Cell and Molec exam 4 Flashcards
1
Q
Why do cells need to have signaling molecules?
A
Help mediate cell proliferation and survival, cell response to nutrients, and organization and cooperation of cells and tissues
2
Q
5 types of extracellular signals
A
amines
peptides and proteins
steroids
fatty acids (eicosanoids)
gases
3
Q
Many signaling molecules are ___
A
small peptides or proteins
4
Q
They then are trafficked through the _____ and then secreted
A
ER and golgi,
4
Q
These signaling molecules are encoded by genes, transcribed, and translated into _____
A
peptides or proteins
5
Q
Function of elicosanoids
A
regulating inflammation, vasodilation, smooth muscle contraction, etc.
6
Q
Steroids bind ___ hormone receptors, which can activate expression of various genes.
A
nuclear hormones
7
Q
Gases can act like signalling molecules
A
Have no effect on neurotransmission but can act as a vasodilator
8
Q
Juxtacrine signals are mediated by interactions between
A
two transmembrane proteins.
The ligand is something that never leaves from the cell that produces it.
9
Q
Paracrine cells
A
signal acts on neighbors
10
Q
Autocrine signals
A
Are self stimulatory
11
Q
Endocrine signals are released at a distance and travel to target tissues
A
released from glands
can be released from cells that are not in glands
travel to target cells via circulatory systems
12
Q
Cell surface receptors
A
G protein-linked receptors
Receptors that contain enzymes, or that directly recruit enzymes
receptors that are ligand-gated ion channels
13
Q
Different receptors can result in different responses to the ____ ligand
A
same
- can do this because of different receptors, different downstream signaling proteins, or different concentrations or duration of molecules
14
Q
What influences receptor-ligand interactions
A
specificity
affinity
saturation
physiological response
15
Q
Binding assays
A
reveals receptor number and affinity
16
Q
Agonist
A
exerts its effects by direct alteration of the functional properties of the receptor with which it interacts (binds receptor and mimics effects of hormone).
17
Q
Antagonist
A
Agent that exerts its effects by inhibiting the action of a specific agonist (often, though not always, acts by competing with agonist for binding site).
18
Q
ways signal transduction can happen
A
Allostery
covalent modification
induced proximity
19
Q
Cross talk
A
Signals from different receptors are intergrated
20
Q
Hormone
A
chemicals that coordinate different functions in your body by carrying messages through your blood to your organs, skin, muscles and other tissues. Are signaling molecules.
21
Q
neurotransmitter
A
A neurotransmitter is a signaling molecule secreted by a neuron to affect another cell across a synapse. The cell receiving the signal, or target cell, may be another neuron, but could also be a gland or muscle cell.
22
Q
Neurohormone
A
a hormone (such as vasopressin or norepinephrine) produced by nerve cells and secreted into the circulation.
23
Q
Ligand
A
A molecule that binds a signal
24
receptor
receptors are chemical structures, composed of protein, that receive and transduce signals that may be integrated into biological systems.
25
Second messenger
are small molecules and ions that relay signals received by cell-surface receptors to effector proteins. They are normally small molecules
26
Allostery
Changing the shape and the ability of proteins to act with other substrates. How GTPases work
27
Effector
A small molecule that selectively binds to a protein to regulate its biological activity can be called an effector.
28
How generally does an extracellular signaling molecule trigger changes in gene expression?
Mostly steroid receptors. Requires the signal to get into the cell either through a transporter or through diffusion of the membrane
29
How do scaffold proteins lead to increased efficiency of signal transduction?
Reduces the amount of time for diffusion and concentrating proteins with second messengers
30
How is an initial signal (e.g. from epinephrine) amplified?
- The receptor activates Adenylyl Cyclase which produces cAMP (the second messenger)
- this then activates many different kinases which can produce many different products
- Have a huge application with a small amount of signal
31
How can the same ligand (e.g. acetylcholine) produce different responses?
