Lecture 21: Cell Signaling Flashcards by Tami Ojewole

examples of cellular signals

growth factors, cytokines, hormones

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Signals are received by ______ where?

receptors
at the cell surface (or inside cell)

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signals are transduced to where? and do what?

to the nucleus
they change the activity of transcription factors

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Changes in gene expression can lead to a wide variety of responses including ____

progression or halting of cell cycle
modification of metabolic pathways differentiation
changes in cell shape or movement.

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Some receptors can transduce signals through _____

more than one pathway

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Many genes are regulated by multiple transcription factors each of which can be activated by ______

more than one type of signal

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There can be ____ between signaling pathways

crosstalk

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signal transduction (def.)

process of converting signal into cellular response

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Steps in signal transduction

release or exposure of signaling molecule by signaling cell
binding of signal to receptor (on target cell)
Initiation of intracellular signal transduction pathway by activated receptor
Specific change in cellular function, metabolism or development
Removal of signal

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examples of effector proteins + effect

-caspase -> death
-metabolic enzyme -> altered metabolism
-transcription regulatory protein -> altered gene expression
-cytoskeletal protein -> altered shape or movement

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Four types of extracellular signaling

1) contact dependent -> signaling cell binds to receptor
2) paracrine -> close signaling cell
3) synaptic
4) endocrine -> hormone over long distance

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contact dependent signaling example

Apoptosis (FasL/Fas)

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Paracrine signaling example

Interferon response

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Endocrine signaling example

Insulin

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Cellular responses to signals can be _____

very fast or slow

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______copies of a specific cell surface receptor/cell

1,000-50,000

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Receptors comprise _____ of plasma membrane proteins

< 0.01%

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Most receptors are_____ that require _____for extraction from cellular material

transmembrane proteins
detergents

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Two ways to isolate and identify receptors

1) Affinity purification/chromatography
2) Expression cloning

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Affinity purification/chromatography requires ______

highly purified ligand

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Expression cloning doesn’t require ___

highly purified ligand

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Affinity purification/chromatography steps

1) attach ligand to inert matrix
2) incubate with solubilized membranes (with receptors and other proteins)
3) wash away unbound proteins
4) elute bound proteins with excess ligand
5) identify all proteins in fractions (receptor will be in fraction with ligand)

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expression cloning is used for receptors that can’t be _____ or ______

isolated by chromatography
are not abundant enough for direct characterization

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expression cloning steps

1) isolate mRNA from cells that express the receptor
2) make cDNA from mRNA
3)insert cDNA into expression plasmid
4) transfect plasmid into cells that don’t express the receptor
4) select cells that have plasmid (ex. antibiotic)
5) identify cells with receptor on cell surface with fluorescent ligand
6) sequence cDNA insert to identify receptor

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3 classes of receptors

1) ion-channel coupled receptors 2) G-protein coupled receptors 3) enzyme-coupled receptors

GPCRs stands for ____

G protein-coupled receptors

GPCRs regulate ______

vision, taste, smell, immune system, nervous system, metastasis

Components of GPCR pathway

1) A trans-membrane receptor 2) A hetero-trimeric G-protein 3) An effector regulated by the “activated” G-protein 4) Production of second messengers (cAMP, cGMP, DAG) 5) Second messengers act on downstream target(s) to yield response

G proteins are ____ with subunits named ____

-Heterotrimeric GTP binding proteins a,b, and g

a-subunit of G proteins does what?

binds and hydrolyzes GTP

___ subunits of G protein are ____ allowing for membrane association

a & g lipidated

a-subunit of G-protein bound to GTP is ___

a-subunit of G-protein bound to GDP is ___

off

when a-subunit of G protein is active/GTP-bound , ____

it dissociates from B and g subunits

There are several ______ of recognizing different receptors and effectors

a, b, and g subunits

GPCRs steps

1) Binding of hormone= conformational change in receptor 2)activated receptor binds to Ga subunit of inactive/GDP trimeric G protein 3) receptor causes conformational change in Ga = dissociation of GDP 4) GTP binds to Ga = dissociation of Ga from receptor and Gbg 5) Hormone dissociates from receptor from receptor, Ga-GTP binds to effector =activation 6) Hydrolysis of GTP = Ga dissociate from effector and re-associate with Gbg

FRET stands for ____

Fluorescence (or Förster) resonance energy transfer

_______ can be used to study interaction between proteins in real time

Loss or gain of FRET

In order to have FRET, you need two things?

