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z:Cell 300 > Lecture 21: Cell Signaling > Flashcards

Lecture 21: Cell Signaling Flashcards

1
Q

examples of cellular signals

A

growth factors, cytokines, hormones

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2
Q

Signals are received by ______ where?

A

receptors
at the cell surface (or inside cell)

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3
Q

signals are transduced to where? and do what?

A

to the nucleus
they change the activity of transcription factors

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4
Q

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

A

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

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5
Q

Some receptors can transduce signals through _____

A

more than one pathway

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6
Q

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

A

more than one type of signal

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7
Q

There can be ____ between signaling pathways

A

crosstalk

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8
Q

signal transduction (def.)

A

process of converting signal into cellular response

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9
Q

Steps in signal transduction

A
  • 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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10
Q

examples of effector proteins + effect

A

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

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11
Q

Four types of extracellular signaling

A

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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12
Q

contact dependent signaling example

A

Apoptosis (FasL/Fas)

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13
Q

Paracrine signaling example

A

Interferon response

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14
Q

Endocrine signaling example

A

Insulin

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15
Q

Cellular responses to signals can be _____

A

very fast or slow

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16
Q

______copies of a specific cell surface receptor/cell

A

1,000-50,000

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17
Q

Receptors comprise _____ of plasma membrane proteins

A

< 0.01%

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18
Q

Most receptors are_____ that require _____for extraction from cellular material

A

transmembrane proteins
detergents

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19
Q

Two ways to isolate and identify receptors

A

1) Affinity purification/chromatography
2) Expression cloning

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20
Q

Affinity purification/chromatography requires ______

A

highly purified ligand

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21
Q

Expression cloning doesn’t require ___

A

highly purified ligand

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22
Q

Affinity purification/chromatography steps

A

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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23
Q

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

A

isolated by chromatography
are not abundant enough for direct characterization

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24
Q

expression cloning steps

A

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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25
Q

3 classes of receptors

A

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

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26
Q

GPCRs stands for ____

A

G protein-coupled receptors

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27
Q

GPCRs regulate ______

A

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

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28
Q

Components of GPCR pathway

A

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

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29
Q

G proteins are ____ with subunits named ____

A

-Heterotrimeric GTP binding proteins
a,b, and g

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30
Q

a-subunit of G proteins does what?

A

binds and hydrolyzes GTP

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31
Q

___ subunits of G protein are ____ allowing for membrane association

A

a & g
lipidated

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32
Q

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

A

on

33
Q

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

A

off

34
Q

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

A

it dissociates from B and g subunits

35
Q

There are several ______ of recognizing different receptors and effectors

A

a, b, and g subunits

36
Q

GPCRs steps

A

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

37
Q

FRET stands for ____

A

Fluorescence (or Förster) resonance energy transfer

38
Q

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

A

Loss or gain of FRET

39
Q

In order to have FRET, you need two things?

A

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

40
Q

Using FRET to use to study GPCR pathway

A

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

41
Q

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

A

you lose FRET

42
Q

If ligands are close together, what happens to fret?

A

you gain FRET

43
Q

RTKs stands for ___

A

Receptor Tyrosine Kinases

44
Q

EGF stands for ___

A

Epidermal growth factor

45
Q

EGFR stands for ___

A

Epidermal growth factor receptor

46
Q

EGFR is what type of receptor?

A

RTKs

47
Q

EGFR binds ___ and then activate _____

A

growth factors
cellular proliferation pathways

48
Q

EGFR is important in ___

A

human cancer

49
Q

EGFR/Her2 phenotype in cancer cells

A

overproduction of EGFR or always active EGFR

50
Q

RTK and cancer therapy

A

anti-RTK therapies

51
Q

RTK cytoplasmic domains have what type of activity?

A

intrinsic kinase activity

52
Q

RTKs all have this domain?

A

tyrosine kinase domain

53
Q

in the absence of ligand, RTK has ____

A

low kinase activity

54
Q

ligand binding on RTK leads to ____

A

trans-auto phosphorylation of receptor cytoplasmic domain

55
Q

the trans-auto phosphorylation generates ____

A

binding sites for signaling proteins which rely signal downstream

56
Q

activated RTK steps

A

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

57
Q

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

A

SH2/PTB
phosphotyrosine residues

58
Q

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

A

SH3
proline rich sequences (PXXP)

59
Q

GEF protein turns Ras protein from __ to __ -bound

A

GDP
GTP

60
Q

example of adaptor protein

A

Grb2

61
Q

example of Ras-GEF

A

Sos

62
Q

_____ (active) signals downstream through additional effectors

A

GTP-Ras

63
Q

Ras can signal through ____

A

MAP kinase pathways

64
Q

Ras protein is associated with ___

A

PM

65
Q

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

A

MAP kinase kinase kinase
ATP
MAP kinase kinase
ATP
MAP kinase

66
Q

MAP KKK (ex.)

A

Raf

67
Q

MAP KK (ex.)

A

Mek

68
Q

MAP K (ex.)

A

Erk

69
Q

Raf (MAP KKK) is recruited to membrane by ___

A

activated Ras

70
Q

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

A

active Raf (MAP KKK)
MEK (MAP KK)

71
Q

MEK (MAP KK) phosphorylates and activates _____

A

MAP K

72
Q

active MAP K does what?

A

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

73
Q

Evidence that Ras functions downstream of RTKs:

A

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

74
Q

anti-RTK monoclonal antibodies can do what?

A

block ligand binding or dimerization

75
Q

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

A

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

76
Q

wildtype drosophila eye

A

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

77
Q

mutant with no Sev drosophila eye

A

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

78
Q

mutant with no Sev but always active Ras drosophila eye

A

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

79
Q

Crosstalk between signaling pathways allows cells to _____

A

differentially transmit signals from cell surface

z:Cell 300 (17 decks)

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