Principles of Cell Biology (BIOL2060)

Department of Biology
Memorial University of Newfoundland

Signal Transduction Mechanisms:  II Messengers and Receptors

Cell communication occurs through chemical signals and cellular receptors by either the
1) direct contact of molecules on two cell’s surfaces or the
2) release of a "chemical signal" recognized by another cell (near or far).
Hormones are carried by the circulatory systems to many sites.
Growth factors are released to act on nearby tissues.
Ligands are signals that bind cell surface receptors (as observed with insulin (a ligand) and the insulin receptor) or that can pass into the cell and bind an internal receptor (such as the steroid hormones).

Signal Transduction

Signal transduction is defined as the ability of a cell to change behaviour in response to a receptor-ligand interaction.
The ligand is the primary messenger.
As the result of binding the receptor, other molecules or second messengers are produced within the target cell.
Second messengers relay the signal from one location to another (such as from plasma membrane to nucleus).
Often a cascade of changes occur within the cell which results in a change in the cell’s function or identity.
The signal transduction pathway can act to amplify the cellular response to an external signal.
Messenger molecules may be amino acids, peptides, proteins, fatty acids, lipids, nucleosides or nucleotides.
Hydrophilic messengers bind to cell membrane receptors.
Hydrophobic messengers bind to intracellular receptors which regulate expression of specific genes.

A ligand binds its receptor through a number of specific weak non-covalent bonds by fitting into a specific binding site or "pocket".
In situations where even low concentrations of a ligand will result in binding of most of the cognate receptors, the receptor affinity is considered to be high.
Low receptor affinity occurs when a high concentration of the ligand is required for most receptors to be occupied.
The dissociation constant (Kd,) is the concentration of ligand required to occupy one half of the total available receptors.
This measurement of receptor affinity is often in the range of 10-4 to 10-9 mM.

With prolonged exposure to a ligand (and occupation of the receptor) cells often become desensitized.
Desensitization of the cell to a ligand depends upon receptor down-regulation by either
1) removal of the receptor from the cell surface (receptor-mediated endocytosis) or
2) alterations to the receptor that lower the affinity for ligand or that render it unable to initiate the changes in cellular function (such as phosphorylation).
Desensitization may lead to tolerance, a phenomenon that results in the loss of medicinal effectiveness of some medicines that are over prescribed.
Receptor binding activates a "preprogrammed" sequence of signal transduction events that make use of previously dormant cellular processes.

G Proteins

Activiated G Proteins bind to enzymes or other proteins and alter the target protein’s activity.
G Proteins are guanine-nucleotide binding proteins.
G Protein-linked Receptors have an extracellular N-terminus and a cytosolic C-terminus separated by seven transmembrane alpha helices connected by peptide loops.
One of the extracellular segments has an unique messenger-binding site.
The cytosolic loop between the 5th and 6th alpha helices specifically binds a particular G protein.

G Proteins bound to GTP are active, those bound to GDP are not.
The two classes of G Proteins are large heterotrimeric G Proteins and small monomeric G Proteins.
In heterotrimeric G proteins (G alpha, beta, gamma), when a messenger binds the G Protein-linked receptor, the receptor changes conformation to allow association of the trimeric G Protein with the receptor.
G-alpha subunit binds the guanine nucleotide (GDP or GTP).
This interaction causes the G alpha subunit to release the GDP, pick up a GTP and detach from the complex.
Depending upon the G protein in question, either the GTP-G alpha complex, the G beta- G gamma complex or both bind target protein(s).
The G alpha will remain an activating messenger until the GTP is hydrolyzed by the G alpha subunit (GTP -> GDP +Pi).
The "inactive" GDP-G alpha will then reassociate with the G-beta:G-gamma complex to rapidly turn down this pathway when the original stimulatory signal is removed.

Large numbers of G proteins provide diversity for signal transduction events.
Some bind potassium or calcium ion channels in neurotransmitters.
Some activate kinases (enyzmes that phosphorylate).
Some cause either the release or formation of major second messengers such as cyclic AMP (cAMP) and calcium ions.

cyclic AMP is a second messenger used by a major class of G proteins.
cyclic AMP (cAMP) is generated by adenylyl cyclase which is embedded in the plasma membrane with the enzymatic activity in the cytoplasm.
Adenylyl cyclase is activated by binding an activated alpha subunit of the Gs G-protein (GTP-Gs).
Phosphodiesterase continally degrades cAMP so in the absence of the ligand and active G-Protein, cAMP levels are reduced.
Protein kinase A (PKA), a cAMP-dependent kinase, is the main intracellular target of cAMP.
PKA phosphorylates a number of proteins that bear the key short stretch of amino acids, the PKA phosphorylation site (PKA PO4 site).
PKA transfers a phosphate from the ATP to a serine or threonine in the PKA PO4 site.
cAMP activates the catalytic subunits by causing the release of the negative regulatory subunits.

