linkedreceptor

2. Protein kinase receptor

Figure 16-18

All GPCRs have a similar structure

7 transmembrane -helices

e.g., adenylyl cyclase

e.g., cAMP

e.g., glucagon receptor

G protein-coupled receptors (GPCRs)

GPCR are largest superfamily of proteins in animal genome(e.g., nematode has 19,000 genes of which 1000 are GPCRs)

Target of ~40% of modern medicinal drugsSee Figure 16-19

Heterotrimeric G proteins

1. signal causes shape changein receptor

2. G protein binds to receptor

3. GTP binds to G protein,causing shape change

4. subunit and subunitsseparate

G-protein activation

Fig. 16-19

Signal relay to effector

activated subunitchanges effectorshape (on)

Fig. 16-20

1. GTP hydrolyzed and subunit changes shape(off)

2. G protein no longerbinds effector, butreforms trimer

3. Effector changesshape (off)

Ending the responseTurning off theeffector

Fig. 16-20

Ending the response - II

1. GRK (G protein-coupled receptor kinase)phosphorylates receptor

2. arrestin binds, blocks G protein binding3. receptor endocytosed from PM

Receptordesensitization

G-protein signaling

--Shows that response can be ended by GTP hydrolysisand endocytosis of receptor

Specificity of G protein-coupled responses

not all parts of signal transduction machinery identical inall cells

multiple forms of receptors (e.g., 9 different isoformsof epinephrine receptors) with different ligand and Gprotein affinities

multiple G proteins ( 20 G; 5 G; 11 G ); variouscombinations

Gs stimulatory and Gi inhibitory

G proteincoupled receptors

--some G proteins directly regulate ion channels

16_19_open_K_chan.jpg

Some G proteins directly regulate ion channels

subunits activeslows heart beat

K+

See Fig. 16-21

G proteincoupled receptors

--some G proteins activate membrane-bound enzymes

Figure 16-20

Enzymes activated by G proteins catalyze production ofsmall intracellular signaling molecules

e.g. cAMP; IP3/DAG

e.g. Adenyl cyclase and phospholipase C

Glucose is stored in animal cells asglycogen (polymer of glucose)

(glycogen) phosphorylase makes more glucose

glycogen synthase makes more glycogen

Glucagon & epinephrine

Insulin

Glucagon (pancreas)

glucagon and epinephrine blood glucose byinhibiting glycogen synthase and activatingphosphorylase

the 2 hormones bind to different receptors, yetlead to same intracellular response, How?? Bothuse the same secondary messenger cAMP.

Adenylyl cyclase

integral membrane proteinthat makes cAMP

an effector

See Fig. 16-23

Figure 16-25

Adrenaline stimulates glycogen breakdown inskeletal muscle cells

Protein kinase A (PKA) isactivated by cAMP

PKA also phosphorylatesglycogen synthase, whichinhibits glycogen synthesis

Figure 16-26

Adrenaline in the same cells also stimulates glucose synthesis

Enzymes needed forgluconeogenesis

cAMP can activategene transcription

cAMP Signaling

Signal amplification

Each GPCR activatesmultiple G proteins

Each active adenylylcyclase make many cAMPs

Each active PKA canphosphorylate multiplephosphorylase kinases

Etc..

Reversal of signalphosphatase-1 removes thephosphates that were added byPKA

cAMP is destroyed by cAMPphosphodiesterase

Figure 16-23

Phosphatidylinositol (PI) and phosphoinositides (PIPs)

Numberingthe carbons ininositol

Phosphorylationof OH in inositol

PI PIPs

Named according toring position (inparentheses) andnumber of phosphategroups (in subscript)

Animal cells have several PI and PIP kinasesand phosphatases

PI headgroups are recognized by proteindomains that discriminate the different forms

Proteins are recruited to regions of themembrane where these PIs are present

Figure 16-20

Enzymes activated by G proteins catalyze production ofsmall intracellular signaling molecules

e.g. cAMP; IP3/DAG

e.g. Adenyl cyclase and phospholipase C

Lipid-derived second messengers

phospholipases --hydrolytic enzymes thatsplit phospholipids

PLA -- phospholipase A

PLD -- phospholipase D

PLC -- phospholipase C

Phosphatidylinositol-derivedare best studied

inositol

phospholipase Ccleaves PIP2 into DAGand IP3

Phosphatidylinositol (PI)- mediated responses

See Fig. 16-25

DAG IP3

How does PLC get activated?

See Fig. 16-25

Diacylglycerol (DAG)

lipid molecule remains in PM

recruits and activates protein kinase C (PKC)

PKC is a serine/threonine kinase that is imp. in manycellular events (e.g., cell growth, differentiation,metabolism and transcriptional activation)

phorbol esters -- plant compounds that mimic DAGand activate PKC; cells lose growth control andbehave as malignant cells

IP3 (inositol 1,4,5-trisphosphate)

small, water soluble

binds to a Ca++ channel receptoron smooth endoplasmic reticulum(SER); Ca++ release

Figure 16-27

Phospholipase C releases lipid-derived second messengers

Ca++ as an intracellular messengerCa++ release from intracellular stores acts as a secondmessenger

First messenger: hormones, neurotransmitters, electricalactivation (muscle)

Calcium Signaling

Ca++ is not made enzymatically [Ca++] controlled by pumps and channels Free intracellular [Ca++] ~ 0.1 M [Ca++] within lumen of ER ~ 1 mM (10,000-fold

higher)

channels intocytoplasm normallyclosed

out of cytoplasmpumps usually active

Where are Ca++ release channels found?

