What are second messengers?
Molecules that relay signals received at receptors on the cell surface to target molecules in the cytosol and/or nucleus. In addition, second messengers serve to greatly amplify the strength of the signal
Major classes of second messengers
- cyclic nucleotides (e.g. cAMP, cGMP)
- inositol triphosphate (IP3) and diacylglycerol (DAG)
- calcium ions (Ca2+)
- arachidonic acid (AA)
Cyclic nucleotides
- cAMP and cGMP: derivatives of ATP and GTP respectively
- diffuse freely in cytoplasm, act by binding reversibly to specific target proteins
- cAMP to protein kinase A
- cGMP to protein kinase G
- inactivated by phosphodiesterases (break cyclic nucleotides to 5’monophosphates)
Adenylyl cyclase
- 12 membrane spanning segments
- at least 8 isoforms known
- 2 catalytic domains
- calmodulin binding site is regulated by Ca2+
- 1 G protein activates 1 cyclase
- 1 cyclase produces 100-1000 cAMP
Actions of cAMP
- activates Proetin Kinase A (phosphorylates ion channels, and enzymes) - rapid action
- activates cyclic nucleotide gated channels (CNGC, change membrane potential) - rapid action
- activates transcription factors and changes gene expression - slow action
Signal transduction - cAMP-PKA
E.g. flight or fight response
Reception:
- Binding of adrenaline to beta receptor
Transduction:
- Inactive G protein is activated (G alpha s subunit dissociates)
- Inactive adenylyl cyclase becomes active adenylyl cyclase (one G protein activates one cyclase)
- ATP is converted to cAMP (one cyclase produces 100-1000 cAMP)
- Inactive phosphorylase kinase becomes activate phosphorylase kinase (increase by factor of 10)
- Inactive glycogen phosphoylase becomes active glycogen phosphorylase (increase by factor of 10)
Response:
Glycogenolysis: Glycogen produces glucose-1-phosphate (increase x100) which is converted to glucose-6-phosphate
Signal transduction - cAMP-PKA
general
- G alpha s subunit
- increased adenylyl cyclase
- increased cAMP
- increased protein kinase A
- increased protein phosphorylation
- G alpha i subunit
- reduced adenylyl cyclase
- reduced cAMP
- reduced protein kinase A
- decreased protein phorphorylation
Signal transduction - IP3/DAG
- G alpha q subunit
- production of phospholipase C
- IP3 and DAG production
- IP3 causes Ca2+ release, DAG causes protein kinase C production and AA production
- AA converted to eicosanoids (released as hormones)
- Ca2+ released and protein kinase C cause increased protein phosphorylation, activate calcium-binding proteins
cAMP-PKA and cardiac muscle contraction
- ATP converted to cAMP, activating PKA causing Ca2+ influx, enters SR, causing increased Ca2+ efflux from SR leading to contraction
cAMP-PKA and smooth muscle relaxation
Normal pathway:
- Ca2+ influx which combines with Ca2+ released from ER to form calmodulin (Ca2+)4 radical
- leads to myosin light chain kinase (MLCK)
- when inactive smooth muscle relaxation occurs
- when activate, myosin-(PO4)2 is produced leading to smooth muscle contraction
- when PKA is activated myosin light chain kinase signalling is inhibited, meaning the MLCK is not active, leading to smooth muscle relaxation
cAMP-inducible gene expression - CREB
- cyclic AMP response element binding protein (CREB)
- CREB is a transcription factor which is activated by cAMP-dependent PKA
- CREB binds to certain DNA sequences called cAMP response elements (CRE)
- Genes with either (TGACGTCA) or (CGTCA) CRE motif in the promotor are activated by CREBS
- PKA consists of paired regulatory (R) and catalytic (C) subunits
- cAMP binds to the R subunits, leading to release of C subunits
- liberated C subunits migrate into the nuclear compartment by passive diffusion
- C subunits phosphorylate CREB
- Phosphorylated CREB promotes the recruitment of the co-activator CREB-binding protein, which in turn mediates transcriptional activation through association with RNA polymerase to regulate mRNA expression
- CREB is activated in response to a great array of physiological stimuli and is associated with numerous cellular functions
Guanylate cyclase and cGMP
- nitric oxide
- cGMP activates protein kinase G
- -> myosin phosphatase –> smooth muscle relaxation
- -> VASP –> platelet inhibition
- -> transcription factors –> changes in gene transcription
IP3 and DAG
- G-alpha-q protein couples to phospholipase C (PLC)
- PLC is an enzyme that hydrolyses phosphatidylinositol 4,5-biphosphate (PIP2) to form inositol triphosphate (IP3 and diacylglycerol (DAG)
- DAG activates protein kinase C
- IP3 signals the release of Ca2+ ions from the ER stores
PIP2
- PIP2 is a minor component of cell membrane, localised to the inner phospholipid layer
- cleavage of PIP2 by phospholipases produce various active mediators
