Download Lecture 13 - Columbia University

Lecture 13
C2006/F2402 '14 OUTLINE OF LECTURE #13
(c) 2014 Dr. Deborah Mowshowitz, Columbia University, New York, NY. Last update 03/05/2014 02:28 PM. Handouts: 13A -- Overview of Signaling -- the Biological Big Bang Theory
13B -- Types of Signals -- Modes of communication between cells
13C -- Combinatorial Control of Hormone Response
Some Interesting/Useful Links
Discovery of G proteins. The 1994 Nobel Prize was awarded to Gilman and Rodbell for discovering G proteins and the
processes of signal transduction. The "Press Release" describes their scientific findings, and there are neat illustrations
and biographical notes. Several animations of Signaling are at the McGraw Hill Web site for the Raven text book.
I. Introduction to Signaling (Chap. 7 of Sadava, Chap. 14 of Becker. Detailed references below.)
A. Big Issues
1. Why bother with signaling? What is it for?
It's needed so events in a multicellular organism can be coordinated.
It's not enough to regulate what one cell does!
2. What are the major issues in all cases?
How are messages sent from one cell to another? -- What are the signals?
How are the signals received? -- What are the receptors? Where are they?
How do signals produce a response in the target cell? -- How does signal transduction work?
3. What are the important issues in signal transduction? How does a small amount of signal from outside
produce a large response inside the target cell?
How do signals cross membranes?
How is amplification of signal achieved?
4. What is the end result in a specific tissue? What is the cell response? This depends on the cell type and
signal; several examples will follow.
B. Summary of important aspects/factors of signaling to be discussed -- See handout 13A.
1. Signals -- lipid soluble vs water soluble
2. Receptors -- cell surface vs internal (Sadava, fig. 7.4 (7.5))
3. Amplification -- 3 basic methods
4. Specific cellular responses -- some examples
5. Signal Transduction -- How can a signal from outside produce a response inside the target cell?
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A. How to Classify Signal molecules -- two useful ways
1. By chemical properties -- hydrophobic (lipid soluble) or hydrophilic (water soluble)? The two types
work in different ways, as explained below & diagrammed on handout 13A.
2. By type of cell that makes them and/or target location. Where do the signals come from, and where do
they go? (endocrine, paracrine, etc) This is outlined on handout 13B.
Good way to study this: Make a table summarizing info on handout 13B. Include name of type of signaling, source of
signal, type or location of target cell, any other important features, and an example of each.
B. How do secreted signal molecules work at molecular level? Overview of hydrophobic vs hydrophilic
signals. See handout 13A, bottom.
1. Signal Molecules
a. Signals are evolutionarily conserved. Same signal molecules used by different organisms
or cell types for different purposes.
b. Two main kinds of signal molecules with different mechanisms of action
(1). water soluble -- may be either proteins or small molecules
(2). lipid soluble -- mostly steroids or TH (thyroid hormone = thyroxine)
Question: Which types of signals can be made in advance and stored until needed?
2. Receptors.
a. First step in signaling is binding (noncovalent) of signal to receptor, causing
conformational change in receptor.
b. Locations of Receptors -- intracellular and on cell surface. See Sadava fig. 7.4 (7.5).
(1). Intracellular -- for lipid soluble signals. All similar, all TF's -- details below
(2). On Cell Surface -- for water soluble signals. See Becker fig. 14-2.
(a) Structure: All cell surface receptors are transmembrane proteins
with an extracellular binding domain for signal.
(b). Terminology: Cell surface receptors are sometimes called
"extracellular receptors" but only ligand-binding domain is
extracellular, not the entire protein.
(c). Ligand binds to extracellular domain on the outside; effect ('big
bang') is inside the cell.
Signal Type
Example
Receptor Type
Effect
Lipid Soluble
Thyroxine, steroids
Intracellular**
Gene activity
Water Soluble
Peptide hormones, GF's
Cell Surface
Protein activity (usually)
**Note: Some lipid soluble signals have cell surface receptors in addition to their intracellular receptors.
One such case is in the problem book. Cell surface receptors for lipid soluble signals have been discovered
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relatively recently, and will largely be ignored in this course.
C. Modes of Communication Between Cells -- Overview of Classification Scheme by type of signaling. See
handout 13B.
III. Amplification -- The Biological Big Bang A. Why do you need amplification?
1. Not many signal molecules reach target cell. One cell secretes signal molecules that bind to a receptor on (or
inside) a target cell. Signal molecules are usually at low concentration; Only a few signal molecules bind to receptors
on target cell. 2. Usually get a big effect. All signals produce a big effect in target cell from a small concentration of signal.
Therefore all signals require amplification.
