Second messengers in cell signaling

Second messengers in cell signaling

Simple definition: Second messengers are molecules that relay signals from receptors on the cell surface to target molecules inside the cell.

  • The first messengers in cell signaling are usually extracellular signal molecules.
  • Third messengers are usually the large protein kinases and phosphatases that are recruited to the plasma membrane.
  • Second messengers are signaling intermediates connecting events taking place at the plasma membrane with the intracellular signaling that eventually converts the signal into a cellular response.
  • These molecules help to spread a signal through the cytoplasm by altering the behavior of certain cellular proteins.
  • They arise from easily available substrates and only have a short half-life.

Earl Wilbur Sutherland: discovered second messengers (won the 1971 Nobel Prize)

  • Second messengers serve to amplify the strength of the signal.
  • Binding of a ligand to a single receptor at the cell surface may end up causing massive changes in the biochemical activities within the cell.
  • The concentration of these intracellular messengers is regulated by hormones, neurotransmitters, and other extracellular signals.

Types of second messengers in cell signaling

There are 4 major types of second messengers:

1) Cyclic nucleotides: (cAMP and cGMP) – Hydrophillic in nature

2) Membrane lipid derivatives: Inositol trisphosphate (IP3) – hydrophillic in nature

Diacyl glycerol (DAG) – hydrophobic in nature

3) Calcium ions (Ca2+) – Hydrophillic in nature

4) Gases: NO (nitric oxide) or CO (Carbon monoxide) – Can diffuse both through cytosol and across cellular membranes.

Hydrophobic signaling molecules: water insoluble molecules which are membrane associated and diffuse from the plasma membrane into the inter membrane space where they can reach and regulate membrane associated effector proteins.

Hydrophilic signaling molecules: water soluble molecules that are located within the cytosol.

A) cAMP (cyclic 3’- 5’ adenosine monophosphate)

Compositions of cAMP: Adenine base + Ribose sugar + 3’, 5’-cyclic phosphate

Chemical formula: C10H11N5O6P

Molar mass: 329.206 g/mol

Synthesis and Degradation of cAMP:

cAMP is synthesized from ATP by the action of the enzyme adenylyl cyclase which is located on inner side of plasma membrane .

  • Adenylate cyclase activity is regulated by G proteins (Gs and Gi) which in turn are controlled by extracellular signals.
  • Ca2+– calmodulin also activates specific adenylate cyclases.

cAMP decomposition into AMP is catalyzed by the enzyme phosphodiesterase.

  • Phosphodiesterase are Inhibited by: Methylxanthines (Example: caffeine).
  • Insulin activates the esterase and thereby reduces the cAMP level.
Figure 1: A) Formation of cAMP from ATP catalysed by Adenylyl cyclase B) Degradation of cAMP into AMP catalysed by phosphodiesterase enzyme.

Functions of cAMP

1) cAMP as a second messenger:

  • cAMP is a cyclic nucleotide which serves as an intracellular and in some cases extracellular secondary messenger.
  • It is involved in transmitting signal from outside the cell to the interior via the process of binding of hormones like glucagon and epinephrine or other signal molecules to cell membrane receptor.
  • It is involved in the activation of protein kinases and regulates the effects of adrenaline and glucagon.
  • cAMP also binds to and regulates the function of ion channels such as the HCN channels and a few other cyclic nucleotide-binding proteins such as Epac1.

2) Regulation of protein kinase A by cAMP

  • The most important function of cAMP in animal cell is regulation of protein kinase A activity.
  • cAMP is an allosteric effector of protein kinase A (PKA).
  • Protein kinase A is found primarily in inactive form in the cell.
  • In the inactive state: PKA is a hetero tetramer (C2R2). The catalytic subunits are blocked by regulatory units (auto inhibition).
  • Binding of cAMP to the regulatory subunits induces a conformational change that leads to dissociation of the catalytic subunits.
  • It leads to the formation of enzymatically active form of protein kinase A.
  • Active PKA phosphorylates serine and threonine residues of more than 100 different proteins, enzymes, and transcription factors.

