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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
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

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