- cleavage by PLA2 yields arachidonic acid
- cleavage by PLC yields IP3 and DAG
- cleavage by PLD produced phosphatidic acid (PA)
IP3-Ca2+ and smooth muscle contraction
- G alpha q
- IP3 produced
- IP3 receptor activated, releasing Ca2+ from ER stores
- Ca2+ combine with Ca2+ ions that have influxed from extracellular stores to produce calmodulin radicals (Ca2+)4 which activated myosin light chain kinase
- causes myosin-(PO4)2 to be released
- leads to filament sliding muscle contraction
Targets of IP3
Direct effects
- cell motility
- contraction of muscle cells
- secretion
- activation of regulatory enzymes
- Ca2+ activated K+ channels
Effects through calmodulin
- activation of phosphorylase kinase
- activation of cAMP phosphodiesterase
CaM kinase II: Calcium/calmodulin-dependent protein kinase II is a serine/threonine-specific protein kinase
Calcium as second messenger
- Ca2+ very important in regulating cellular and physiological responses
- extracellular concentrations are 2mM (e.g. blood) and levels in cytoplasmic vesicles and the ER can reach up to 10mM
- Baseline cytosolic Ca2+ conc. is around 100nM in resting cells
- high gradient makes this a very fast and sensitive signalling system - only sligh changes in membrane permeability will result in dramatic changes in the concentration of [Ca2+]
Sources of calcium - intracellular
Intracellular compartment (calcium is released from intracellular apparatus into the cytoplasm)
- Ca2+ is stored in mM concs. in ER and SR
- two types of calcium channels (IP3 receptors and ryanodine receptors) on the ER and SR membrane control the release of Ca2+ from these stores
Intracellular compartment: calcium release mechanisms
- IP3R - IP3 produced through receptor activated PLC pathway. IP3 diffuses through cytoplasm and binds IP3R. IP3R is a ligand-gated ion channel. No specific blocking agents for IP3R (heparin can be used by intracellular injection)
- Ryanodine receptors (RyR) - named after sensitivity to ryanodine. Very important in skeletal muscle - mediated Ca2+-mediated Ca2+ release. Also activated by cyclic ADP ribose. Activated by caffeine, blocked by ryanodine.
Sources of calcium - extracellular
- voltage gated calcium channels
- ligand gated calcium channels
- store operated calcium channels
- sodium-calcium exchanger
eicosanoids
- eicosanoids are a class of lipids including prostaglandins, prostacyclins, thromboxanes and leukotrienes, that acts in autocrine and pararcrine signalling
- prostaglandins and leukotrienes are important inflammatory mediators
PLC/PLA2 converts diacylglycerol or phospholipid to arachidonic acid (AA)?
second messengers as drug targets
Erectile dysfunction
- glyceryl trinitrate is a NO donor, used in treatment of angina and ischaemic heart disease
- phosphodiesterase type 5 (PDE5) is found predominately in the corpus cavernosum
- PDE5 inhibitors (sildenafil, vardenafil, tadalafil) - first line drugs for males with erectile disfunction (potential for pulmonary hypertension)
calcium channel blockers
- main clinical use to treat hypertension
- some can also be used as anti-anginals
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Pharmacogenetics0
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Transcription Factors/Nuclear Receptors0
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Enzyme Modulation0
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Voltage Gated Ion Channels0
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Ligand Gated Ion Channels0
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Catalytic Receptors21
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GPCR Families17
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GPCR Structural Motifs17
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Second Messengers21
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G-Proteins26
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G-Protein Beta-Gamma Subunits20
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Regulation of GPCR Signalling26
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Receptor Internalisation and Alternative Signalling Pathways22
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Allosteric Modulators23
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Constitutively Active Receptors and Inverse Agonists19
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Advanced Pharmacodynamics0
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Signalling Bias0
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Receptor Theory I0
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Receptor Theory II0
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Orphan Receptors0
