Download BIOL 201: Cell Biology and Metabolism

BIOL 201: Cell Biology and Metabolism
WEEK 11
Lipid Soluble Hormones:
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Hormones are molecules that are made by cells that act through specific receptors
and confer information to a target cell so that it responds correctly to some
stimulus
Different Types of Intercellular Signaling:
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Endocrine Signaling: Have a cell that secretes a hormone, which is released into
circulation and acts on cells far away (global signals)
Paracrine Signaling: Have a cell that secrets a hormone which acts on a nearby
cells
Autocrine Signaling: Cell releases a hormone that acts on itself. Typical of a lot
of tumor cells that produce growth factors, inducing there growth
Signaling between Attached Receptors: The receptors interact between two
cells, and are important for organizing a cellular response in all the cells in a
given tissue
Intracellular Signal Response Systems in Cells:
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Receptor signaling pathways are so few, but yet carry out most of the diversity
that is seen in tissues in humans
A number of the receptor signaling pathways are conserved among eukaryotic
organisms
Pathways work from receptors all the way to changes in transcription
o Respond to the extracelluar environment, go to the nuclease and change
the genetic output
Going to focus on the G-Protein Coupled receptors
Lipid Soluble Hormones – Steroids:
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They don’t require receptors on the surface of the cell, but because they can go
through the cell membrane, can interact with intracellular receptors (can be in the
cytoplasm or in the nucleus)
When these molecules interact with the receptors, there ultimate change will be at
the level of the genetic output
Steroid Hormone Structure:
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They are all based on a cholesterol backbone
They are very hydrophobic
They interact with receptors that have similar structures
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Have major effects on growth and behavior
Nuclear Hormone Receptors:
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Maintain same structure:
o Variable N-Domain, always followed by DNA
binding domain, then followed by ligand
domain
The top 3 are initially found in the cytoplasm
(Plus androgen)
The bottom 3 are always located in the nucleus
(Plus Vitamin D)
Steroid Hormone Receptors always homodimerzie
Nuclear Hormone Receptors act as heterodimerzes
The Zinc Finger Motif:
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When it forms a homodimer, forms a point of symmetry
Steroid Hormone Receptors:
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The way the steroid hormone receptors bind to the DNA, they need
to bind to Reverse Palindromes
Nuclear Hormone Receptors:
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These receptors interact with direct repeats in the DNA
Mechanism of Steroid Hormone Activity:
o The hormone receptors are always found in the cytoplasm when they are
not activated
o There is an inhibitor protein HSP 90. It binds to the ligand-binding
domain of the receptors in the cytoplasm, until the hormone comes in.
When it comes in, it binds to the LBD, and causes a conformational
change in the receptor protein which kicks out the HSP 90
o This then allows the receptor to go into the nucleus
o When its in the nucleus, finds homodimers, binds to the
DNA, and the activation domains will induce RNA Pol II
transcription complex to turn on the genes
Mechanism of Nuclear Hormone Activity:
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Under no ligand circumstances, the receptors bind to their
specific genes but block the transcription of those genes
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As soon as the hormone comes in and acts with its appropriate receptor, it turns
on the transcription of the gene by increasing the levels of Histone acytliation
(when there is no hormone, recruits histone deacetlase, keeping the chromatin
condensed)
RAR Receptor Function in Development:
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Retinoic Acid can lead to posterior changes in the spinal chord to take on a more
anterior structure if taking at the inappropriate times (causing deformities)
Environmental Mutagens Mimic RA Activation:
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Tadpoles and frog development are sensitive to environmental conditions
Turns out that the sunlight leads to the production of Methoprenoic Acid by
acting on an insecticide that found its way into the water leading to deformities
o This can bind to Retinoic acid receptors because they have similar
structures, leading to growth
G-Protein Coupled Receptors and cAMP:
Dissociation Constants (K0) And Binding Assays:
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An easy of way to quantify a number of receptors on a cell. Take the known
ligand and combine it with a radioactively labeled form of the ligand
With the mixture, can introduce into a cellular environment, wash away the stuff