3. Example: 1 molecule of epinephrine can cause release of 104 -108 molecules of glucose from a liver cell ! (See
Becker 14-3 for one calculation -- Sadava fig. 7-18 (7-20) -- gets a different number.)
B. Three basic Amplification Methods -- (See 'Big Bang' handout -- 13A.) Types of amplification:
1. Opening ion channels -- opening a few (ligand gated channels) triggers opening of more (voltage gated
channels), which causes a big change in ion concentrations and protein activity.
2. Cascades of modifications -- for example, adding phosphates to enzymes that are kinases or
phosphatases, activating them, so they modify more proteins, and so on.
3. Affecting transcription &/or translation -- make more of target protein.
C. Details & Examples of each method
Note: Specific examples are given here primarily for reference. More details of each specific example will
be discussed below or in later lectures.
1. By opening (ligand-gated) channels
a. General Idea:
ligand
open a few ligand
a little
hit threshold
open many (voltagebig change in ion
big
→ →
→
→
→
→
binds
gated channels
ion flow
voltage
gated) channels
concentrations
effect b. Specific example: Acetyl choline (AcCh) effects on muscle. Signaling by AcCh is
important in both muscle & nerve responses. AcCh receptor is a Na+ channel in plasma
membrane opened by AcCh.
AcCh
→ binds
open a few ligand
gated Na +
channels
a little
open many
big change in
cell becomes less Muscle
(voltage-gated)
→
→ inside; hits threshold →
→
→
Na +
contracts voltage
Na + channels
flows in
concentrations
Na +
2. By cascades of modification → lots of (pre-existing) protein is modified→ big effect.
a. General idea:
ligand (1st
messenger)
→
activate receptor
activate protein inside cell (usually a
→
→
in membrane
chain of activations = cascade*)
activate a lot of target
protein (enzyme, or TF,
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→
lots of
product
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binds
etc.)
b. Specific Examples: TSH & epinephrine (2 hormones). TSH stimulates release of thyroid
hormone (thyroxine of TH) from thyroid gland. Epinephrine stimulates glycogen breakdown.
Many water soluble hormones work in this way. Details of receptors, cascades etc. later.
* The first example for this type of modification cascade to be discovered was the breakdown of glycogen, stimulated
by the hormone epinephrine (adrenaline). For details on the extent of amplification, see Becker fig. 14-3 or Sadava fig.
7.18 (7.20) .
3. By affecting transcription/translation → lots of new protein made → big effect
a. General Idea
ligand
binds →
activate a
transcribe a
→
→
TF
gene
make lots of mRNA
molecules/gene
→
mRNA
translated
→
many new protein
molecules/mRNA
b. Specific Examples: Thyroid hormone (also called thyrotropin or TH) & steroid hormones.
Most lipid soluble hormones act in this way. Receptor is itself a TF.
Question: How does the speed of signaling compare for the three types of amplification? Which is the fastest? The
slowest?
Try problems 6-12 & 6-13.
IV. How do Intracellular Receptors Work? See Sadava fig. 7.8 (7.9) or Becker fig. 14-24 (23-27) or 23-25.
A. What sorts of ligands (signal molecules) use intracellular receptors? What are the properties of the ligands?
1. All lipid soluble ligands use intracellular receptors -- Steroids, thyroxine (TH), retinoids (vitamin A), and
vitamin D.
2. Lipid soluble ligands cannot be stored -- Can pass through membranes, so must be made from soluble
precursors as needed. 3. Hormone binding proteins are needed in blood -- Are not water soluble, so all lipid soluble ligands travel in
blood bound to soluble proteins.
B. All intracellular receptors are Transcription Factors
1. Effect on transcription. Some activate and some repress transcription of target genes. (Examples below and on
handout 13C)
2. HRE -- hormone response elements
All intracellular receptors bind to cis acting regulatory elements upstream of start of transcription.
Binding site for receptor/TF usually called a hormone response element or HRE. See Becker fig. 2325 (23-26).
HRE's are usually proximal (close) to the core/basal promotor -- can be considered part of (core)
promotor, or as separate proximal upstream sites.
C. All these receptors are similar -- All members of same gene/protein family. (Note: Not all TF's are members of
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the same family, but all hormone receptor TF's are related.)
D. These receptors have (at least) three domains
1. Transcription activating (or inhibiting) domain -- also called transactivating domain (for an activator).
Binds to other proteins and activates or inhibits transcription.
2 DNA binding domain -- binds to HRE (different HRE for each dif. hormone)
3. Ligand binding domain -- binds particular steroid (or thyroxine, etc.)
4. Other domains -- Receptors also need NLS, and region that allows dimerization -- these may be
separate or included in domains listed above.
E. What (usually*) happens when TF receptors bind their lipid soluble ligands -- how receptors are activated
& affect transcription
1. Binding -- Receptor binds its ligand. Causes conformational change of at least one domain of protein
receptor.
