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CELL SIGNALING 3Second messengers are the intracelluar signals which serve as downstream signaling elements that propagate and amplify the signals initiated by ligand binding.

Second messenger molecules are chemically diverse - e.g. inorganic ion (Ca2+) cyclic nucleotides (cAMP, cGMP) lipids (diacylglycerol; DAG) gas (nitric oxide)

Different second messengers function in discrete sub-cellular compartments e.g. Ca2+ in cytosol or diacylglycerol (DAG) in lipid bilayer.

Messages are encoded by the change in concentration of the second messenger (rise or fall).

Second messengers convey signals by binding to, and altering the conformation and behavior of, selected signaling molecules and effector proteins.

The major targets for activated trimeric G-proteins

adenylyl cyclase phospholipase C K+ ion channel

Targets for activated trimeric G-proteins

Adenylyl cyclase - an effector enzyme that produces the second messenger cAMPAdenylyl cyclase (AC) is a large, multiple transmembrane domain protein.Catalytic domain is located on cytosolic face of membrane.Gs stimulates AC activity, while Gi forms of GPCR inhibits ACGs subunit stimulates AC by mediating a conformational change in catalytic domains of AC

cAMP pathwaycAMP is produced from ATP via action of adenylyl cyclase

HOW DOES cAMP THEN ACT?

Active protein kinase A then acts on serine residues on target proteinIt activates protein kinase A

Activation of protein kinase A (PKA) by cAMP

In some cells increase in cyclic AMP activates the transcription of specific target genes that contain a regulator sequence called cAMP response elementIn some animal cells an increase in cyclic AMP activates the transcription of specific genes. In cells that secrete the peptide hormone somatostatin, for example, cyclic AMP turns on the gene that encodes this hormone.

The regulatory region of the somatostatin gene contains a short DNA sequence, called the cyclic AMP response element (CRE), that is also found in the regulatory region of other genes that are activated by cyclic AMP.

This sequence is recognized by a specific gene regulatory protein called CRE-binding (CREB) protein. When CREB is phosphorylated by A kinase on a single serine residue, it is activated to turn on the transcription of these genes; the phosphorylation stimulates the transcriptional activity of CREB without affecting its DNA-binding properties.

If this serine residue is mutated, CREB is inactivated and no longer stimulates gene transcription in response to a rise in cyclic AMP levels.

Signal carried from cytoplasm to nucleus by catalytic subunit of protein kinase A

Protein kinase then phosphorylates a transcription factor called CREB

Leads to coactivators and transcription of cAMP inducible genes.

Activation of target gene expression via PKA / CREB Note - PKA can have many additional effects on the cell, not just CREB !The targets for PKA differ depending upon the specific cell type.CRE - cAMP response element; CREB - protein that binds to CRE sequences.

When cAMP is elevated in skeletal muscle and liver cells, the breakdown of glycogen is stimulated. In cardiac muscle, the elevation of cAMP strengthens heart contraction, whereas in smooth muscle contraction is inhibited. In intestinal epithelial cells, it causes the secretion of salts and water into the lumen of the gut.REGULATION OF PROTEIN PHOSPHORYLATION

cAMP can directly regulate ion channels independent of protein phosphorylation.This pathway is involved in sensing smellWhen it directs the opening of the Na+ channels it causes depolarization of membrane & initiation of nerve impulsecGMPGuanylyl cyclases are activated by NO, CO and peptide ligands.Guanylyl cyclase increases the level of cGMPcGMP then mediates its action through cGMP dependent protein kinases.Page 19

Two cyclic nucleotide monophosphates adenosine 3',5'-cyclic monophosphate (cAMP) guanosine 3',5'-cyclic monophosphate (cGMP)Well characterized role of cGMP is seen in the vertebrate eye.Important for converting visual signals received as light to nerve signals.

Phototransduction is the process through which photons, elementary particles of light, are converted into electrical signals.

Visual phototransduction occurs in the retina through photoreceptors, cells that are sensitive to light.

