C&T: Signaling & G-Protein-Coupled Receptors

Intracellular Domain of GPCR

In order to desensitize a cell to G-Protein linked signaling, Arrestin binds to this phosphorylated structure and targets it for internalization.

cAMP

In the GPCR signaling pathway of adenylyl cyclase, this is a secondary messenger.

GEF (nucleus)

Small GPCR (e.g. RAN) are monomeric, and they need accessory proteins to cycle them in and out of the cell. This is the name of number 2. (bonus: this is the location of number 2)

Effector enzymes

Adenylyl cyclase and phospholipase C

Agonist

Compounds that stimulate signal transduction

Intracellular receptors

DIRECT ACTION - These are nuclear structures that are effected by small and HYDROPHOBIC ligands (steroids/ hormones - cholesterol derivatives). They do not have a cell surface receptor, but unliganded forms exist in the cytoplasm and nucleus. Then liganded forms act in the nucleus. They are most easily defined by their conserved DNA binding domain.

Integral, single, alpha, hydrophilic

Describe the structure of the GPCR: 1) Integral or Peripheral 2) single or multiple polypeptide 3) 7 spanning alpha or beta regions 4) Hydrophilic or Hydrophobic loops

GPCR kinase (arrestin)

Following binding of ligand and activation of the GPCR, this enzyme is responsible for phosphorylating Ser and Thr residues on the cytosolic side, which begins the cascade of events leading to cellular effect. (Bonus: this protein can stop the cascade by binding the phosphorylated Ser and Thr)

Rapid, Slow

G-protein coupled receptors have two major pathways of effect. One, by regulating the concentration of intracellular second messengers, and, two, by activating kinase signaling cascades that effect gene expression. State how the pathways differ according to the character of their changes.

Extracellular receptors

INDIRECT ACTION - These structures receive polar ligands on the surface of the cell. Then act via secondary messengers.

Nitric Oxide

If calcium levels were locally in high concentration, calmodulin would bind to NO synthetase, which reacts with arginine to produce this compound.

Increase cytosolic Ca, no gene expression

If phospholipase C was able to create IP3 but could not create diacylglycerol due to a defect in the fatty acid chains of the lipid monolayer, one would expect this cellular response.

Acetylcholine (contraction)

In addition to activated calmodulin, this ligand binds to the endothelial cell surface receptor in order to promote the NO synthase cascade, which ultimately results in the rapid relaxation of the smooth muscle cell. (Bonus: responses to signalling molecules differ depending on cell type, so, for example, the same ligand in question has this effect on skeletal muscle)

GAP (cytosol)

Small GPCR (e.g. RAN) are monomeric, and they need accessory proteins to cycle them in and out of the cell. This is the name of number 1. (bonus: this is the location of number 1)

Protein Kinase A (phosphorylation)

Once adenylyl cyclase is activated by Gs, it produces 4+ cAMP. This is the destination of the cAMP. (bonus: this is the ultimate effect of the enzyme on the cellular protein of interest)

Amplification

One molecule of signaling ligand binds to the GPCR. Then each activated receptor protein may activate many molecules of Gs protein, each of which liberates an alpha subunit that can activate an adenylyl cyclase molecule for a prolonged period of time. Each activated adenylyl cyclase molecule generates many cAMP. The cAMP can activate many protein kinase A. Each PKA then can phosphorylate and thereby activate many copies of enzyme X. Each copy of enzyme X produces many molecules of product. This long cascade of signal transduction includes this a very important characteristic.

G-Protein interaction with effector

One would not see amplification in 1:1 interactions. This is an example of a 1:1 interaction.

Increase cytosolic Ca

Phospholipase C is another effector enzyme that catalyzes the production of IP3 and diacylglycerol. Ultimately, the purpose of phospholipase C action is to begin a cascade that does this.

Slow

Protein kinase can effect different types of pathways: rapid and slow. This picture shows PKA acting on CREB to effect gene transcription, which would create change at this rate.