that didn’t bind, and get an idea of the number of counts that bind to the cell. Will
give you an idea of how much radiolabled ligand is bound to the receptors. It is an
indicator of all the known labeled ligand bound
Get Total Binding. However, a lot of binding is Nonspecific Binding
o Subtraction of Total Binding and Nonspecific Binding give a
curve for Specific Binding: Can say how many moles of
receptor are on the surface
Must repeat the experiment with lots of none-labeled ligand
This count will give the affinity of the ligand to receptor
R + L  RL: Largely depending of the affinities of the two molecules:
If half the binding sites are occupied, the [Free Receptors] = [RL]. Therefore,
the [L] = to KD
Signaling Through Cell Surface Receptors:
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Most signaling happens when a hormone is released, it eventually interacts with a
receptor that is specific. This receptor then illicits a response in the cell. Has to
transfer the signal into the cell. Does this by interacting with intracellular
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molecules (Second Messenger Molecules, where the first messenger is the
ligand)
Activation of the receptor give rise to short term and long term responses
Afterwards, the receptor can’t always be activated. Therefore, there is a means to
regulate the receptors activity, getting rid of the ligand in the receptor
Second Messengers – Intracellular Mediators of External Signals:
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They are activated downstream of the receptor-ligand binding. They vary a lot:
o Cyclic AMP, Cyclic GMP, Diacylglycerol, Inositol 1,4,5Trisphosphate
G-Protein Coupled Receptors (GPCR):
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Starts with the binding of epinephrine to its receptor
The receptor for epinephrine is made up a membrane spanning (7) receptor. Its NTerminal is in the extracallular space, C-Terminal domain in the cytosol
Has loops on intracellular space: They interact with Heterotrimeric G-Proteins
o They are important for downstream transduction signaling
There are non-covalent interactions of the amino
acids in the transmembrane domain
Mechanism of Action of GPCR:
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The G-Proteins consists of 3 subunits:
o G-Alpha
o G-Beta
o G-Gamma
Epinephrine interacts with the G-Protein coupled
receptor. Immediately, G-Alpha recognizes a major
conformation change that is in the receptor itself
G-Alpha is in a GDP bound state. When it interacts with the activated receptors, it
undergoes its own conformational change, kicking out GDP from the binding site
o Activated receptor acts as a Guanine Exchange Factor
This allows GTP to interact with G-Alpha. When GTP binds, the G-Protein falls
apart, whereas Beta/Gamma stick together. The 2 complexes move away. GAlpha finds an Effector Protein (Adenylyl Cyclase for epinephrine). Interacts
with this protein and modifies it so that there is a change in its activity
The Effector Protein will carry out the appropriate downstream event that are
typical of this receptor (activating the formation of second messengers (cAMP
for epinephrine) or directly effect ion channels)
o Also acts as a GAP
Helps G-Alpha protein to hydrolyze GTP back to GDP, therefore inactivating GAlpha, so that it can recomplex with G-Beta/Gamma, forming the Heterotrimeric
Complex
Different effector proteins for all the different hormones/receptors
Cyclic AMP – Regulation of Protein Kinase A:
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Cyclic AMP is formed from the activity of Adenylyl Cylase: Converts
AMP to Cyclic AMP
This can then interact with Protein Kinase A, and activates it
o PKA has 2 Catalytic subunits and two regulatory subunits. For every
regulatory subunits that it has, 2 cAMP binds. Therefore, when cAMP
binds it releases the Catalytic subunits, which are the activated Protein
Kinase A
PKA then phosphorylates a number of intracellular targets
Protein Kinase A Affects Gene Expression:
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PKA makes its way into the nucleus, and phosphorylates key
transcription factors (CREB), which gives rise to long term
changes by changing transcription of its specific genes
G-Protein Activity Regulation:
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The receptor acts as a GEF for the G-alpha
The Effector protein acts as a GAP for G-Alpha
G-Protein Switching Mechanism:
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There are two key amino acid residues that interact with the gamma
phosphate of GTP: Gly-60 and Thr-35. They change the
conformation of the protein
Bidirectional G-Protein Effects:
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End up altering the conformation of the Heterotrimeric G-Protein such that you
end up stimulating Adenylyl Cyclase (Stimulatory G-Alpha)
In some cases, get a different situation, where you don’t have a Stimulatory active
G-Alpha, but an Inhibitory active G-Alpha. Instead of G-Alpha stimulating the
effector protein, it inhibits it
G-Protein Function and Glycogen Metabolism:
GPCR – Other Effector Proteins:
