Signal Transduction and Molecular circuits.

I. Signaling proteins a. 3 domains

i. Extracellular, Transmembrane, IntracellularII. Primary Messenger -> The hormone/ligand that binds the receptor on the extracellular portion

a. Generates a second messenger on the inside of the cell.b. Net result if amplification, fidelity, diversity.

1.) 7 helix receptor (GPCR) a. Has 7 trans-membrane alpha helices that are made up of hydrophobic amino acids

segments = >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 second messenger.d. The GPCR is a GEF (Guanine Exchange Factor) it changes the state of Heterotrimeric

protein G-Protein -> anchored to plasma membrane, has three subunits (alpha, beta, gamma) -> Causes it to exchange GTP for GDP thereby turning the G protein on.

i. GTP = on, GDP = off.

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 GDP(off) and exchange for GTP(on). (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 cAMP, or other enzymes such as phospholipase C, which cleaves phospholipids to generate second messanger molecules DAG and IP3.

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 regulatory subunits which inhibit the activity of the two catalytic subunits.

o Two cAMP bind to each regulatory subunits, and the regulatory subunits dissociate and release the catalytic 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 Monomeric (Ras) or trimeric (Gαβγ) o Bind GTP and become active, o Hydrolyze GTP -> GDP and become inactive. (GTPase)

Also known as molecular switches. ‘ON’ and ‘OFF’o G-Proteins by themselves are very slow 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 accelerate the rate of hydrolysis.

o GAPs (GTPase Activating proteins) -> other proteins that activate the GTP hydrolysis thereby turning G-proteins off.

o GEFs (Guanine Exchange factor) ->Other proteins Exchange GDP for GTP, thereby turning G-proteins on.

Ras -> A monomeric 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 GTPase activity -> require the assistant of GAP’s (GTPase Activated Proteins)

o Common in cancer tumors -> for example lack GTPase 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 Mg2+ for hydrolysis.

Mg2+ 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 water molecules that will be used for hydrolysis.

o Structure : Alpha/beta barrel -> 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 carboxy end of the beta strands with the N-terminal end of the alpha helices.

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 GTPase domain -> very similar to Ras Alpha-Helix 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, Thr177 H-bonds to the g-phosphate of GTPSwitch II: alpha-helix 2 rotates so G199 can H-bond to g-phosphate, which pulls beta-strand 3 away from beta-strand 1 and toward beta-strand 2. 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 OH- to attack g-phosphate

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

• Mechanism involves the direct hydrolysis without the formation of covalent intermediate like we saw with serine proteases.

• Glutamine acts as the base when it its carboxyl accepts a proton from the water molecule and the glutamine nitrogen atom donates its proton to the y phosphate.

• Arginine together with a Thr stabilizes the negative charge on the Gamma phosphate.

• Note: Ras does not have the arginine in its active site, therefore it has virtually no GTPase activity on its own, it requires the assistance of a GAP -> GTPase hydrolyzing protein to provide this arginine, and increase the rate of hydrolysis.

• Also RGS molecules that accelerate the rate of hydrolysis in Gα proteins work by binding to and stabilizing the active site, they do not provide an arginine.

III. Structure of the βγ dimer a. Beta -> The Gβ a beta propeller with 7 blades, each blade consisting of 4 antiparallel

strands -> 7 blades coordinate to form a circular superbarrel -> also contains one N-terminal alpha helix

b. Gamma folded into two alpha helices in an extended arrangement, with the N-terminal helix forming a coiled-coil with the helix on the Beta 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 photonso Activates G-protein called transducin o Transducin activates cGMP phosphodiesterase degradation of cGMP o One photondegradation of over 100,000 cGMP molecules!!o To dampen the sensitivity, protein phosducin reduces transducin’s activity

Binds Gby 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 Ser 73 reduces the stability of the Phosducin-Gβγ complex and allows Gβγ to rebind with Gα.

d. Cholera -> Produces an AB toxin that targets Gα protein.i. A peptide = Enzyme

ii. B peptide = Pore that transports enzyme into the celliii. 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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phosducin

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Tyrosine Kinase Receptors

• Growth Hormone Receptor Growth hormone is a peptide hormone, structure is a four helix bundle -> up, up, down,

down. o The receptor is made of two subunits that upon binding the growth hormone ligand come together and

dimerize. o Each subunit has single transmembrane alpha helix domain and an extracellular domain arranged in two

immunoglobulin like domains (beta barrels) joined by a linker. The loop regions (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 growth hormone, this will allow the two monomers to come together and form a dimer. The intracellular domains on the dimer will know cross-phosphorylate each other on tyrosine 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 phosphotyrosine binding domain a. Recognizes 2 Parts

i. Recognizes phosphotyrosine ii. 3-5 specific residues 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 poly proline residues on another protein.a. The SH3 domain recognizes a poly proline motif -> proline fold into a special helix

conformation that SH3 recognizes and binds -> 3 helix turn. b. Structure -> Five up up-down anti-parallel strands twisted into a barrel (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 MHC protein binding an unfolded peptide segment.