Final: Ch 15 Signal Transduction & Assays Flashcards Preview

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

Flashcards in Final: Ch 15 Signal Transduction & Assays Deck (83):
1

pheremones (single-celled organisms)

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

2

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

receptors

3

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

yes

4

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

bind cell-surface receptors (integral membrane proteins)

5

3 domains of cell-surface receptors

extracellular

transmembrane

intracellular

6

what happens when a ligand binds the cell-surface receptor

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

7

signal transduction

process of converting extracellular signals into intracellular responses

8

what is the most numerous class of receptor

G-protein coupled receptor (GPCR)

~900

9

structure of a GPCR

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

subset of G-protein (7 transmembrane passes)

10

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

cell function

11

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

alteration of a cell's pattern of gene expression

12

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

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

13

what do transcription factors do

stimulate or repress gene expression in the nucleus

14

why is it called a signal transduction pathway

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

15

can signaling molecules act locally or at a distance?

yes

16

3 types of signaling from extracellular molecules

endocrine

paracrine

autocrine

17

endocrine signaling

signaling molecules synthesized and secreted by signaling cells

transported through circulatory system

act on distant targets

18

hormone is what type of signaling?

endocrine

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

19

paracrine signaling

the signaling molecules released only affect nearby cells

ex. nerve released nt, muscle cell

20

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

growth factors

21

autocrine signaling

cells respond to substances that they themselves released

ex. tumor growth factors, cultured cells

22

binding specificity of a receptor refers to

its ability to bind or not bind closely related substrates

23

ligand binding depends on

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

molecular complementarity

24

examples of molecular complementarity

growth hormone

ACh

insulin (IGF-1 and IGF-2)

25

organisms have evolved to use a single ligand to...

stimulate different responses in cells

26

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

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

27

2 examples of effector specificity

ACh released onto muscle induces contraction

ACh released in heart muscle slows the heart rate

28

what 2 enzymes are used in all signaling pathways?

protein kinases

phosphatases

act as switches (on/off)

29

what does a kinase do

covalently adds a phosphate

30

what does a phosphatase do

removes a phosphate

31

can a receptor posses intrinsic kinase activity?

yes, or it is rightly bound to a cytosolic kinase

32

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

inactive

33

what does binding of ligand do to the kinase

changes conformation of the receptor, which activates the kinase

the kinase phosphorylates the monomeric inactive form of a TF

34

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

TF dimerizes and moves from cytosol to nucleus

TF activates gene transcription

35

what would a phosphatase in the nucleus do

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

36

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

downstream

37

most important group of intracellular switch proteins

GTPase superfamily

bound GTP = on

bound GDP = off

38

conversion of inactive GTPase to active is triggered by...

a signal (hormone binding receptor)

mediated by guanine nucleotide exchange factor (GEF)

39

GEF causes...

release of GDP from the switch protein and replacement with GTP

40

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

accelerate GTP hydrolysis --> GDP

active --> inactive

41

many regulators of G protein activity are controlled by ...

extracellular signals

42

trimeric (large) G proteins

directly bind to and activated by cell-surface receptors

43

monomeric (small) G proteins

not bound to receptors

play roles in pathways of cell division/apoptosis

ex. Ras

44

what are second messengers

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

how to detect 2nd messenger Ca2+ changes

fluorescent dyes

ex. in muscle, rise in Ca = contraction

46

cAMP

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

47

protein kinase A (PKA)

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

48

how can GPC hormone receptors induce signal amplification?

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

49

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

adenylyl cyclase molecules --> catalyze synthesis of many cAMP molecules

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

50

2 functions of G proteins

signal transduction

signal amplification

51

______ activate G proteins

hormones

52

40% of medicines act...

biochemically by pituitary gland (GPCR pathway)

53

3 subunits of a trimeric G protein

alpha binds GTP

beta/gamma binds GDP

activation = release from receptor & beta/gamma subunits

54

adenylyl cyclase (enzyme)

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

55

2 effects of 2nd messengers

amplification

integration - combining multiple signals (inhibitory or excitatory)

56

cAMP can bind to ...

protein kinase C

57

protein kinase C (PKC)

releases catalytic subunits when activated

allosteric effect in binding cAMP

58

catalytic subunits

activate glycogen degrading enzymes to produce glucose

59

what is the dissociation constant a measure of

the affinity for a receptor for its ligand

measure of bound and unbound receptor

units = molarity

60

Kd =

[R] [L] / [RL]

concentration of ligand where 1/2 receptor is bound

61

the lower the Kd...

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

62

does a lower Kd = tighter binding?

yes

63

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

1/2

~10^-10 M

64

what is TNF alpha (tumor necrosis factor alpha)

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

abnormal levels cause inflammation in autoimmune disease

65

treat excess TNF alpha disease with chimeric fusion protein

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

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

binding assay

see how much fluorescent or radioactive ligand is attached to receptors

67

what does a competition assay measure

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

68

how to perform a competition assay

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

what is competitive binding used to study usually

synthetic analogs of natural hormones

70

agonist

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

71

antagonist

binds to receptor but doesn't induce a response

can block binding of natural hormone or agonist

72

beta-blocker

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

used to slow heart contractions

73

how is the sensitivity of a cell to signals determined?

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

desensitization

reduction in a cell's sensitivity to a ligand

75

regulation of receptors

change affinity for ligand

change # of receptors present

76

________ of cell-surface receptors can lower the cellular response

endocytosis

77

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

recombinant DNA techniques

78

immunoprecipitation of kinases

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

how to determine kinase function unsing immunoprecipitation

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

80

what use do monoclonal Ab have?

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

81

what is a pull-down assay used for

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

82

what does a pull-down assay use

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

how to purify receptors that still bind ligand

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