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GPCR can also affect ion channels using an alternate mechanism
Acetylcholine binds, causing conformational change. This is
transduced by G-Beta/Gamma
They translocated, and their effector proteins are Ion Channels, activating and
opening them
GPCR – Other Effector Proteins:
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Another mechanism is involved in vision
This happens in Rod cells (help in low light vision)
There are a number of molecules: Rhodopsin
Opsin combined with 11-cis-Retinal form Rhodopsin (the
protein that responds to photons)
o Will respond to even a single photon. The photon causes the
conformational change. It turns 11-cis-Retinal to an
all-trans-Retinal
o This conformational change causes a major change in Rhodopsin. The
change in Rhodopsin is transduced by another Heterotrimeric G-Protein
complex; G-Alphat (Very specific)
o Following activation of the Rhodopsin receptor, end up chaning the
conformational. G-Alphat becomes activated, transloacting through the
membrane, interacting with Cyclic GMP Phosphodiesterase
o CGMPP: Converts Cyclic GMP (second messenger) back to GMP. In
doing this, it inactivates the second messenger
o Under normal circumstances, the membrane in the cells are depolarized in
dark situations. When you shine light on them, leads to lower level of
cGMP. This shuts of the cGMP gated ion channels, hyperpolarizing the
cell. The brain then interprets this as light
Intracellular Amplification:
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One single molecule of activated Rhodopsin on the membrane
can single up to 500 molecules of G-Alphat
Once this is activated, can make major changes in the cell
It is a typical way of how G-Protein Coupled Receptors
leads to rapid and major changes within the cell from a
little amount of hormones
Functional Role of G-Protein Regulators:
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Want to ensure that you can turn off all the amplification signaling after the
stimulus is terminated
The GAP activity is a major regulator
There are molecules that help get back to the ground state
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Very hard to get back into the ground state if these are knocked out
Cyclic AMP – Synthesis and Breakdown;
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Phosphodiesterases are another way that help get back to the ground state
Turn the Second messenger back to a deactivated form. Work on cAMP or CGMP
o cAMP to AMP by cAMP phosphodiesterase
Protein Kinase A is Localized to the Nuclear Membrane of Heart Muscle Cells:
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In the heart, bring PKA in close proximity to the
phosphodiesterase so that you can:
o Maintain PKA close to where it acts
o To help bring back to ground state quickly
(Turns it off quickly)
G-Protein Regulators:
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GPCR gets activated, and there are Protein Kianses that recognize that it gets
activated. These phosphorylate the C-Terminal loop on the cytosol site. This is
recognized as a binding site for Beta-Arrestin. It interacts with this region and
AP2 and Clathrin, which are important for endocytosis
The receptors become endocytosis so they are no longer
involved in signaling (can then either go back or be degraded)
It is all dependent on Beta-Arrestin
Second Messengers – Intracellular Mediators of External Signals:
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Diacylglycerol (DAG): Membrane bound
Inositol 1,3,5-triphosphate (IP3): It is fully diffusible
Formation: Phosphatidylinositol goes through phosphorylations that make it
become very energetic. Gives rise to PIP2. It is a major substrate of
Phospholipase C. This cleaves it to DAG and IP3
Ca2+ are another second messenger
o Induces secretion of cargo from hormone containing vesicles in
neuroendocrine cells
o Enhances neurotransmitter release from neurons
o Binds to EF/Hand protein Calmodulin to affect protein targets
 Binds to 4 molecules of calcium, causing a major conformational
change, allowing it to interact with protein effectors
DAG and IP3 effects are associated with changing the levels of intracellular Ca
Regulation of Calcium Levels in Cells and G-Protein Activation:
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Get an interaction with a hormone and a receptor causes a conformational change in
Phospholipase C (the Effector Protein) through G-Alpha. This leads to DAG and IP3
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IP3 will open up ion channels, changing the conformation,
causing Ca to leave the Endoplasmic Reticulum
When the intracellular levels of Ca increase, the calcium binds
to Protein Kinase C. This will go to the membrane, and
interact with the DAG that is up there. This interaction will
activate PKC. The PKC will then interact with a number of
important targets leading to: cell growth, cell division and
changes in cytoskeleton
This is seen a lot in the Pituitary
Calcium and Fertilization:
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When sperm fertilizes the egg, leads to a spike in calcium, filling the occytes