- It can bind to different receptors
- different concentration gradients
- Different pathways (different downstream signaling proteins called effectors)
32
What does the dissociation constant in receptor-ligand interactions refer to?
- How we characterize how tightly the ligand binds
- Ligands can come on and off the receptors
- Saturation is when the receptor has a limited number of binding sites. If there is no saturation then it is a non-specific interaction site
- concentration at which half of the sites are filled. With a low affinity, it will be hard to fill up the sites.
- baseball analogy
33
A higher dissociation constant = a _____ affinity between receptor and ligand.
lower
34
Nuclear receptors are ...... transcription factors
ligand-activated
35
Nuclear receptors share a ... domain structure
common
36
Type I nuclear receptor
Nuclear receptor (NR) in inactive complex in the cytoplasm
Hormone binding leads to release of chaperone
Nuclear receptor dimerizes
Nuclear receptor enters nucleus and binds DNA response element (RE)
37
Type II nuclear receptor
Nuclear receptor already “docked” on DNA
Hormone binding leads to release of co-repressors and recruitment of co-activators
38
Many steroid receptors bind ___
steroids
39
Xenobiotic
a chemical substance found within an organism that is not naturally produced by that organism
- are stable in the environment
- toxic
- modified by gut microbes and by human cells
40
Why do we have receptors
Similar to chemicals produced by fungi, bacteria and plants that we might need to detect and detoxify
This diagram shows PXR binding to zenobiotics; the aryl hydrocarbon receptor (AHR) also commonly binds them.
41
Orphan receptors
- Have similar protein structures to other nuclear receptors.
- Ligands are not known.
- Some might be constitutively
active (no ligand needed).
42
The receptors are inactive in the cytoplasm.
Ligand-binding changes shape of receptor and...
recruits transcriptional activators
43
Nuclear receptors bind to specific DNA sequences called
response elements
- Helices of PPAR-gamma and RXR-alpha contact the major and minor grooves of the DNA helix.
44
Many steroid receptors binds DNA using a
zinc finger
45
Ligand-bound nuclear receptors can recruit
coactivators
-Coactivators like histone acetyl transferases (HAT) are recruited.
- Nucleosomes repositioned/chromatin becomes more accessible
-Other transcription factors also join in (as do other surprising proteins).
-RNA polymerase recruited
46
Nuclear receptors can also repress gene expression
-Block access by binding to DNA and preventing something else binding
-Competition for limiting amounts of co-activators
-Recruiting co-repressors like histone de-acetylases
-Recruiting nucleosome remodeling complexes like SWI/SNF
47
How to run an experiment to see if your transcription factor bonded to sequence you identified
Do CHIP and then sequencing/ pcr
48
nuclear receptors
They are ligand-activated transcription factors. They bind ligand and activate gene expression. Required for steroid hormones. And respond to lot of different chemicals
49
cytochrome p450s
Metabolic enzymes which add OH groups to soluble compounds
response element (RE)
50
zinc finger
Used by many steroid receptors to bind DNA. Is on top of the response element and has an affinity for specific nucleotides- the spacing and the orientation are different
51
Give an example xenobiotic. Why do we have receptors for these?
- Are things that are found in the environment but are not naturally produced by us. Can be made by other things. Common sources are when things are burned, herbicides, plastics
- Lots of microbes produce similar compounds that can be detected by us
- If we eat these things they can be detected by us and detoxify them.
- bpa is an example
52
Xenobiotic receptors often lead to activation of cytochrome P450s, like CYP3A4, enzymes that hydroxylate small molecules. Name a possible beneficial effect of hydroxylation and a potential deleterious effect.
- Help detoxify them from us (benefit) We want to add hydroxyl groups to these compounds so that they become more soluble and that we can pump them out of the cell
- Adding hydroxyl groups makes them more active and sometimes makes them activate DNA etc.