2. Disassociation -- Receptors disassociate from inhibitory proteins. 3. Dimerization -- Receptors dimerize -- form pairs.
4. Location -- If receptor is in cytoplasm, NLS is uncovered, and receptor moves to nucleus.
5. DNA binding -- Activated Receptor (dimerized & bound to ligand) binds to HRE on DNA.
6. Effect on Transcription -- Activated receptor binds to other proteins associated with the DNA (other
TF's and/or co-activators or inhibitors), and stimulates or inhibits transcription.
*Details vary somewhat with different hormones (& corresponding receptors)
F. Example -- Estrogen (a steroid) -- How do you get different responses in different cell types?
1. Example of some proteins controlled by E -- controls production of receptors for other hormones. For
example, during pregnancy controls production of receptors for oxytocin (in uterus) and prolactin (in
breast). Oxytocin controls birth contractions; prolactin controls milk production.
a. In uterus: estrogen binding → activates transcription of gene for oxytocin receptors → production of new receptors for oxytocin = up regulation of oxytocin receptors. Receptors
needed to allow response to contraction signal (oxytocin) → contractions → birth.
b. In breast: estrogen binding → inhibits transcription of gene for prolactin receptors → down regulation of prolactin receptors; at birth, estrogen level falls and inhibition stops → transcription of gene for prolactin receptors → synthesis of prolactin receptors → response to
lactation signal (prolactin).
Summary of Effects of Estrogen
Target Organ
Uterus
Breast
Affects Receptors
for
oxytocin ( → contractions)
prolactin ( → lactation)
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Receptors are
up-regulated
down-regulated
Effect
Response to oxytocin
increases
No response to prolactin
Result
Contractions & birth possible
Lactation only after birth (when
estrogen levels fall)
Note: Prolactin is need to produce milk; oxytocin is need to cause contractions so breast can release the
milk.
2. Basic Mechanism.
E → binds to estrogen receptors → complex
Complex binds to estrogen response elements (EREs) in regulatory regions of (multiple) target
genes
Binding of complex → increased transcription of some genes (genes activated) and/or decreased
transcription of others (genes repressed).
3. How do you get different results (different patterns of transcription) in different tissues -- in response to
the same lipid soluble hormone? See handout 13C.
a. May be different hormone receptors in different tissues. Many hormones/signals have
multiple types of receptors. (Examples another time.) That's not the explanation here -- in this
case, receptors in both cell types are the same (Note this receptor = a TF).
b. May be different combinations of TFs (& factors that affect state of chromatin) in
different tissues. Usually more than one TF is required to get proper transcription of each gene.
Hormone signal acts as trigger, by binding to receptor/TF.
Type of hormone effect (what is triggered) depends on the combination of TFs present
(not just on presence or absence of TF that is the hormone receptor.)
4. Why do you get different results from different genes? (Even if the TFs are the same?)
a. Different genes have different cis-acting regulatory sites. Therefore different genes
respond differently to the same combination of TFs.
b. Reminder: All cells have the same DNA. Therefore
All cells (except immune system) have the same cis-acting regulatory sites -- the same
HRE's, enhancers, etc.
It's the trans-acting factors such as hormone receptors and other TFs that vary, between
cells, not the cis-acting regulatory sites
The cis-acting sites do vary between genes
All cells have the same genes for the trans-acting factors, receptors etc., but different
genes are used (expressed) to make different regulatory proteins in different cells.
Other examples of how hormones can give different results in different tissues will be discussed later. In general, what
any hormone does depends on combination of proteins (enzymes, receptors, TF's, etc.) already in target cell.
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Try problem 6-19. By now you should be able to do 6-12 to 6-15.
V. How do Cell Surface (Transmembrane) Receptors Work? (See 13A)
A. The problem -- Signal Transduction: How does a signal produce a response using a cell surface receptor? Two ways to put it:
1. How does the signal cross the cell membrane when the signal molecule does not? 2. How does signal molecule binding to the extracellular domain of a cell surface receptor trigger a
response inside a cell?
B. The solutions
1. Some receptors are themselves (parts of) channels. See AcCh receptor above and Sadava fig. 7.5 (7.6)
How signal work to open channels, either directly or indirectly (see below), will be discussed at a later
date.
2. Some receptors work by activating an intermediary that in turn triggers amplification through one of the
3 methods
a. Type 1 -- activates a G protein -- active form of G protein triggers an amplification
cascade, opens or closes channels, etc. (G proteins are described below; more next time.)
b. Type 2 -- activates an enzyme (in or linked to cytoplasmic domain of receptor) -active form of enzyme triggers a cascade of modifications and/or transcription.