Disruption of G Protein signaling causes several human diseases.
Vibrio cholerae (causes cholera) secretes the cholera toxin which alters salt and fluid in the intestine normally controlled by hormones that activate Gs G-Protein to increase cAMP.
The cholera toxin enzymatically changes Gs so that it is unable to convert GTP to GDP.
Gs can not then be inactivated and cAMP levels remain high causing intestinal cell to secrete salt and water.
Eventually dehydration can lead to death (cholera).

Many G proteins use inositol triphosphate and diacylglycerol as second messengers G Protein-linked Receptor.
The Gp G-Protein is activated by a ligand binding its G Protein-linked receptor to activate phospholipase C.
Phosphatidylinositol-4,5-bisphosphate (PIP2) is cleaved by phospholipase C into two molecules cytosolic inositol-1,4,5-triphosphate (InsP3) and membrane-bound diacylglycerol (DAG).
The InsP3 receptor, a ligand-gated calcium channel in the endoplasmic reticulum, binds InsP3 and calcium ions are released into the cytosol.
Calcium binds a protein known as calmodulin, and the Ca-calmodulin complex act to activate an number of processes.
DAG remains membrane-bound and activates protein kinase C (PKC).
 

Calcium as a signal

The release of calcium ions is a key event in many signaling processes.
Intracellular concentrations can be followed by injection of calcium indicator fluorescent dyes.
Presence of ligand or increase in InsP3 and monitoring the increase in fluorescence
The calcium ionophore releases calcium from the intracellular stores that mimics effect of InsP3 activation.
Calcium ions act to regulate many cellular functions.
Calcium levels in the cytoplasm is normally kept low (10-4) by calcium pumps in the plasma membrane (out of the cell) and
by sodium-calcium exchangers a) out of the cell, b) into the endoplasmic reticulum (ER) lumen and c) into the mitochondrion.
Calcium stores can be released from the ER by the InsP3 receptor channel and ryanodine receptor channel which opens in the presence of calcium itself (calcium-induced calcium
release).

Although other proteins bind calcium to control activity, most often binding to the protein calmodulin, to form a calcium-calmodulin complex, is an intermediate step.
When calcium ions are present, two bind each globular end (4 in total), the helical arm region then changes conformation (the active complex) and then the wraps around the
calmodulin-binding site of target proteins.
These are often protein kinases and protein phosphatases which vary depending upon the target cell (different cells have different responses).

Fertilization of animal eggs reveals an important example of calcium-mediated signal transduction after a receptor-ligand interaction.
Initially the sperm binds the egg’s surface at the membrane and within 30 seconds, a wave of calcium release spreads from the site of sperm contact.
Two main events in fertilization rely on calcium release.
1) Calcium stimulates the fusion of the cortical granules with the egg’s plasma membrane to alter the coat surrounding the egg to help prevent the binding of another sperm cell to the egg (slow block to polyspermy).
2) Calcium initiates egg activation, the resumption of metabolic processes.
 

Nitric oxide as a Signal

Nitric oxide (NO) is a toxic, short-lived gas molecule and has been found to be a signaling molecule in the cardiovascular system.
Nitric oxide couples G protein-linked receptor stimulation in endothelial cells to relaxation of smooth muscle cells in blood vessels.
NO synthase converts arginine to citrulline and NO.
The binding of acetylcholine causes the release of NO in vascular endothelial cells that causes the relaxation of the vascular smooth muscle (vasodialator).
1) binding of acetylcholine to G protein receptors causes InsP3 production.
2) InsP3 releases calcium ions from endoplasmic reticulum.
3) ca++ ions and calmodulin form complex which stimulates NO synthase to produce NO.
4) NO (g) diffuses from endothelial cell into adjacent smooth muscle cells.
5) In smooth muscle cell, NO activates guanylyl cyclase to make cyclic GMP (cGMP).
6) cGMP activates protein kinase G which phosphorylates several muscle proteins to induce muscle relaxation.
...

Receptor tyrosine kinases

When the ligand binds a protein kinase-associated receptors, the kinase activity is stimulated and a cascade of phosphorylation transmit the signal.
The best studied examples are the receptor tyrosine kinases (RTKs).
Protein kinases add a phosphate group to amino acids that have a hydroxyl (OH) group containing side chain. (aa-OH to aa-PO4).
Receptor tyrosine kinases aggregate and undergo autophosphorylation to start chain reactions that lead to cell growth, proliferation or differentiation.
The structure of receptor tyrosine kinases often have one transmembrane (TM) domain, an extracellular ligand-binding domain and a cytosolic tail that contains tyrosine residue targets of the tyrosine activity.
The RTK can be comprised of either one protein or two proteins: a receptor and a tyrosine kinase.
The separate kinase is a nonreceptor tyrosine kinase.
Activation of the receptor tyrosine kinases is started by ligand binding causing receptor aggregation, often as dimers.
Once clustered the tyrosine kinase activity phosphorylates other RTK of the same type (autophosphorylation).
With phosphorylation, cytosolic adaptor proteins bind to receptors phosphorylated tyrosine residues.
The adaptor proteins recognize short stretches of amino acids which include a
phosphotyrosine through specific recognition domains such as the SH2 domain.
RTKs can activate several signal transduction pathways at once, including inositol-phospholipid-calcium pathway and the Ras pathway.