53
Name 2 reasons a receptor might be classified as an orphan receptor.
1. We don’t know their ligand
2. Some might not need ligands – might be constitutively active
54
Name 2 important characteristics of response elements that help to confer specificity of receptor binding.
1. orientation
2. spacing
55
You make a chimeric receptor with the ligand binding domain of GR and the DNA binding domain of ER. This receptor will bind _____ and activate ______responsive genes (glucocorticoid, estrogen).
glucocorticoid and activate estrogen
56
Name 1 way nuclear receptors like GR can activate gene expression
- Coactivators like histone acetyl transferase (HAT) are recruited
- Nucleosomes repositioned/ chromatin becomes more accessible
- Other transcription factors also join in (as do other surprising proteins)
- RNA polymerase is recruited
- GR recruits the complex which leads to the activation of genes
57
Name 1 way nuclear receptors like RXR can repress gene expression.
- Block access by binding to DNA and preventing something else from binding
- Recruiting co-repressors like histone deacetylases
- can recruit nucleosome remodeling complexes
- Depends on the cellular context and what receptors are activated whether they activate or inactivate gene expression
58
You are studying the promoter and enhancer regions of a gene. By sequence analysis, you think the region contains three EREs (estrogen response elements). Suggest a possible approach for determining if estrogen regulates the expression of this gene. Suggest a possible approach for determining which (if any) of these three EREs are required for the response to estrogen.
- You can measure gene expression by using ChIP. Can run a PCR to see what genes it is actually interacting with
- Could do a western blot for the estrogen receptor to see if you actually pulled out. Could also run a komassi stain to see if you have protein in your gel
59
Briefly describe how a ChIP experiment works.
A CHIP experiment works by having an immunoprecipitation specific antibody-protein interaction to isolate and quantify a specific protein of interest
- is used to identify the relative abundance of a specific protein or a specific protein modification at a certain region in the genome.
60
Basic structure of G proteins
Seven transmembrane α-helices
Three extracellular and three intracellular loops
An extracellular amino-terminal segment
The carboxy-terminal tail
61
Diversity of physiological responses to GPCR stimulation
Different extracellular signals can activate GPCRs.
This activates the signal transduction pathways, and in turn, causes alteration in cellular responses.
Hence, GPCR stimulation can cause a variety of physiological responses to occur in the body.
multiple GPCRs often recognize the same ligand
62
Receptor activation of heterotrimeric G-proteins.
Upon ligand binding to the receptor inactive G protein heterotrimers dissociate into separate, active Gα and Gβγ subunits.
63
Gαs
- activates adenylyl cyclase, which in turn stimulates cAMP production.
- Increase in cAMP in turn activates protein kinase A (PKA).
PKA is a serine/threonine kinase that phosphorylates many different substrates, including other kinases and transcription factors.
64
Gαi
inhibits adenylyl cyclase activity
65
Gαq
activates phospholipase C β (PLCβ), which produces IP3 and DAG from the hydrolysis of PIP2.
- which breaks down PIP2 into two different second messengers, IP3 and DAG
66
Gα12/13
activates Rho guanine nucleotide exchange factor (RhoGEF), which activates Rho GTPases.
67
Gβγ heterodimers
regulate K+, Ca2+ channels activity
recruit proteins to the plasma membrane as a scaffold, including PI3K, P-Rex-1, and β-adrenergic receptor kinase.
68
Regulation of systemic functions by signaling through G protein pathways
-Many extracellular agents, such as hormones, neurotransmitters, chemokines , and local mediators signal to the 4 main G protein families to regulate cellular functions such as metabolic enzymes, ion channels.
- Modulation of the activities of the cellular machines in turn gives rise to altered cellular functions such as changes in glucose metabolism in liver and muscle or altered activities of pacemaker cells in the heart.
-contribute to the regulation of large-scale systems such as organismal homeostasis and learning and memory.
