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Flashcards in Molecular Signalling Deck (31)
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1

Essential components of intercellular communication

-signalling cell
-signal
-receptor
-target molecule
-response

2

Forms of intercellular communication

-synaptic
-paracrine
-endocrine

3

Types of signals mediating intercellular communication

-signalling molecules can be
-cell-impermeant (cant diffuse through cell - need receptors on post cell)
-cell permeant (can diffuse through post cell)
-cell-associated (signalling molecule is attached to pre cell)

4

Mechanisms of target protein regulation

-protein targets of signalling cascades are often phosphorylated on SERINE, THREONINE, or TYROSINE residues
-by Ser/Thr kinases and Tyrosine kinases

-phosphorylation leads to changes in protein structure or ability of protein to bind other proteins (changes its function)

-dephosphorylation by protein phosphatases reverses change

5

Ionotropic receptors

-ligand gated ion channels
-binding of ligand causes channel to open
-channel is ion selective
-diffusion of ions in/out of cytoplasm elicits change in membrane potential
-if ion channel permeable to calcium, calcium signalling initiated

6

Ionotropic receptor structure

-4-5 subunits grouped around a central pore
-charged amino acid residues at pore entrance form ion selectivity filter
-gate in pore center opens with ligand binding
-DESENSITIZATION: closing when ligand is bound for long time

7

Metabotropic receptors

=GPCRs
-bind heterotrimeric G proteins
-binding of ligand = signal and activated the G protein
-G protein regulates enzymes and ion channels

8

Metabotropic receptor structure

-common structure: 7 transmembrane receptors
-ligand binding leads to conformational change in receptor that leads to replacement of GDP for GTP in G protein

9

Enzyme linked receptors

-have an intracellular domain that is enzymatically active
-Receptor Tyrosine Kinases
-Ser/Thr kinases
-Tyrosine and S/T phosphatases
-Guanylyl cyclases

-signalling binding to extracellular domain activates enzyme activity

10

Enzyme linked receptor: RTK

-activation of RTC involves:
1. Ligand binding
2. Receptor DIMERIZES (2 halves come together with NGF dimer as ligand
3. Autophosphorylation
4. Binding of effectors, phosphorylation of other proteins

11

Intracellular receptors

-activated by lipophilic signalling molecules that diffuse across PM
-binding of signalling molecule causes disinhibition of receptor (activates it) by dissociation of inhibitory protein
-cytosolic receptors translocate the receptor to the nucleus
-the activator receptors bind co-activator proteins and/or DNA to induce GENE TRANSCRIPTION

12

Properties of G Proteins

-able to bind and hydrolyze GTP
-regulate effectors (enzymes or ion channels)
-only interact with effectors when GTP bound
-inactive in GDP bound form

13

Classes of G proteins

-heterotrimeric:
-composed of 3 subunits (a, B, Y)
-activated by METABOTROPIC receptors

-small monomeric:
-single polypeptides
-activation by RECEPTOR TYROSINE KINASES

14

Activation/Inactivation of heterotrimeric G proteins

1. ligand binds to GPCR
2. GPCR promotes exchange of GDP for GTP
3. G protein a subunit dissociated from BY subunit and leaves receptor
4. Both a and BY subunit can interact with effectors
5. GTPase activating protein (GAP) facilitated GTP hydrolysis (turning a unit back to GDP bound)
6. Subunits reassociate with GPCR

15

Types and targets of heterotrimeric G proteins

Gs: activates adenylate cyclase
-increases cAMP production

Gi: inhibits adenylate cyclase
-decrease cAMP production

Gq: activates phospholipase C (PLC)
-increase diacyclglycreol and IP3 production

Gt (transducin): activates cGMP phosphodiesterase (PDE)
-decrease cGMP

16

Activation of small monomeric G proteins

1. Guanine nucleotide exchange factor (GEF) facilitates replacement of GDP by GTP
-activates G protein
2. GTPase activating protein (GAP) facilitates hydrolysis of GTP
-inactivates protein
3. GEFs can be activated by active RTKs
4. GAPs can be regulated by upstream signalling

17

Targets of monomeric G proteins

-ras: functions in cell proliferation, differentiation, survival
-effector: MAP kinase pathway