3) Regulation of Cyclic AMP inducible gene expression via protein kinase A:

  • In many animal cells, increase in cAMP activates the transcription of specific target genes that contain a regulatory sequence called the cAMP response element, or CRE.
  • The signal is passed from the cytoplasm to the nucleus by the catalytic subunit of protein kinase A.
  • Catalytic subunit can enter the nucleus after its release from the regulatory subunit.
  • Within the nucleus, protein kinase A phosphorylates a transcription factor called CREB (CRE-binding protein), leading to the activation of cAMP-inducible genes.
  • Thus such type of regulation of gene expression by cAMP plays significant role in controlling proliferation, survival, and differentiation of a wide variety of animal cells.

Figure 2: Cyclic AMP inducible gene expression. 

4. Role in eukaryotic cells:

  • cAMP and its associated kinases play key role in numerous biochemical processes including the regulation of glycogen, sugar, and lipid metabolism.
  • The olfactory system transduce signal via cAMP second messenger.
  • The olfactory receptor protein is coupled to a membrane protein called a G protein,
  • Binding of an odorant molecule to an olfactory receptor protein activates a G protein and adenylate cyclase, resulting in the production of cAMP.
  • Cyclic AMP opens sodium ion (Na+) channels and Na+ ions enter the olfactory receptor. The resulting depolarization may generate an action potential, which propagates along the axon of the olfactory receptor.
Figure 3: Olfatory transduction

5. Role in bacteria: Positive regulation of Lac operon

  • cAMP level varies depending on the medium used for growth of bacteria (the carbon source).

High glucose → Low cAMP

Low glucose → High cAMP

  • CAP is only active when glucose levels are low (cAMP levels are high).
  • Without cAMP, CAP cannot bind to the cap binding site on DNA and is inactive. That means CAP binding is regulated by levels of cAMP.
  • When cAMP binds to CAP its shape changes and then it is able to bind DNA and promote transcription.
  • With a high glucose concentration, the cAMP concentration decreases, and the CAP disengages from the lac operon.
Figure 4: Positive regulation of lac operon by glucose repression coupled to enhance level of cAMP.

6) In some slime moulds:

  • In some slime mold species such as Dictyostelium discoideum, the chemotactic movement of cells is organized by periodic waves of cAMP that propagates through the cell.
  • The waves are the result of a regulated production and secretion of extracellular cAMP and a spontaneous biological oscillator that initiates the waves at centers of territories.

Mechanism of Regulation of cAMP:

  • When stimulatory hormone binds to GPCR, GPCR activates the G protein that contains stimulatory alpha subunit which in turn activates membrane bound adenylyl cyclase that facilitates the conversion of ATP to cAMP.
  • When inhibitory hormone binds to GPCR, GPCR activates the G protein that contains inhibitory alpha subunit which binds to enzyme and inhibits cAMP production.
  • The elevated cAMP level is regulated by degradation pathway which takes place by cAMP phosphodiesterase enzyme.

B) Nitric oxide (NO)

  • Nitric oxide (NO) acts as a second messenger because it is a free radical that can diffuse through the plasma membrane and affect nearby cells.
  • It is synthesized from arginine and oxygen by the NO synthase.
  • NO works mainly through activation of its target receptor, the enzyme soluble guanylate cyclase which in turn produces another second messenger cGMP.
  • NO can also act through covalent modification of proteins or their metal cofactors.
Figure 5: NO mediated cell signaling pathway
  • It is toxic in high concentration.
  • NO serves multiple functions includes: Relaxation of blood vessels, Regulation of exocytosis of neurotransmitters, Cellular immune response. It also relaxes smooth muscle tissues.
Figure 6: NO mediated smooth muscle relaxation via cGMP

C) cGMP (Cyclic guanosine monophosphate)

  • It is a cyclic nucleotide derived from guanosine triphosphate (GTP).

Molecular formula: C10H12N5O7P

Molar mass: 345.2 g/mol.

Composition of cGMP:  Guanine nucleotide base, ribose sugar and cyclic phosphate between 3’ and 5’ positions of ribose sugar.