69
Protein kinase A (PKA)
-Protein kinase A (PKA) regulates a diverse set of biological functions downstream of cyclic AMP (cAMP).
-It is a tetramer consisting of two catalytic subunits (PKA-C) and two regulatory subunits (PKA-R).
-The two PKA-C and two PKA-R subunits are bound together in the inactive state of the enzyme.
-When cAMP binds the PKA-R subunits, the PKA-C subunits are released and interact with downstream effectors.
- Kinases can also affect transcription, by phosphorylating transcription factors
70
The effects of kinases are terminated by _____
(remove phosphate from target proteins)
phosphatases
71
Different extracellular agents can activate the ___ receptor, regulate the ___ pathway to bring about changes in different effectors!!!
same
- glycogen and epinephrin
72
Why is the signaling mechanisms so complicated?
- signalling amplification
- timing
- cross talk
73
Crosstalk between GPCR and RTK
- The transactivation of GPCRs by RTK agonists can occur in a ligand-independent manner and requires the formation of GPCR–RTK complexes.
- RTK stimulation triggers GPCR transactivation through the formation of a molecular protein complex.
- This event is crucial for β-arrestin recruitment, which, in turns, promotes the internalization of the RTK/GPCR complex and intracellular signaling cascades.
74
How to turn off G protein signaling
- Second messenger is degraded by cyclic nucleotide phosphodiesterase
- G protein inactivates itself (GTP hydrolyzed to GDP by GTPases)
True of all G proteins, not just heterotrimeric Gs
-Substrates of protein kinase A are dephosphorylated by phosphatases
- Receptor is phosphorylated and internalized
75
Turning off GPCR signaling
Short-term desensitization
– occurs in minutes
- Requires G-protein coupled receptor kinase (GRK) and β-arrestin
Long-term desensitization
Occurs over hours to days
- Internalizes receptor for recycling or degradation by lysosomes
76
Turning off GPCR signaling – short-term
- GPCR phosphorylated by GRKs
- The phosphorylated GPCR recruits β-arrestin
- Binding of β-arrestin to GPCR prevents the dissociated G proteins from interacting with the arrestin-GPCR complex
- This inhibits further downstream sinaling
-Arrestin binding targets GPCR for clathrin coated pits in cell membrane for internalization
77
How are the following steps in GPCR signaling TERMINATED?
Receptor activation – GRK phosphorylation of GPCR, transactivation of GPCR by RTK, degradation by arrestin and clathrin coated pits
Activation of heterotrimeric G-protein – hydrolysis of GTP
Calcium release – activation of sarcoplasmic reticulum to take in Ca2+, inhibit PLC-beta, inhibit Gq
Cyclic AMP - phosphodiesterase
Phosphorylated proteins - phosphatases
78
Second Messenger
molecules that bring signals downstream of the signal cascade
heterotrimeric G protein
79
epinephrine (aka adrenaline)
small molecule that increases blood glucose level
80
Be familiar with the general mechanism by which GPCRs work:
ligand binding, association with heterotrimeric G protein, GEF activity activates G alpha, Galpha separates from Gbeta and Ggamma subunits, Galpha activates effector, Gbeta+Ggamma can also activate effectors, signal is amplified, various signaling cassettes (sets of proteins working in series) can be activated.
81
Give two examples (from this lecture) where phosphorylation activates the target protein, and two examples where phosphorylation inactivates the target protein (hint: turning off GPCR signaling).
a. Inactivation:
i. Substrates of protein kinases A are dephosphorylated by phosphatases
ii. A receptor is phosphorylated and internalized
b. Activation:
i. For gene expression, CREB is phosphorylated to stimulate gene transcription
ii. PKA phosphorylates other kinases to lead to different things being produced
82
Name two ways the original signal detected by the GPCR is amplified.