Both rods and cones contain opsin, a G protein-coupled receptor.

Opsin is bound to a light-absorbing chromophore, 11-cis-retinal

Rhodopsin is present in rods and transduces dim light while photopsins are present in cones and generate color vision

Regulation of cGMP phosphodiesterase - vision depends on a GPCR that regulates a cyclic nucleotide-gated ion channel.

PHOSPHOLIPIDS &Ca2+Phosphatidyl inositol 4,5 bisphosphate is known as PIP2

This is a present on the inner leaflet of the plasma membrane.

PIP2 can be hydrolyzed by phospholipase C

This producers two distinct second messengers called DAG and IP3

What activates the hydrolysis if PIP2?

G protein coupled receptorsProtein tyrosine kinases

PLC - PLC -

IP3 is a small polar molecule that is released into the cytosol where it acts to signal the release of Ca2+ from intracellular stores

Calcium level in the cell then increases from 0.1 to

This affects the activities of various target proteins including kinases and phosphatatses.

How does Ca2+ mediate its effects?When the concentration of Ca2+ increases to 0.5 microns, calcium binding protein CALMODULIN is activated

Ex : myosin light chain kinaseProteins activated by this complex are CaM kinase familyIntersections between calcium and cAMP signaling pathwayCaM phosphorylates transcription factors --- CREBCa2+/ Calmodulin regulates adenlyl cyclase and phosphodiesterasecAMP regulates calcium channels

Thus these two pathways function coordinately to regulate many cellular responsesDiacylglycerol DAG activates the serine threonine kinases belonging to protein kinase C family

Ca2+ in electrically excitable cellsNeurons triggers the release of neurotransmitters

Muscle triggers muscle contraction

Phospholipase C produces two independent second messengers, inositol 1,4,5 triphosphate (IP3) and diacylglycerol (DAG), by cleavage of phosphoinositol 4,5 bisphosphate (PIP2).DAG is a hydrophobic lipid that can translocate laterally through the inner leaflet of the lipid bilayer.IP3 is a hydrophilic molecule that can diffuse rapidly through the cytosol.IP3 can bind to IP3 receptors on surface of ER and mediate rapid release of Ca2+.In turn, Ca2+ and DAG each bind to, and activate PKC.PKC phosphorylates targets on serine and threonine residues.Cells contain different kinases and lipases that produce discrete signaling molecules from phosphoinositides

Several different phospholipases (e.g. -C, -D, -A2) can cleave PIP2 at different locations to generate different molecules that affect additional signaling pathways (-C = DAG+IP3; -D = phosphatidic acid; -A2 = arachidonic acid).Arachidonic acid is important intermediate in synthesis of prostaglandins that play important roles in inflammation.

ADRENERGIC HORMONESWhen secreted into the bloodstream, epinephrine and norepinephrine stimulate changes in many different tissues or organs, all aimed at preparing the body for dangerous or stressful situations

Overall, the adrenergic hormones trigger increased cardiac output, shunting blood from the visceral organs to the muscles and the heart, and cause dilation of arterioles to facilitate oxygenation of the blood.

In addition, these hormones stimulate the breakdown of glycogen to supply glucose to the muscles.

Adrenergic hormones bind to a family of G protein linked receptors known as adrenergic receptors.

The adrenergic receptors are located on the smooth muscles that regulate blood flow to visceral organs. This activates Gq (trimeric G-protein), which in turn activates phospholipase C (effector), which produces IP3 and DAG (second messengers) from PIP2.

Contraction of smooth muscle hence constriction of blood vessels and reduced blood flowThe adrenergic receptors are found on smooth muscles associated with arterioles that feed the heart, smooth muscles of the bronchioles in the lungs, and skeletal muscles

It activates Gs which activates the cAMP signal transduction pathway

Leading to relaxation of smooth muscle.

In addition, these hormones stimulate the breakdown of glycogen to supply glucose to the muscles