IP3Rs: present onsmooth ER of mostcell types

Unlike cAMP, Ca++ modulates a broad rangeof effectors

cAMP acts on kinases; primarily PKA

Ca++ can activate kinases, ion pumps,proteases, phosphatases, Ca ++-bindingproteins, etc.

Calmodulin -- best known Ca ++-bindingprotein

Calmodulin (CaM)

binds Ca++ only instimulated cells(affinity too low)

Ca++ binding changesconfirmation andaffinity for otherproteins

Activated CaMactivates Ca++ pumpto reduce cytosoliclevels

Figure 16-29

Calmodulin

Role of GPCR in vision

Receptor - rhodopsin

G protein -transducin

Effector - cGMPphosphodiesterase

response 20 msec(20/1000th of a sec)

cGMP keepsopen

See Figure 16-30

16_29_amplifies_light.jpg See Fig. 16-31

2 alternate types ofsignal transductionpathways

1. G protein-linkedreceptor

2. Protein kinase receptor

transmembrane receptorsenzymes that adds phosphate groups totyrosine aa on proteinseach RTK monomer crosses PM once (GPCR7Xs) > 50 different RTKs have been identified

Receptor tyrosine kinases (RTKs)

Typical RTK

1. Inactive receptor monomers

2. Ligand induces dimerization andautophosphorylation

3. Signaling protein bind to specificphosphorylated Tyr

Figure 16-32

phosphotyrosine motifs -- onlytyrosines surrounded by certain aa arephosphorylated

SH2 domains (src homology 2) andPTB domains (phosphotyrosine binding);sites in proteins that recognize thephosphorylated tyrosines; found inmany cell signaling proteins

Important concepts

Interaction between SH2 domain andphosphotyrosine

~ 100 aa long

Found in ~110 different human proteins

SH2 compact plug-in module can be inserted nearlyanywhere in a protein sequence and not disturb proteinfolding

Role of RTK in cellular activities

many types, but best studied:

-- hormones - e.g. insulin, growth hormone-- growth factors - epidermal growthfactor (EGF), fibroblast growth factor(FGF), platelet-derived growth factor(PDGF)

tetramer of two and two polypeptide chainsinsulin binding to changes conformation of activated receptor phosphorylates self(autophosphorylation) and soluble proteins (insulinreceptor substrates; IRSs)

Insulin receptor

PTB domain of IRS-1binds to insulin receptor

receptor phosphorylatesdocking sites on IRS-1

IRS-1 (insulin receptor substrate-1)

pTyr binding oninsulin receptor

docking sites

SH2 domain

RTK activate PI-3-kinase

Fig. 16-33

phosphorylated inositol rings bind to modules on manyproteinsrecruits specific protein to cytoplasmic face of PMPH: pleckstrin homology domains targets proteins tomembranes by binding PIP2 or PIP3 when they areproduced

PI PIP PIP2

Phosphoinositides direct major signaling cascades

PIP3 -- PI(3,4,5)

-can act as docking sites and as secondary messengers

PIP3 Produced in Response to Stimulation of a RTK

-GFP fused to a PH (PIP3 binding domain from a tyr kinase)-PDGF causes the production of PIP3 at the plasma membrane

Dr. Seth Field

e.g., PDGF or Insulin

PH = pleckstrin homology

PI-3-kinase and PKB activation

Akt = Protein kinase BFig. 16-33

PI3K generates PIP2 and PIP3protein kinase B (PKB)binds/partially-activated by PIP2and PIP3 via its PH domainsfully activated by anotherkinase(s)signal reversed bydephosphorylating receptorInc. protein syn.Inc. glucose uptakeInc. glycogen syn.

thus, insulinleads to lowerblood glucose

PH domains --pleckstrin homologydomain -- bindsPIP2/PIP3

The importance of Ras

Ras is a monomeric G protein held to PM bylipid group

Monomeric G proteins mimic G in function

nearly all RTK activate Ras

30% of human tumors have a mutated ras (orras-like) gene

Figure 16-33

Most RTK activate the monomeric G protein Ras

Ras mutations leading to tumorsprevent GTP hydrolysis; Ras alwayson

Ras GEF MAP kinasepathway

Grb2Sos

Ras

MAP kinase cascadeMitogen-activated proteinkinase

growth factor binding leads toinc transcription of genesinvolved in growth responses

Ras GTP hydrolysis is inducedby a GTPase activating protein(30 min dec to 1/10 sec)

Ras mutations leading totumors prevent GTP hydrolysis;Ras always on

16_32_MAP-kinase.jpg

MAP kinase pathway

See Fig. 16-34

Signals that originate from ECM

integrins transmit signals thatinfluence cell growth, shape,migration, differentiation and survival

normal cells do not grow insuspension

Signals induce cell growth

1. clustering ofintegrins inducesprotein binding

2. P-tyr activation ofsrc and FAK kinases

3. Ras activated

4. MAP kinase cascadeinc. transcription ofgenes involved ingrowth

FAK = Focal adhesion kinasesrc is an oncogene

Actin

P-tyr proteins

Extracellular signals bind to cell-surface or intracellularreceptors

Carrier protein

Figure 16-8

Role of nitric oxide as an intracellular messenger

Mode of action for Viagra

NO (nitric oxide) released by nerve and endothelial cells in penis

NO --> higher cGMP levels --> muscle relaxation and increasedblood flow --> erection

Viagra inhibits PDE5 (form of cGMP phosphodiesterase; found onlyin penis) keeping cGMP levels high

smooth muscle cellaround arterialblood vessels

Animal cells get multiple signals

Figure 16-6

Figure 16-40

Signaling cascades are not just linear

convergence - 2 ligands, 1 pathdivergence - 1 ligand, many paths

convergence divergence

Scaffolds help organize cascades

structural not enzymatic components

provide spatial localization & substratespecificity