Signal Transduction and Molecular circuits.
I.
Signaling proteins
a. 3 domains
i. Extracellular, Transmembrane, Intracellular
II.
Primary Messenger -> The ______________ that binds the receptor on the extracellular portion
a. Generates a _______________________ on the inside of the cell.
b. Net result if amplification, fidelity, diversity.
1.) 7 helix receptor (GPCR)
a. Has 7 trans-membrane ________________ that are made up of
_______________________ = >bind adrenalin (Adrenergic beta Receptors), and other
peptide hormones.
b. When the hormone binds it alters the interaction between the a.a. on the outside and
changes the structure, transmits the change to the inside of the protein.
c. Hormone binding changes the conc. of _______________________.
d. The GPCR is a GEF (Guanine Exchange Factor) it changes the state of
____________________ -> anchored to plasma membrane, has three subunits (alpha,
beta, gamma) -> Causes it to exchange _____ for ________ thereby turning the G
protein on.
i. GTP = ____, GDP = _____.
Mechanism -> hormone binds receptor, causes conf change, and now it can bind a G-Protein trimer and
interact with it -> binding causes the Gα and the G(βγ) to dissociate and the Gα will release ________
and exchange for __________ (This can be a step of amplification; one GPCR can activate many G
proteins) -> The Beta/gamma remain together and can diffuse in some causes and effect some ion
channels, like K+ or Ca+ channels. Gα can go on to inactive or active adenylate cyclase to generate
second messanger ___________, or other enzymes such as phospholipase C, which cleaves
phospholipids to generate second messanger molecules _________ and __________.
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There is different kinds of Gα subunits.
Gs -> will increase adenylyl cyclase activity.
Gi -> will inhibit adenylyl cyclase.
Gq -> Stimulate phospholipase Cβ ( which cleaves the head group and the product is
diacylglycerol (DAG), and the IP3 .
Gαs - activates adenylate cyclase will catalyze the reaction of ATP - cAMP + Pi
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cAMP will bind enzyme Protein Kinase A – a tetramer with 2 _________________ subunits
which inhibit the activity of the two ________________subunits.
o Two cAMP bind to each _____________subunits, and the regulatory subunits
__________ and release the _________________subunit.
o The catalytic subunit will phosphorylate other enzymes, for some it a stimulation, and
the other enzymes it is the inhibition of the activity. This depends on the enzyme and
the type of protein Kinase A. PKA can also go into the nucleus to activate transcription.
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Amplification : one GPCR can activate many G-proteins, one G-protein can activate many AC,
and one AC can make a lot of cAMP, can active a lot of PKA and PKA can phosphorylate many
proteins.
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G-Proteins -> GTP binding proteins with GTP hydrolyzing activity.
o Can be ________________ or _________________
o Bind GTP and become ________________,
o Hydrolyze GTP -> GDP and become _______________. (GTPase)
 Also known as __________________. ‘ON’ and ‘OFF’
o G-Proteins by themselves are very _________ GTPases -> switching off the G-proteins is
normally accelerated by regulatory molecules, known as RGS (regulators of GTP
hydrolysis -> which bind to Active G-Proteins (GTP) and ___________ the rate of
hydrolysis.
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______________________ -> other proteins that __________ the GTP hydrolysis
thereby turning G-proteins _________.
________________________ ->Other proteins Exchange GDP for GTP, thereby turning
G-proteins ______.
Ras -> A _______________ G-Protein, regulator of signal transduction processes leading to cell
multiplication and differentiation. Known as molecular switches, which are activated in response
to tyrosine kinase receptors.
o GTP binding proteins with essentially no _______________-> require the assistant of
GAP’s (GTPase Activated Proteins)
o Common in ____________________-> for example lack ____________ activity can lead
to constitutive activation and cell growth. -> mutation can be with the Ras protein or
the GAP protein in the cell.
o Like all nucleotide triphosphate hydrolyzing enzymes (including Gα)-> requires
assistance of _________for hydrolysis.
 _____________ is required for proper positioning of the y phosphate and for
weakening the P-O bond that is split during catalysis, as well as maintain
stability of the complex -> Mg2+ is also important for coordinating the
_____________ molecules that will be used for hydrolysis.
o Structure :
 _____________________ -> a central beta sheet consisting of 6 beta strands, 5
of which are parallel, surrounded by 5 alpha helices on both side of the beta
sheet.
o Active site
 Loops connecting the _________________ end of the _______________strands
with the ________________ end of the ___________________.
 P-Loop -> essentially for positioning phosphate groups β and α.
 G3 loop -> binds Mg2+ and the γ phosphate.
 Switch regions -> Switch I and Switch II, undergo a large conformational change
upon binding GTP or GDP.