-rho: functions in actin dynamics
-effector: ROCK kinase

-rab: function sin membrane trafficking
-effector: various

18

MAP kinase pathway

-activated RTK causes GEF to facilitate exchange of GDP for GTP bound Ras
-Ras (membrane bound) activates MAPKKK
-MAPKKK phosphorylates (activates) MAPKK
-MAPKK phosphorylates (activates) MAPK
-MAPK phosphorylates and regulates many proteins

19

cAMP

-cAMP = cyclic adenosine monophosphate
-generated by adenylyl Cyclades (activated by Gs, inhibited by Gi)
-activates protein kinase a (PKA)
-binds and modulates conductance of cyclic nucleotide-gated ion channels

-cAMP degrades by phosphodiesterases (PDE)

20

Protein kinase A

-is a serine/threonine kinase
-phosphorylates proteins involved in synaptic transmission, glucose, and lipid metabolism

-2 catalytic, 2 regulatory subunits

-cAMP bind to regulatory subunits, relieving inhibition of the catalytic subunits (so they are then active)

21

cGMP

-cGMP = cyclic guanosine monophosphate
-generated by guanylyl cyclase
-activates protein kinase G (PKG)
-binds and modulates cyclic nucleotide gated ion channels

-cGMP degraded by Gt (transducin) activated phosphodiesterases (PDE)
-cGMP important second messenger in photoreceptors

22

IP3 and diacylglycerol

-PIP2 is a phospholipid in PM
-cleavage of PIP2 by phospholipase C (PLC) yields diacylglycerol (DAG - membrane bound 2 messenger) and inositol trisphosphate (IP3 - 2 messenger in cystol_
-multiple PLC activated by Gq proteins, tyrosine kinase, and calcium

-IP3 binds to and activates IP3 receptors (ligand gated Ca2+ channels in ER membrane)
-Ca2+ released from ER stores is 3rd messenger
-initiates Ca2+ dependent signalling
-DAG (and Ca2+) activates PKC (Ser/Thr kinase)

23

Maintenance of basal cytoplasmic Ca2+ concentration

-Basal Ca2+ conc in cytoplasm is low (<100nm) due to:

1. Extrusion by plasma membrane Ca2+ ATPase (PMCA)
2. Uptake into ER by sarco/edoplasmic reticulum calcium ATPase (SERCA)
3. Buffering by Ca2+ binding proteins
4. Mitochondrial calcium uptake

24

Sources of Ca2+ signals

-transient, specially restricted calcium signal through opening of:

1. Voltage gated calcium channels
-ligand gated calcium channels
-IP3 receptors
-Ryanodine receptors (from ER, Ca2+ gated)

25

Ca2+ effectors

-transient, local calcium elevations activate Ca2+ effectors:

-Ca2+ activated Calmodulin (CaM)
-Ca2+/CaM binds and modulated kinases, calcium channels...
-modulation of synaptic transmission by Ca2+/CaM dependent Kinase 11 (CaMKII)
-which is a Ser/thr kinase

26

Locally restricted Calcium signalling

-due to efficient calcium buffering, extrusion: calcium signalling often local rather than cell-wide

-compartmentalization of calcium signals in dendritic spines

27

Regulation of gene expression by signal transduction pathways

-synthesis of new mRNA and proteins regulated by signal transduction pathways
-slow onset (>30min), long lasting
-gene transcription requires binding of transcriptional activator proteins to DNA near promoter region of target gene
-binding of transcriptional activator allows formation of RNA polymerase complex, transcription of gene

28

CREB signalling

-cAMP responsive CREB needs to be phosphorylated to have transcriptional activity

-phosphorylated by PKA, MAPK (from ras) and Ca2+/CaM kinase

-activated CREB stimulates transcription of specific genes

29

C-fos signalling

-c-fos: transcriptional activator that is present in unstimulated cells at low concentration

-c-fos: is immediate early gene: stimulus by MAPK, PKA, CaMK directly elicits transcription of c-fos

-synthesized c-fos protein then stimulates transcription of other genes

-CREB activates synthesis of mRNA from c-fos gene, which is turned into a protein which works as a transcriptional activator

30

Divergent signal transduction: NGF

-NGF = neurotrophic growth factor
-TrkA = NGF receptor (RTK receptor)
-required by sensory and sympathetic neurons for survival, differentiation, neurite outgrowth
-activation of multiple signalling pathways: PLC, ras/MAPK