1) Spectrum of Em (donor)-emission overlaps with Ex -excitation (acceptor) b) The two molecules need to be closed in distance

Using FRET to use to study GPCR pathway

1) Measure extent of fluorescence resonance energy transfer (FRET) before and after addition of ligand 2) Loss of FRET signal occurs very soon after ligand addition

If ligands aren't close together, what happens to fret?

you lose FRET

If ligands are close together, what happens to fret?

you gain FRET

RTKs stands for ___

Receptor Tyrosine Kinases

EGF stands for ___

Epidermal growth factor

EGFR stands for ___

Epidermal growth factor receptor

EGFR is what type of receptor?

RTKs

EGFR binds ___ and then activate _____

growth factors cellular proliferation pathways

EGFR is important in ___

human cancer

EGFR/Her2 phenotype in cancer cells

overproduction of EGFR or always active EGFR

RTK and cancer therapy

anti-RTK therapies

RTK cytoplasmic domains have what type of activity?

intrinsic kinase activity

RTKs all have this domain?

tyrosine kinase domain

in the absence of ligand, RTK has ____

low kinase activity

ligand binding on RTK leads to ____

trans-auto phosphorylation of receptor cytoplasmic domain

the trans-auto phosphorylation generates ____

binding sites for signaling proteins which rely signal downstream

activated RTK steps

1) adaptor protein binds to activated RTK 2) Ras-GEF protein binds to adaptor protein 3) GEF activates Ras protein 4) Downstream signals

____ domains on adaptor protein binds to ____ on RTK cytoplasmic domain

SH2/PTB phosphotyrosine residues

____ domains on adaptor proteins binds to ______ on Ras-GEF proteins

SH3 proline rich sequences (PXXP)

GEF protein turns Ras protein from __ to __ -bound

GDP GTP

example of adaptor protein

Grb2

example of Ras-GEF

Sos

_____ (active) signals downstream through additional effectors

GTP-Ras

Ras can signal through ____

MAP kinase pathways

Ras protein is associated with ___

active Ras activates ___ which uses ___ to activate ____ which uses ___ to activate ___

MAP kinase kinase kinase ATP MAP kinase kinase ATP MAP kinase

MAP KKK (ex.)

Raf

MAP KK (ex.)

Mek

MAP K (ex.)

Erk

Raf (MAP KKK) is recruited to membrane by ___

activated Ras

Hydrolysis of GTP by Ras releases ____ which phosphorylates and activates _____

active Raf (MAP KKK) MEK (MAP KK)

MEK (MAP KK) phosphorylates and activates _____

MAP K

active MAP K does what?

translocates to nucleus and phosphorylates transcription factors -> changing gene expression

Evidence that Ras functions downstream of RTKs:

1) Microinjection of neutralizing antibodies to Ras blocks proliferation of cells stimulated with growth factors (EGF) 2) Expression of constitutively active Ras mutant causes cells to proliferate in absence of growth factors

anti-RTK monoclonal antibodies can do what?

block ligand binding or dimerization

Drosophila eye is composed of many ____ Each _____ is composed of _____ The _____ gene encodes an ___ that is required for _____

ommatidia ommatidium 8 tubular photoreceptors (R1-8) sevenless RTK development of the R7 receptor

wildtype drosophila eye

1) R8 cell makes surface ligand (Boss) 2) R7 receptor/RTK (Sev) binds Boss 3) R7 precursor with RTK becomes R7 neuron

mutant with no Sev drosophila eye

1) R8 cell makes surface ligand (Boss) 2) no signal transduction in R7 cell 3) R7 precursor without RTK becomes cone cell

mutant with no Sev but always active Ras drosophila eye

1) R8 cell makes surface ligand (Boss) 2) always active Ras -> signal transduction 3) R7 precursor without RTK becomes R7 neuron

Crosstalk between signaling pathways allows cells to _____

differentially transmit signals from cell surface

Lecture 21: Cell Signaling Flashcards

(79 cards)