Gα protein Transducin
o 2 domains
 ____________ domain -> very similar to Ras
 ________________ domain not found in Ras
o Contains an extra switch III
o Conformational change 3 main areas of G-protein change conformation:
Switch I: Moves closer to Guanine when active, __________ H-bonds to the -phosphate
of GTP
Switch II: alpha-helix 2 rotates so ___________ can H-bond to -phosphate, which pulls
beta-strand __ away from beta-strand 1 and toward beta-strand __. This breaks old
hydrogen bonds and makes new ones.
Switch III: interacts with switch II which propagates its structural changes to it
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Mechanism of hydrolysis
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General idea:
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generate __________ (what base/nucleophile) to attack -phosphate
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Have to neutralize the ____________ charge on the phosphate -> this reaction
does not happen spontaneously in water because a catalyst is required to
______________________________________.
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Mechanism involves the direct hydrolysis without the formation of covalent
intermediate like we saw with serine proteases.
Glutamine acts as the __________ when it its carboxyl accepts a
_______________ from the __________ molecule and the glutamine nitrogen
atom donates its _____________ to the ____________.
____________ together with a Thr stabilizes the ___________charge on the
Gamma phosphate.
Note: Ras does not have the __________ in its active site, therefore it has
virtually no ____________ activity on its own, it requires the assistance of a
___________-> ________________ to provide this arginine, and ____________
the rate of hydrolysis.
Also RGS molecules that accelerate the rate of hydrolysis In _______ proteins
work by binding to and ______________ the active site, they do not provide an
arginine.
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III.
Structure of the βγ dimer
a. Beta -> The Gβ a ________________ with ___________, each blade consisting of ____
antiparallel strands -> 7 blades coordinate to form a circular superbarrel -> also
contains one N-terminal ________________
b. Gamma folded into two ______________ in an extended arrangement, with the Nterminal helix forming a _____________ with the helix on the _____________ subunit.
c. The By dimer binds inactive Gα at the Gα active site.
Notice the Alpha from the Beta subunit and the alpha from
the Gamma subunit. Also notice where the By binds to the alpha subunit.
In rod cells:
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Rhodopsin absorbs ___________________
Activates G-protein called ________________
Transducin activates __________________ degradation of cGMP
One photondegradation of over 100,000 cGMP molecules!!
To dampen the sensitivity, protein ________________ reduces transducin’s activity
 Binds ____________and pulls it into the cytoplasm where it is inactive -> this
prevents reassociation with Gα and thereby reducing signal amplification by
transducin.
 Phosphorylation of Phosducin at _____________ reduces the stability of the
Phosducin-Gβγ complex and allows Gβγ to rebind with Gα.
d. Cholera -> Produces an AB toxin that targets _________protein.
i. A peptide = ___________________
ii. B peptide = ___________________
iii. Epithelial cells in the GI. Toxin Enzyme targets Gα, ribosylates it and inactivates
its GTPase activity. What will happen? The Gα will remain permanently on,
prevents it from turning off.  AD will continue to be active, increase in cAMP
concentration and this will stimulate a membrane protein CFTR (cystic fibrosis
transmembrane regulator). This protein is a cAMP gated Ion Channel which
allows the passage of Chloride.
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Tyrosine Kinase Receptors
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Growth Hormone Receptor
 Growth hormone is a peptide hormone, structure is a ____________________ ->
______________ ( up – down?)
The receptor is made of _______subunits that upon binding the growth hormone ligand come together and
___________.
Each subunit has single transmembrane ____________ domain and an extracellular domain arranged in two
___________________ like domains (beta barrels) joined by a linker.
 The _________________ (just like a antibody) from each Ig domain form the hormone binding site.
 Note: the two monomers are identical and bind the hormone with essentially the same a.a.
residues, however they bind to different regions on the hormone and vary in the number of
interactions, one monomer generally will bind hormone with a higher affinity than the other.
First 1 monomer will bind ___________________, this will allow the two monomers to _________________
and form a dimer. The intracellular domains on the dimer will know _________________ each other on
____________ residues, or recruit soluble tyrosine kinases to cross phosphorylate (depending on the type of
tyrosine kinase, growth hormone receptors will recruit soluble tyrosine kinases). The binding sites on the
two monomers are different and form different interactions with the Growth hormone.
Small Protein modules form adaptors for a signaling network
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These modules function as adaptors that bring together a kinase domain with its proper targets.
IV.
V.
SH2 Domain : highly conserved ____________________ domain
a. Recognizes 2 Parts
i. Recognizes __________________
ii. _____________________ on the carboxyl terminal side of the phosphotyrosine
residues, generally -> p-Tyr-Glu-Glu-Ile
iii. Structure is essentially a antiparallel beta sheet flanked by two alpha helices.
SH3 Domain : domain that binds _____________________ residues on another protein.
a. The SH3 domain recognizes a poly proline motif -> proline fold into a
_________________ conformation that SH3 recognizes and binds -> 3 helix turn.
b. Structure -> __________________________ twisted into a _______________(3 strands
+ 3 strands orthogonal to each other.) forms a peptide landing site which is wide an
extensive.
c. Gives opportunity for polyproline to bind, analogous to the _____________________ an
unfolded peptide segment.