lec 4 - cell-cell interactions: TGF-β and FGF signaling

cells recieve info from their environment to make ____ decisions

cell fate decisions

information from environment → cell behaviors + progressive specialization/restriction of cell potential towards specific differentiated cell types

what is the most common type of signaling during development

paracrine

paracrine signaling

a cell produces a signal → acts on nearby cells to produce a response

signal transduction

• the conversion of a signal from one form to another

• the process by which a signal is recieved and initates a series of intracellular molecular events that lead to a response

3 stages of signal transduction

reception, transduction, response

TGF-β ligands are ___meric

dimeric

two subfamilies of the TGF-β super family

TGF-β-like family

BMP-like family

types of ligands in the TGF-β-like family

TGF-βs

Activins

Nodal

types of ligands in the BMP-like family

BMPs

GDFs

AMH

role of TGF-βs

important roles in ECM + cell division

• implicated in tumors

role of activins + nodal

• differentiation of mesoderm

• left-right asymmetry in the early embryo

what does BMP stand for

bone morphogenic protein

role of BMPs

bone formation + embryonic development

what does canonical mean

the main pathway

the ____ subfamily uses Smad 2/3

the ____ subfamily uses Smad 1/5/8

• TGF-β-like

• BMP-like

describe the TGF-B signaling pathway

1. dimeric TGF-β ligand binds to a type 2 TGF-βR to form the ligand-receptor complex

2. type 2 receptor recruits/activates type 1 receptor via phosphorylation of serines/threonines in the GS domain

3. type 1 receptors phosphorylate Smad proteins, which act as secondary messengers

4. pSmad 2/3 (or 1/5/8) link to Smad 4 to create the phosphorylated Smad complex

5. the Smad complex translocates from the cytoplasm to the nucleus

6. the Smad complex binds upstream TGF-β target genes → activates transcription of the genes → cell response

what are the components of the ligand-receptor complex formed during TGF-β signaling

1. dimeric TGF-β ligand

2. 2 type two receptors

3. 2 type one receptors

receptor oligomerization

describes the process of assembling the ligand-receptor complex

TGF-β signaling - what kind of receptors are type 2 and type 1 receptors

2 - serine/threonine kinases (STKs)

1 - receptor tyrosine kinases (RTKs)

how do type 2 receptors activate type 1 receptors

bind to serines/threonines in GS domains

BMP signaling is controlled via

inhibition

notable BMP inhibitors

chordin, noggin, follistatin

what type of inhibitors are BMP inhibitors

ligand traps

ligand trap

inhibitor binds to ligand to inhibit it

why is BMP signaling controlled via inhibition

crucial to making sure pathway activation only occurs in the correct time and place

how can we image BMP pathway activation

use an antibody against pSmad 1/5/8 - specifically phosphorylated Smad

design an experiment to analye TGF-β receptor ffunction

use genetic engineering to create DNA that encodes a mutated receptor (LOF or GOF) and then express it in cells to interfere with signaling

how to create a LOF mutated receptor

change an amino acid in the kinase domain

what does a GOF mutation do to a receptor

makes it constitutively active

design an experiment to understand the role of Nodal signaling

• Nodal is a TGF-β ligand

• squint/cyclops are zebrafish Nodal ligands

• oep (one-eyed pea) is a mutant for Cripto. Cripto is an essential Nodal co-receptor.

1. WT zebrafish

2. squint/cyclops KO

3. Cripto → oep

what happens if you KO squint and cyclops in a zebrafish

mesoderm? gone

(Nodal helps pattern the mesoderm)

what happens if you change Cripto → oep?

mesoderm? gone

just like squint/cyclops, bc Cripto is a Nodal co-receptor (if Cripto doesn’t work, then Nodal can’t bind)

Nodal helps pattern the mesoderm

how to rescue oep mutants

inject constitutively activated type receptor RNA into the oep embryo → allows for Smad phosphorylation/mesoderm production without ligand binding

RTKs play essential roles in

RTK defects can cause

fundamental cell processes

diseases, cancer

RTKs mediate signals from ___, which are [diffusable/non-diffusable] because they are ____. These ligands use ____ signaling.

growth factors

non-diffusable

associated with the ECM

mostly paracrine (except for hormones)

are RTKs specific or can they interact with many ligands

both

most RTKs exist as ____ when unbound, except for ___

monomers

insulin receptor

RTK domains

extracellular, transmembrane, intracellular

RTK extracellular domain

• variable or conserved?

