Signal Transduction and Molecular circuits.

I. Signaling proteins a. 3 domains

i. Extracellular, Transmembrane, IntracellularII. 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 __________.

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

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.

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.

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.

o ______________________ -> other proteins that __________ the GTP hydrolysis thereby turning G-proteins _________.

o ________________________ ->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 IIIo Conformational change 3 main areas of G-protein change conformation :

Switch I: Moves closer to Guanine when active, __________ H-bonds to the g-phosphate of GTPSwitch II: alpha-helix 2 rotates so ___________ can H-bond to g-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

• Mechanism of hydrolysis

– General idea:

• generate __________ (what base/nucleophile) to attack g-phosphate

• Have to neutralize the ____________ charge on the phosphate -> this reaction does not happen spontaneously in water because a catalyst is required to ______________________________________.

• 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.

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 N-terminal 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:

o Rhodopsin absorbs ___________________o Activates G-protein called ________________o Transducin activates __________________ degradation of cGMP o One photondegradation of over 100,000 cGMP molecules!!o 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.

G

phosducin

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Tyrosine Kinase Receptors

• Growth Hormone Receptor Growth hormone is a peptide hormone, structure is a ____________________ ->

______________ ( up – down?) o The receptor is made of _______subunits that upon binding the growth hormone ligand come together and

___________. o 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.

o 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

• These modules function as adaptors that bring together a kinase domain with its proper targets.

IV. 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.

V. 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.