Figure 7: structure of cGMP

Synthesis and degradation of cGMP

  • cGMP is synthesized from the nucleotide GTP using enzyme guanylyl cyclase.
  • Cyclic nucleotide signals are degraded by phosphodiesterases enzymes that cleave phosphodiester bonds and convert cGMP into GMP.
  • There are now several drugs which function as phosphodiesterase inhibitors and can restore Nitric Oxide / cGMP signaling. The best known of this class of drugs is Viagra.

The guanyl cyclase is generally found in cell in two forms (isozymes):  soluble form and membrane bound form. Both forms participate in signal transduction.

Soluble guanylyl cyclase: Activated by intracellular nitric oxide (NO).

  • NO is non polar and can easily cross the plasma membrane of target cell without any carrier.
  • Then it binds to the heme group of guanylyl cyclase and activates the cGMP production.
  • This form is found in many tissues including smooth muscle of the heart and blood vessels.

Membrane bound guanylyl cyclase: Activated by their extracellular ligands (Example: atrial natriuretic factor or ANF)

  • Receptors in cells of the renal collecting ducts, smooth muscle of blood vessels and receptors in intestinal epithelial cells when activated, stimulate cGMP synthesis via adenylyl cyclase.
Figure 8:Two types (isozymes) of guanylyl cyclase that participate in signal transduction.

Functions of cGMP:

1) Activation of protein kinase G

In animals most of the actions of cGMP are supposed to be mediated by cGMP dependent protein kinase which is also called protein kinase G (abbreviated as PKG).

  • PKG (protein kinase G) is a dimer consisting of one catalytic and one regulatory unit on a single polypeptide chain. A part of regulatory domains fit comfortably in substrate binding site on the catalytic domain and block the active site.
  • cGMP binds to sites on the regulatory units of PKG and activates the catalytic units, enabling them to phosphorylate their substrates.
  • When PKG is activated the catalytic and regulatory units do not disassociate (Different from other Protein kinases).
  • After activation of PKG by cGMP, phosphorylates Ser and Thr residues in target proteins.
  • Activation of PKG by cGMP leads to activation of myosin phosphatase that cause conformational change in myosin.This in turn leads to relaxation of the smooth muscle cells.
  • PKG can also have other effects in cells. For example by activating a number of transcription factors which can lead to changes in gene expression which in turn can alter the response of the cell to a variety of stimuli.
Figure 9: cGMP mediated activation of protein kinase G and cellular effects mediated by PKG

2) Mediates biological responses:

  • In blood vessels: Relaxation of vascular smooth muscles leads to vasodilation and increased blood flow.
  • Relaxation of smooth muscle tissues.

3) It serves as the second messenger responsible for converting the visual signals received as light to nerve impulses in vertebrate eye.

  • Change in cGMP level in retinal rod cells is translated to a nerve impulse by a direct effect of cGMP on ion channels in the plasma membrane.
Figure 10: Visual Signal Transduction (The light-induced activation of rhodopsin leads to the hydrolysis of cGMP, which in turn leads to ion channel closing and the initiation of an action potential).

4) In kidney: Membrane bound guanylyl cyclase is activated by the hormone atrial natriuretic factor (ANF), which is released by cells in atrium of the heart when the heart is stretched by increased blood volume.

On binding to this receptor ANF causes relaxation of blood vessels via cGMP which increases the blood flow with decreasing blood pressure.

5) cGMP is a common regulator of ion channel conductance, glycogenolysis, and cellular apoptosis.

6) Signal transduction in olfactory cells

  • like cAMP, cGMP gets synthesized when olfactory receptors receive odorous input.
  • cGMP is a secondary messenger in phototransduction in the eye.
  • cGMP is produced slowly and has a more sustained life than cAMP (long term cellular responses to odor stimulation)
  • cGMP in the olfactory is synthesized by both membrane guanylyl cylcase (mGC) as well as soluble guanylyl cyclase (sGC).

D) Calcium ion

  • Calcium ion is a widely used second messenger.
  • The free concentration of calcium ions (Ca2+) within a cell is very low because ion pumps in the plasma membrane continuously use ATP to remove it.
  • For signaling purposes, Ca2+ is stored in cytoplasmic vesicles such as the endoplasmic reticulum or accessed from outside the cell.
  • When signaling occurs, ligand-gated calcium ion channels allow the higher levels of Ca2+ that are present outside the cell (or in intracellular storage compartments) to flow into the cytoplasm, which raises the concentration of cytoplasmic Ca2+.
  • The response to the increase in Ca2+ varies, depending on the cell type involved.