Can activate multiple G proteins
Can phosphorylate multiple cellar proteins
83
How are the following steps in GPCR signaling stopped:
i. Receptor activation – GRK phosphorylation of GPCR, transactivation of GPCR by RTK, degradation by arrestin and clathrin-coated pits
ii. Ca2+ release – inhibit PLC-beta and Gq, and activation of sarcoplasmic reticulum to take in CA+2
iii. PKA activity- phosphodiesterase
84
How growth factor signaling generally works
Ligand binding
Receptor dimerization
Receptor phosphorylation
Binding of adaptor proteins
Signaling cascade of kinases
Alteration of transcription factors or other effectors
85
Intracellular actions of receptor tyrosine kinases
Binding of growth factors to their receptors leads
to receptor dimerization and autophosphorylation
86
Receptor phosphorylation generates multiple binding sites
Can do this by
1. localizing protein that will pass the signal to downstream effectors
2. Localizing proteins near the plasma membrane (scaffolding)
87
Proteins containing ____ domains can bind to phospho-tyrosine
SH2
88
An SH2-domain-containing protein commonly activated by growth factor receptors is ____
Grb2
- Grb2 activates SOS
- SOS is an activator for Ras
- activated RAS begins a kinase chain
- Binds to phosphotyrosine and the signal can be traveled down into the cell
89
MAP kinase cascade
- Raf (kinase)
- MEK (kinase)
- ERK (kinase) -> ERK phosphorylates and activates transcription factors that
regulate, among other things, the initiation of cell division
90
Receptor tyrosine kinases can stimulate _____
other pathways – it is not just about activating MAPK!
91
Some receptors don’t contain their own tyrosine kinase, but instead _____
recruit and activate cytosolic tyrosine kinases, or other enzymes
92
Turing off RTK signaling
Usually by de-phosphorylation
Or in the case of Ras by hydrolysis of GTP
Inhibitory proteins: phosphatase, GAP, or proteins like 14-3-3 that
Bind and sequester phosphorylated proteins
93
Common methods for studying signaling pathways:
Phospho-specific antibodies to detect active states of signaling proteins (example: Active ERK)
Chemical inhibitors that block specific enzymes in the pathway (example: blocking the receptor)
Kinase assays (in vitro interaction of kinase and substrate)
Genetics
94
Basic concept of a kinase assay
substrate + ATP = ADP + substrate
- Substrate can be protein, peptide, lipid, or sugar depending on the kinase.
95
________are small molecules that block the action of kinases – often by competitively blocking the binding of ATP
Kinase inhibitors
96
Kinase assay
Treat cells with various concentrations of compound.
Purify kinase.
Add substrate.
Add radiolabeled ATP.
Measure labeled substrate.
- A kinase assay works by simply measuring the activity of the kinase.
97
autophosphorylation
Autophosphorylation is a type of post-translational modification of proteins. It is generally defined as the phosphorylation of the kinase by itself.
98
adaptor
An adaptor molecule is a double-stranded, chemically synthesized oligonucleotide that is used for the ligation of DNA. The adaptors have one sticky and one blunt end. The blunt end is ligated with the blunt end of the target DNA producing a DNA fragment with sticky ends.
99
phospho-specific antibody
detect only the phosphorylated forms of proteins in a complex protein mixture within cells.
100
How do adaptor proteins distinguish between phosphorylated tyrosines?
- different amino acids around the tyrosines
101
Why is it advantageous to have many of the proteins involved in RTK signaling localized to the plasma membrane?
- it gives multiple advantages to regulate them
- Localized proteins that will pass signal downstream effectors (activating signaling)
- Localizing proteins near the plasma membrane (scaffolding) GAP inhibit GTPases like Pho, Ras (these are peripheral membrane proteins)
102
Suggest an experiment to determine if your chosen growth factor activates (leads to phosphorylation of) ERK1,2.