C. Types of Cell Surface receptors that are not channels -- details
1. Type 1: G protein Linked Receptors.
a. Terminology: Called G protein linked receptors, or G Protein Coupled Receptors (GPCRs).
For a generalized case, see Sadava fig. 7.7 (7.8).
b. What are they receptors for? Many hormones such as TSH & epinephrine use GPCRs.
c. Significance -- a very large proportion of medically important drugs work by binding to
GPCRs.
(1) Antagonists -- block the action of the normal ligand -- blocks signaling even
in presence of normal ligand.
(2) Agonists -- mimic the action of the normal ligand -- causes signaling in
absence of normal ligand.
d. How do they work? Activate a G protein, which acts as a switch to trigger amplification
(details below & next time). G proteins usually do one or both of the following: (1). Activate enzymes that generate second messengers. See Sadava fig. 7.7
(7.8).
First messenger = signal molecule itself; may remain outside the cell (if
signal is water soluble).
Second messenger = small molecule generated inside the cell that activates
or inhibits proteins.
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(2). Open/close ion channels.
e. Structure: All are 7 pass transmembrane proteins with same basic structure; all belong to
same protein/gene family. (See Becker 14-4 -- handout next time)
f. Terminology: Be careful not to confuse G proteins (activated by receptors) & GPCRs (the
actual receptors).
2. Type 2: Enzyme Linked Receptors -- Not linked to G proteins. To be discussed in more detail later
when we get to cell cycle and cancer.
a. Many are (or are linked to) protein kinases. In addition to extracellular, ligand binding
domain, have an intracellular kinase domain, or interact with an intracellular kinase (when
activated).
b. Most well known type: Receptor Tyrosine Kinases (RTKs) -- also called TK linked
receptors.
c. Structure: Usually are single pass proteins that aggregate into dimers when activated.
d. What are they receptors for? Many Growth Factors use TK linked receptors or related
receptors. (See Becker table 14-3 if you are curious).
e. How do they work? These usually generate cascades of modifications, but do not usually
use 2nd messengers. If you want to see an example, see Sadava figs. 7.6 & 7.10 (7.7 & 7.12).
We won't get to details of how these work for a while.
VI. G proteins & 2nd Messengers -- How do they Fit In? What is their significance?
A. What are the important properties of G proteins? (See Becker fig. 14-5 & Handout next time.)
1. Have active and inactive forms
a. Active form is bound to GTP
b. Inactive form is bound to GDP.
2. G-proteins act as switches in many processes (not just signaling)
a. Activation: GDP bound form is converted to GTP bound form
G protein is activated by dumping GDP and picking up GTP in response to some signal.
It is NOT activated by phosphorylation of the bound GDP to GTP.
b. Inactivation: G protein inactivates itself by catalyzing hydrolysis of GTP to GDP.
c. Why is it a switch? The G protein does not stay active for long. "Turns itself off."
B. Typical Pathway -- Role in signaling (detailed handout next time.)
ligand (1st messenger)
activate G protein
activate receptor
→
→
→
in the membrane
binds outside cell
in membrane
activate target
enzyme in
membrane
→
generate small molecule
(2nd messenger) inside cell
Note that the ligand (1st messenger) binds to the extracellular domain of its receptor. The remaining events are inside
the cell.
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C. What are Second messengers? How do they fit in? More details, and examples, will be covered next time.
1. What are they? Small molecules or ions that move through the cell (or membrane) and bind to their target
proteins.
2. The usual second messengers
2nd
Messenger
Where does it come from?
How is it made?
cAMP
ATP
by action of adenyl cyclase
DAG & IP3
membrane lipid
by action of phospholipase C
Ca2+
stored Ca2+ in ER (or
extracellular)
by opening channels (in ER/plasma
memb.)
3. How are are 2nd messengers made? How are they related to G proteins?
Active G protein (subunit) → binds to & activates enzyme in (or associated with) membrane.
Activated enzyme → generates second messenger, in cytoplasm and/or membrane.
Details of cAMP pathway next time. See Becker fig. 14-7 or Sadava fig. 7.12 (7.14).
We will get to IP3 pathway later. If you are curious, see Becker fig. 14-10 or Sadava fig. 7.13
(7.15).
4. What do 2nd messengers do? Bind to and thereby activate (or inactivate) target proteins, such as
kinases, which in turn modify multiple substrates. (Detailed examples next time.)
ligand (1st messenger)
activate receptor
activate G protein in
activate target
generate small molecule (2nd
→ in membrane
→
→ enzyme in membrane →
→
binds outside cell
the membrane
messenger) inside cell
Bind to &
activate some
protein
Next Time: Chemical Communication, continued -- More on G proteins & 2nd messengers, and then how is
signaling used to maintain a multicellular organism?
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