For example:

In the β-cells of the pancreas: Ca2+ signaling leads to the release of insulin

In muscle cells: Increase in Ca2+ concentration leads to muscle contractions.

  • Ca2+ is used in a multitude of processes: muscle contraction, release of neurotransmitter from nerve endings, vision in retina cells.
  • Cells use Ca2+ as a second messenger in both G protein pathways and tyrosine kinase pathways.
  • Various protein pumps transport Ca2+ outside the cell or into the endoplasmic reticulum or other organelles.
  • As a result, the concentration of Ca2+ in the ER is usually much higher than the concentration in the cytosol.

Ca2+ acts as a second messenger in two ways:

1)  It binds to an effector molecule such as an enzyme and activates them.

 2) It binds to an intermediary cytosolic calcium binding protein (eg: calmodulin). 

Calmodulin: small protein (17 kDa) that occurs in all animal cells.

  • The biochemical effects of Ca2+ in the cytoplasm are mediated by special Ca2+binding proteins (“calcium sensors”).These include the annexins, calmodulin, and troponin C in muscle.
  • Binding of four Ca2+ ions converts it into a regulatory protein.
  • The binding of Ca2+ causes profound conformational changes in calmodulin that increases affinity for its effector molecules.
  • Ca2+ – calmodulin interacts with effector proteins and modulates their properties.
  • The Ca2+ /calmodulin complex regulates the activity of many different proteins, including cAMP phosphodiesterase, nitric oxide synthase, and protein kinases or phosphatase that control the activity of various transcription factors.
  • Using this mechanism, Ca2+ ions regulate the activity of enzymes, ion pumps, and components of the cytoskeleton.
  • Calmodulin when activated causes contraction of smooth muscles.

Calcium ion flux:

  •  Calcium ions are probably the most widely used intracellular messengers.
  • It plays role in common signaling mechanism because once it enters the cytoplasm it exerts allosteric regulatory effects on many enzymes and proteins.
  • Calcium is a second messenger produced by indirect signal transduction pathways such as G protein coupled receptors.
  • Normally cytosolic calcium (Ca2+) ions is kept very low (10-7 M) by the action of Ca2+ pumps in the ER, mitochondria and plasma membrane.
  • Hormonal, neural, or other stimuli cause either an influx of Ca2+ into the cell through specific Ca2+ channels in the plasma membrane or the release of sequestered Ca2+ from the ER or mitochondria.
  • These events rise the cytosolic Ca2+ occurs and triggering a cellular response. This phenomenon is called Calcium ion flux.

E) Membrane derived lipids (IP3 and DAG)

  • Type Gq G proteins activate phospholipase C. This enzyme creates DAG and IP3 from the double phosphorylated membrane phospholipid phosphatidylinositol bisphosphate (PInsP2).
  • The products of the cleavage of PIP2 serve as second messengers.
Figure 11: Cleavage of PIP2 by phospholipase C yielding DAG and IP3

a) DAG (Diacylglycerol)

  • Diacylglycerol (DAG) remains in the plasma membrane due to hydrophobic and lipophillic nature.
  • Diacylglycerol stimulates protein kinase C activity by greatly increasing the affinity of the enzyme for calcium ions.
  • Protein kinase C phosphorylates specific serine and threonine residues in target proteins.

Known target proteins include: calmodulin, the glucose transporter, HMG-CoA reductase, cytochrome P450 etc.

        b) IP3

  • This soluble molecule diffuses through the cytosol and binds to receptors on the endoplasmic reticulum causing the release of calcium ions (Ca2+) into the cytosol. The rise in intracellular calcium triggers the response.
Figure 12: Structure of PIP2 cleavage products IP3 and DAG( both of them serve as second messengers).
Figure 13: G protein coupled receptors that activate phospholipase C via two second messengers DAG and IP3

Brief information on different types of second messengers

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