Receptor tyrosine kinases can initiate a Ras/MAP kinase signal transduction cascade
Phosphotyrosine-containing sites bind SH2-containing proteins which result in Ras, a small monomeric G protein, becoming activated.
Once the epidermal growth factor receptor (EGFR) is autophosphorylated in response to the EGF ligand, a complex of GRB2 (SH2 domain-containing) and Sos (guanine-nucleotide release protein: GNRP) binds the receptor.
Sos is thus activated to cause Ras to release GDP that allows Ras to bind a new GTP and become active.
Once active, Ras triggers a cascade (the Ras pathway) which includes the mitogen-activated protein kinases (MAPKs).
Note: a mitogen is a  growth factor signal.
Receptor tyrosine kinases activate a variety ofsignaling pathways
RTK can activate a form of phophlipase C and phosphatidylinositol-3- kinase (PI3K).

Growth Factors

Growth factors act as messengers
In addition to nutrients, cell often need growth factors to grow including
a) Platelet-derived growth factor (PDGF),
b) insulin,
c) insulin-like growth factor 1 (IGF-1),
d) fibroblast growth factor (FGF),
e) epidermal growth factor (EGF) and
f) nerve growth factor (NGF).
These RTK ligands function in much more than growth and cell division.

Disruption of growth factor signaling through receptor tyrosine kinases can have dramatic effects on embryonic development.
The fibroblast growth factors (FGFs) and fibroblast growth factor receptors (FGFRs) function in both embryonic and adult signaling.
FGFRs are important in the development of mesoderm, the embryonic tissue that eventually becomes muscle, cartilage, bone and blood cells.
A mutant receptor that, due to dimerization with normal versions of FGFR, has a dominant inhibitory effect upon the normal activity is a dominant negative (dn) mutation.
A dn mutant version of FGFR mRNA injected into frog eggs cause the failure of mesodermal tissue to develop and produces tadpoles with heads but no bodies.
In humans, defects in FGFRs lead to achondroplsia (dwarfism) and thanatophoric dysplasia severe bone abnormalities (fatal in infancy).

Serine/threonine kinase receptors phosphorylate both serine and threonine residues (not tyrosine) and act to transduce other types of growth factor signals.
Transforming Growth Factor Beta (TGFß) binding to receptor results in the clustering of type I and type II TGFß receptors.
The type I receptors are phosophorylated by type II receptors.
Activated Type I receptors phosphorylate specific receptor-mediated SMADs.
The activated receptor-mediated SMADs bind to co-SMADs and enter the nucleus to interact with DNA binding proteins to regulate gene expression.

Hormones

Chemical signals known as hormones are secreted by one tissue to regulate another tissue, often over a distance.
Hormones are often transmitted by the circulatory system.
Hormones control many physiological functions including growth and development, rates of physiological processes, concentrations of sugars and minerals, and responses to stress.
Hormones can be proteins, peptides, steroids and other molecules.

Hormonal signals can be classified by the distance that they travel to reach their target cells.
An endocrine hormone travels through the circulatory system and a paracrine hormone acts only upon near by cells.
A paracrine hormone is roughly equal to a growth factor.
Endocrine tissues secrete directly into the blood-stream and exocrine tissues into ducts for transport of the secretions to other parts of the body.
The pancreas has both endocrine (insulin and glucagon) and paracrine (digestive enzymes) functions.
Once in the circulatory system, the endocrine hormones will eventually reach their target tissue(s) such as heart and liver (epinephrine) or liver and skeletal muscles (insulin).
In the target tissue, intracellular effects, such as the activation of the cAMP pathway can control a number of cell functions.
One example, is by epinephrine binding to the beta-adrenergic receptor, activation of PKA to cause the stimulation of glycogen breakdown.

Insulin activates a wide range of intracellular effects by the phosphorylation by the insulin receptor complex of its substrate IRS.
This, in turn, leads to activation of 1) the Ras-MAPK pathway and 2) the PI3K/akt kinase pathway that activates (and inactivates) a number of proteins.

Chemical properties of animal hormones
There are four (4) categories of endocrine hormones.
1) amino acid derivatives (epinephrine)
2) peptides (antidiuretic hormone [vasopressin])
3) proteins (insulin)
4) lipid-like hormones including steroids (testosterone)
Paracrine hormones include histamine (a histine derivative) and the prostaglandins (arachidonic acid derivatives).

Notes prepared from Becker's World of the Cell, 8th edition
Hardin, Bertoni & Kleinsmith, 2012
Figures copyright of Pearson Education Inc.
email me at bestave@mun.ca