1. phosphospecific antibodies w/ western blot. This lets us know if it is activated or not
103
Wnt
Wnt signaling regulates embryonic development, cell differentiation, proliferation, and adult stem cell homeostasis
- often dysregular in Cancer
104
Wnt signals ___
EMT
- During EMT, cells lose cell-cell junction connections and polarity
- In epithelial cells, beta-catenin is primarily in the adherens junctions, in mesenchymal cells it is primarily in the
Nucleus where it acts as a transcription factor and induces expression of genes
105
Planar cell polarity refers _____
to the coordinated alignment of a sheet of cells
-Components of the Wnt signaling pathway also regulate planar cell polarity, like these genes which regulate the alignment of “hairs” on the fly wing.
106
The Wnt/Ca++ pathway triggers the release of Ca++ by stimulating _______
phospholipase C
- The relative balance of these factors determines the cell’s response.
107
The Wnt/Ca++ pathway
- Ligand Wnt
- Receptor Fz
- GTPase Gprotein *probably Rho
CDC42
- Kinase PKC PKG CAMKII TAK1 NLK1
- Other enzymes PDE phosphodiesterase; PLC phospholipase
108
Basics of canonical Wnt signaling (no Wnt)
No Wnt situation:
Beta catenin is supposed to be in the adherens junctions, or very low level. Any cytoplasmic beta catenin is targeted for degradation by the destruction complex and degraded by the proteasome
109
Basics of canonical Wnt signaling (Yes Wnt)
Wnt present situation:
LRP is phosphorylated by the destruction complex. The destruction complex is localized to the receptor and inhibited.
Beta catenin levels build up, beta catenin goes in the nucleus, displaces a repressive complex and activates gene expression
110
Structure of the Wnt
- Wnts ‘pinch’ the Fz receptor using their thumb and index finger
- big for signalling molecules
111
Frizzled and LRP5/work together to activate _____
- Wnt signaling
-When these two combine, the destruction complex is inhibited allowing beta catenin levels to increase
112
When Wnt is absent
- When Wnt is absent, the destruction complex is active.
- Axin is the scaffold.
- GSK3 and CK1 phosphorylate beta catenin.
- Phosphorylated beta catenin is “marked for destruction” by ubiquitin ligases (E3)
- Beta catenin is then degraded.
113
biomolecular condensates
- Liquid-like goo-balls of proteins, lipids and RNAs
- Can flow, merge and separate
- Not membrane bound
- Proteins can join and leave
‘phase separate’, like oil in water
114
Functions of biological condensates
Enhancing activity by concentrating enzymes and substrates
Inhibiting activity by sequestration
Influencing protein folding
Facilitating specific subcellular localization
115
Intrinsically disordered regions
- seem important in the formation of lipid droplets
- Many proteins have disordered regions
- Some are induced to fold by binding partners
- Others remain (and function) as disordered regions, helping to cluster other proteins, lipids, RNAs, etc
116
Axin, APC, beta catenin, GSK intertwine in _____
condensates.
117
Beta catenin
is normally found in adherens junctions (Helps to connect cadherin to actin cytoskeleton through binding alpha catenin)
- uses the same binding site to interact with different proteins.
-Can bind axin, e-cadherin, APC, and TCF/LEF
118
DVL
Dishevelled
- becomes active when Wnt binds to frizzled and LRP
- DVL blocks the action of the “Destruction Complex” containing Axin/APC/CK-1/GSK-3
119
Genes activated by beta catenin
- depend on which partner transcription factors it binds
- Beta-catenin doesn’t bind DNA
Directly it always works with partner proteins to activate genes
-Depending on the cell type, different partners are available and different genes will be induced
120
The ligand
is what is binding
121
GSK3
phosphorizes B Catain
122
Axin
is a scaffold
123
Define proteolysis.
The breakdown of proteins or peptides into amino acids by the action of enzymes
124
What would be the result of a double knock-out of DSH and APC? Of GSK3 and β-catenin?
a. There would be no destruction complex
b. DSH is upstream of actin and the beta catin will build up
125
