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Cellular and Molecular Biology > Final: Ch 15 Signal Transduction & Assays > Flashcards

Final: Ch 15 Signal Transduction & Assays Flashcards

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1
Q

pheremones (single-celled organisms)

A

secreted molecules that coordinate the grouping of cells for sexual mating or differentiation

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2
Q

in cells, a signal produces a specific response only in target cells with ______ for the signal

A

receptors

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3
Q

can the signal be a physical stimulus like light, touch, or heat OR a chemical molecule like gases, peptides, and proteins?

A

yes

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4
Q

how can extracellular signaling molecules that are too large and hydrophilic to penetrate the membrane affect intracellular processes?

A

bind cell-surface receptors (integral membrane proteins)

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5
Q

3 domains of cell-surface receptors

A

extracellular

transmembrane

intracellular

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6
Q

what happens when a ligand binds the cell-surface receptor

A

binding of ligand induces a conformational change in the receptor

transmitted through transmembrane domain to intracellular domain resulting in cytosolic activation or inhibition of proteins

proteins/second messengers carry signal to effector proteins

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7
Q

signal transduction

A

process of converting extracellular signals into intracellular responses

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8
Q

what is the most numerous class of receptor

A

G-protein coupled receptor (GPCR)

~900

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9
Q

structure of a GPCR

A

integral membrane receptor coupled to an intracellular G protein that transmits signals into the cell

subset of G-protein (7 transmembrane passes)

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10
Q

signal transduction through GPCRs usually induces short-term changes in ____ _____, such as a change in metabolism or movement

A

cell function

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11
Q

activation of cell-surface receptors that are not GPCR usually results in

A

alteration of a cell’s pattern of gene expression

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12
Q

2 cellular responses that occur when signaling molecules bind to their receptors

A

change in the activity or function of specific enzymes

change in the amount of specific proteins produced by a cell b/c of modification by transcription factors

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13
Q

what do transcription factors do

A

stimulate or repress gene expression in the nucleus

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14
Q

why is it called a signal transduction pathway

A

several intermediates convert the signal’s info from receptor to target

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15
Q

can signaling molecules act locally or at a distance?

A

yes

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16
Q

3 types of signaling from extracellular molecules

A

endocrine

paracrine

autocrine

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17
Q

endocrine signaling

A

signaling molecules synthesized and secreted by signaling cells

transported through circulatory system

act on distant targets

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18
Q

hormone is what type of signaling?

A

endocrine

ex. insulin (pancreas), epinephrine (adrenal glands)

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19
Q

paracrine signaling

A

the signaling molecules released only affect nearby cells

ex. nerve released nt, muscle cell

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20
Q

many protein ______ factors regulating development in multicellular organisms act at short range

A

growth factors

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21
Q

autocrine signaling

A

cells respond to substances that they themselves released

ex. tumor growth factors, cultured cells

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22
Q

binding specificity of a receptor refers to

A

its ability to bind or not bind closely related substrates

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23
Q

ligand binding depends on

A

weak noncovalent forces (van der waals, ionic, hydrophobic interactions)

molecular complementarity

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24
Q

examples of molecular complementarity

A

growth hormone

ACh

insulin (IGF-1 and IGF-2)

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25
Q

organisms have evolved to use a single ligand to…

A

stimulate different responses in cells

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26
Q

2 ways a single ligand can affect cells differently (effector specificity)

A

different cell types may have different receptors for the same ligand

the same receptor is found on multiple cell types, but binding of ligand induces a different response due to the types of proteins within the cell

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27
Q

2 examples of effector specificity

A

ACh released onto muscle induces contraction

ACh released in heart muscle slows the heart rate

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28
Q

what 2 enzymes are used in all signaling pathways?

A

protein kinases

phosphatases

act as switches (on/off)

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29
Q

what does a kinase do

A

covalently adds a phosphate

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30
Q

what does a phosphatase do

A

removes a phosphate

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31
Q

can a receptor posses intrinsic kinase activity?

A

yes, or it is rightly bound to a cytosolic kinase

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32
Q

if ligand is unbound, is the kinase active or inactive?

A

inactive

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33
Q

what does binding of ligand do to the kinase

A

changes conformation of the receptor, which activates the kinase

the kinase phosphorylates the monomeric inactive form of a TF

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34
Q

what happens when the monomeric inactive form of a TF is phosphorylated by the kinase

A

TF dimerizes and moves from cytosol to nucleus

TF activates gene transcription

35
Q

what would a phosphatase in the nucleus do

A

dephosphorylate the TF –> 2 inactive monomers that move back into cytosol

36
Q

do intracellular switch proteins turn upstream or downstream proteins on or off?