• role

• variable

• determines ligand binding specificity

RTK intracellular domain

• variable or conserved

• role

• highly conserved

• tyrosine kinase

canonical RTK receptor signaling pathway

1. ligand usually dimerises - binds simultaneously to two receptors

   1. sometimes ligand remains a monomer - two ligands bind simultaneously to two receptors

2. ligand binding facilitates receptor dimerization

3. receptor dimerization brings together intracellular tyrosine kinase domains

4. cross-phosphorylation of Y sites to activate

5. post-phosphorylation of Y sites → sites are sticky to other proteins → act as docking sites for downstream signaling proteins

how does phosphorylation of Y sites make the receptor sticky?

all RTK-binding proteins have an SH2 domain, which recognizes the phosphopeptide pY-E-E-I, found inside phosphorylated Y sites

proteins that bind to RTKs contain an ___ domain and ____ domain.

the first domain allows it to:

the second domain allows it to:

SH2; SH3

SH2 binds to pY-E-E-I domains, letting it stick to phosphorylated tyrosine sites

SH3 facilitates protein-protein interaction

RTKs are regulated by

Ras

Ras is an

intracellular switch

explain the Ras activation/inactivation cycle

• GDP-Ras = inactive

• GEF proteins exchange GDP for GTP

• GTP-Ras = active

• GAP proteins hydrolyze GTP → GDP

Ras pathway - how does docking lead to signal transduction

1. GRB2 contains both SH2 domains and SH3 domains

2. GRB2 binds to tyrosine kinase domains of the RTK (SH2)

3. GRB2 binds to Sos, a GEF protein (SH3)

4. Sos binds to nearby Ras, and promotes dissociation of GDP from Ras

5. Ras binds to GTP, is activated, and then it dissociates from Sos

6. Ras is now free to phosphorylate secondary messengers

Ras can get inactivated by GAP proteins easily. how does it create a more lasting signal?

binds to MapK signaling pathway

explain the MapK signaling pathway

• Ras → MapKKK/Map3K (usually Raf)

• MapKKK/Map3K/Raf → MapKK/Map2K/MeK

  • via dual phosphorylation on a serine and threonine separated by one amino acid (pS-R-pT)

• MapKK/Map2K/MeK → MapK

  • via dual phosphorylation on a serine and threonine separated by one amino acid (pS-R-pT)

• MapK → downstream proteins/TFs

why does the MapK pathway involve dual phosphorylation

safety mechanism - activation of the MapK pathway will only occur when Ras activates it

how many FGF receptors are there

4

what are the 3 FGF subfamilies

paracrine, intracrine, endocrine

describe the FGF receptor

• extracellular domains

  • Ig (immunoglobulin)-like domains - D1, D2, and D3

  • D2 and D3 are responsible for ligand binding

  • D3 determines ligand specificity

• between D1 and D2

  • acid box

• within D2

  • heparin binding site

    • FGFR activation requires binding with heparing sulfate proteoglycans

• transmembrane domain

• intracellular domain - split into 2 kinase domains

HSPG

heparan sulfate proteoglycan

what are heparan sulfate proteoglycans (HSPGs)

important extracellular modifiers of cell-cell signaling

3 main types of heparan sulfate proteoglycans (HSPGs)

• A - transmembrane syndecans

• B - membrane-tethered glypicans

• C - secreted perlicans

what are HSPGs made of

protein core + long chain of sugars called heparan

how do HSPGs help FGF receptors

each heparan sugar can be modified by sulfation

modification results in a code that creates binding sites for specific FGF ligands

• there are a LOT of FGF ligands - this helps the receptor react specifically to each one

FGF ligand can only bind/activate the RTK with HSPGs

_____ FGFs have a high affinity for HSPGs.

_____ FGFs have a low affinity for HSPGs.

Paracrine; Endocrine

• Paracrine FGFs - high affinity for HSPGs → retained at cell surface, act locally

• Endocrine FGFs - low affinity for HSPGs → diffuse into bloodstream

FGF signaling may trigger different cell behaviors through distinct pathway.

what are the pathways it triggers, what gene does it activate, and what is that gene’s function?

• canonical

  • Mapk - FOS (for cell proliferation)

• noncanonical

  • Akt - FOXO (for cell survival)

  • Calcineurin - NFAT (for cell motility)

name three diseases caused by FGFR mutations

• Apert syndrome, Pfeiffer syndrome, Achondroplasia

  • craniofacial, skeletal, and limb abnormalities

achondroplasia is caused by

mutations in FGFR3

a cell produces a signal → acts on nearby cells to produce a response

paracrine signaling

• the conversion of a signal from one form to another

• the process by which a signal is recieved and initates a series of intracellular molecular events that lead to a response

signal transduction

name the subfamily

• TGF-βs

• GDFs

• AMH

• Nodal

• BMPs

• Activins

• TGF-β-like

• BMP-like

• BMP-like

• TGF-β-like

• BMP-like

  • TGF-β-like

TGF-β-like family uses Smad ____

BMP-like family uses Smad ____

2/3

1/5/8

chordin, noggin, and follistatin fxn

inhibit BMP

Apert syndrome, Pfeiffer syndrome, and Achondroplasia are caused by

FGFR mutations