Rapid

Protein kinase can effect different types of pathways: rapid and slow. This picture shows PKA acting on glycogen metabolism, which would create change at this rate.

Cytosol

The GEF/GAP cycle works so the GPCR can pick up GTP from this part of the cell.

GTP binding (alpha)

The GPCR is integral to the membrane. The G-protein subunits are lipid anchored into the membrane on the cytosolic side. Once ligand binds to the GPCR, it leads to activation of the GPCR, alpha, then beta-gamma complex primarily through this process. (bonus: this G Protein subunit is a GTPase)

Gi activation is prevented (inhibition impossible)

The bacterium Bordetella pertussis causes whooping cough, which inflicts serious harm on Infants. These symptoms result from this effect of pertussis toxin on a G-protein coupled receptor pathway. (Bonus: this is the effect on adenylyl cyclase)

Gs is locked in the active state (overactive)

The bacterium Vibrio cholerae causes the symptoms of watery diarrhea and vomiting. These symptoms result from the this effect of cholera toxin on a G-protein coupled receptor pathway. (bonus: this is the effect on adenylyl cyclase)

Cell surface receptors (polar ligand)

The following are examples of this: 1) Ion-channel-coupled receptors 2) Enzyme coupled (receptor tyrosine kinases) 3) GPCR (bonus: this is the type of polarity of the ligands in these receptors)

all 3

There are three types of intracellular signalling complexes. 1) phosphorylate something else (e.g. phospholipid molecules in the membrane) to make docking sites 2) Use scaffold proteins 3) The receptor is phosphorylated then proteins are added. from the picture, this is the type(s) of intracellular signaling in insulin.

Ligands

These include the following classes: 1) Hormones (diverse categor; some small peptides, other proteins; steroids, thyroids, retinoids, etc.) 2) Growth factors (proteins) 3) Nuerotransmitters (modified AAs) 4) Extracellular proteins (in ECM)

Desensitization (receptor inactivation)

These pictures are mechanisms of this reaction to extracellular signaling molecules. (bonus: this defines the mechanism of proteolytic events)

Gi

This G Protein pathway works to inactivate the adenylyl cyclase, which then stops the cellular response.

Dissociation (effector enzymes)

This action in the subunits follows the binding of GTP by the alpha subunit. (bonus: this is the destination of the subunits)

Affinity (alprenolol)

This characteristic of analogs is important to consider when designing a drug that competes for the binding site with native ligand. (Bonus: this drug is most tightly binding on its target and be the best inhibitor of epinephrine signal transduction.)

Ca-release channel on ER

This is the target of IP3.

Protein kinase C (calcium)

This is the target of diacylglycerol. (bonus: in order to complete activation of this target, the target needs this cofactor to bind)

Steroid hormone (intracellular)

This mechanism of ligand activation could represent this type of ligand. (Bonus: this mechanism is known as this type of receptor.)

Calmodulin

This protein is: 1) calcium-binding protein 2) abundant, ubiquitous 3) allosterically regulated 4) no enzymatic activity 5) Activates dependent protein kinases, cAMP phosphodiesterase, NO synthase

G protein-coupled receptors

This type of cell surface receptor creates RAPID changes in metabolism. It almost always sends secondary messengers to intracellular receptors.

Ser and Thr (glycosylation)

When a ligand binds to the GPCR these C-domain residues are phosphorylated. (Bonus: this modification on the N-linked sites facilitates correct binding of ligand)

cAMP (Gs)

When activated, Adenylyl cyclase creates 4 of this molecule. (Bonus: this G Protein activates adenylyl cyclase)

IP3 and diacylglycerol (Gq)

When activated, phospholipase C creates these two molecules. (Bonus: this G Protein activates phospholipase C - by the way this G protein is not affected by cholera or pertussis toxin)

Auto-phosphorylation

When calmodulin binds calcium it undergoes a conformational change that exposes a hydrophobic region that can wrap around/ bind and activate an effector protein. Bound with calmodulin, this is the name of the mechanism within the effector protein that leads to full activation.