A

downstream

37
Q

most important group of intracellular switch proteins

A

GTPase superfamily

bound GTP = on

bound GDP = off

38
Q

conversion of inactive GTPase to active is triggered by…

A

a signal (hormone binding receptor)

mediated by guanine nucleotide exchange factor (GEF)

39
Q

GEF causes…

A

release of GDP from the switch protein and replacement with GTP

40
Q

GTPase-activating protein (GAP) and regulator of G protein signaling (RGS)

A

accelerate GTP hydrolysis –> GDP

active –> inactive

41
Q

many regulators of G protein activity are controlled by …

A

extracellular signals

42
Q

trimeric (large) G proteins

A

directly bind to and activated by cell-surface receptors

43
Q

monomeric (small) G proteins

A

not bound to receptors

play roles in pathways of cell division/apoptosis

ex. Ras

44
Q

what are second messengers

A

low MW signaling molecules that transmit and amplify signals from many receptors (bind to other proteins)

ex. Ca2+ released from ER stores or imported through channels

45
Q

how to detect 2nd messenger Ca2+ changes

A

fluorescent dyes

ex. in muscle, rise in Ca = contraction

46
Q

cAMP

A

2nd messenger that triggers activation of protein kinase A or opens/closes ion channels

47
Q

protein kinase A (PKA)

A

phosphorylates target proteins to induce changes in cell metabolism / ion channels

48
Q

how can GPC hormone receptors induce signal amplification?

A

a single receptor can activate MULTIPLE G proteins –> activate effector proteins

49
Q

a single epinepherine-GPCR complex can activate up to 100 _____ cyclase molecules

A

adenylyl cyclase molecules –> catalyze synthesis of many cAMP molecules

2 cAMP –> activate 1 PKA –> phosphorylate multiple target molecules

50
Q

2 functions of G proteins

A

signal transduction

signal amplification

51
Q

______ activate G proteins

A

hormones

52
Q

40% of medicines act…

A

biochemically by pituitary gland (GPCR pathway)

53
Q

3 subunits of a trimeric G protein

A

alpha binds GTP

beta/gamma binds GDP

activation = release from receptor & beta/gamma subunits

54
Q

adenylyl cyclase (enzyme)

A

catalyzes rxn of cycling ATP to cAMP (2nd messenger)

55
Q

2 effects of 2nd messengers

A

amplification

integration - combining multiple signals (inhibitory or excitatory)

56
Q

cAMP can bind to …

A

protein kinase C

57
Q

protein kinase C (PKC)

A

releases catalytic subunits when activated

allosteric effect in binding cAMP

58
Q

catalytic subunits

A

activate glycogen degrading enzymes to produce glucose

59
Q

what is the dissociation constant a measure of

A

the affinity for a receptor for its ligand

measure of bound and unbound receptor

units = molarity

60
Q

Kd =

A

[R] [L] / [RL]

concentration of ligand where 1/2 receptor is bound

61
Q

the lower the Kd…

A

the lower the [L] required to bind 1/2 receptors

62
Q

does a lower Kd = tighter binding?

A

yes

63
Q

for a cellular response, usually less than ___ of the receptors need to be activated

A

1/2

~10^-10 M

64
Q

what is TNF alpha (tumor necrosis factor alpha)

A

hormone secreted by immune cells to recruit more immune cells to site of inflammation

abnormal levels cause inflammation in autoimmune disease

65
Q

treat excess TNF alpha disease with chimeric fusion protein

A

contains extracellular domain of TNF alpha receptor

fused to constant (Fc) region of human immunoglobin

binds tightly to TNF alpha to prevent it from binding cell-surface receptors

66
Q

how to detect receptors and determine their affinity and specificity for ligand?

A

binding assay

see how much fluorescent or radioactive ligand is attached to receptors

67
Q

what does a competition assay measure

A

measure weak ligand binding by comparing to a ligand that binds the same receptor with high affinity

68
Q

how to perform a competition assay

A

add increasing amounts of unlabeled low-affinity ligand to a cell sample with a constant amount of labeled high-affinity ligand

calculate inhibitory constant (close to Kd)

69
Q

what is competitive binding used to study usually

A

synthetic analogs of natural hormones

70
Q

agonist

A

mimic function of natural hormone by binding receptor and inducing normal response

71
Q

antagonist

A

binds to receptor but doesn’t induce a response

can block binding of natural hormone or agonist

72
Q

beta-blocker

A

antagonist of B-adrenergic receptors in the heart (increase heart rate)

used to slow heart contractions

73
Q

how is the sensitivity of a cell to signals determined?

A

by the number of surface receptors the cell has for that ligand

also by how high the affinity the receptors have for the ligand

74
Q

desensitization

A

reduction in a cell’s sensitivity to a ligand

75
Q

regulation of receptors

A

change affinity for ligand

change # of receptors present

76
Q

________ of cell-surface receptors can lower the cellular response

A

endocytosis

77
Q

how would you generate a cell with a large # of cell-surface receptor you want to purify?

A

recombinant DNA techniques

78
Q

immunoprecipitation of kinases

A

Ab specific for kinase is reacted with small beads coated with protein A to bind via Fc region

beads mixed with cytosol or nucleus and washed with salt

only kinase and associated proteins are bound to Ab beads

79
Q

how to determine kinase function unsing immunoprecipitation

A

add radiolabeled 32P ATP to mix, and see how quickly the phosphate is transferred to the product by the kinase

80
Q

what use do monoclonal Ab have?

A

use one with selectivity for only phosphorlyated peptide to see if the kinase works with a certain signal

81
Q

what is a pull-down assay used for

A

quantify the activation of a GTP-binding protein (western blotting)

82
Q

what does a pull-down assay use

A

prepare beads with the binding domain of the target protein (of the GTP switch)

add to cell extract to pull-down (capture) GTP-bound form

centrifugation

western blotting with Ab

83
Q

how to purify receptors that still bind ligand

A

like affinity chromatography but ligand is attached to beads instead of Ab

Cellular and Molecular Biology (34 decks)

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