Second Messengers - cAMP
GPCR Activation and G-Protein Signalling:
GPCRs are 7-transmembrane domain receptors.
When an extracellular ligand (e.g. serotonin, adrenaline etc.) binds:
The receptor undergoes a conformational change.
This change activates a heterotrimeric G protein:
Inactive State: α bound to GDP, and βγ tightly associated.
Active State: α exchanges GDP for GTP - α-GTP active.
Both α-GTP and βγ interact with downstream effectors.
Gα Subunit Type determines the effect on downstream signalling:
Gαs stimulates adenylyl cyclase, increasing [cAMP].
Gαi inhibits adenylyl cyclase, decreasing [cAMP].
Other subunits (Gαq, Gα12/13 etc.) link to different pathways, such as phospholipase C, cytoskeletal changes etc..
❗GPCRs are not simple on/off switches! Different agonists can:
Induce different GPCR conformations.
Recruit different G proteins.
Lead to biased signalling, where specific downstream pathways are preferentially activated.
Production and Regulation of cAMP:
cAMP (Cyclic Adenosine Monophosphate):
Produced from ATP by adenylyl cyclase.
Levels increase rapidly in response to Gαs activation.
E.g. in neurones, Serotonin → GPCR → Gαs → AC → cAMP rise.
Degradation:
Phosphodiesterases (PDEs) hydrolyse cAMP to 5’-AMP.
This ensures transient signalling, preventing continuous activation.
Features of a Good Second Messenger:
Low basal levels in resting cells.
Rapid, transient upregulation, which allows repeated responses.
Fast termination which maintains temporal control.
cAMP in Different Cell Types (Functional Specificity):
Same cAMP signal means that there are different cellular responses because of differential expression of downstream effectors. For example:
Thyroid Gland - stimulates thyroid hormone synthesis.
Adrenal Cortex - stimulates cortisol secretion.
Ovary - stimulates progesterone secretion.
Muscle - activates glycogenolysis.
Differences arise because PKA phosphorylates distinct substrates in different cell types.
Protein Kinase A (PKA) - Structure and Activation:
PKA Structure:
Tetramer: 2 Regulatory Subunits (R) and 2 Catalytic Subunits (C).
Inactive: R subunits bind to C subunits, preventing phosphorylation activity.
Activation:
cAMP binds to the R subunit (2 cAMP per R subunit, so 4 total).
Conformational change releases active C subunits.
C subunits phosphorylate serine/threonine residues on target proteins.
Short-Term Effects:
Phosphorylation of cytosolic proteins, leading to rapid functional changes.
Long-Term Effects:
C-subunits translocate to the nucleus, then activation of gene transcription.
Spatial Regulation via AKAPs:
A-Kinase Anchoring Proteins (AKAPs):
Bind PKA regulatory subunits; localising PKA to subcellular compartments.
They bind to:
Organellar membranes.
Cytoskeletal elements (i.e. actin and microtubules).
Other signalling molecules (i.e. PDEs, receptors, etc.).
Functional Significance:
Localised signalling ensures rapid and precise response.
M-AKAP Example:
Anchors PKA to the nuclear envelope.
Anchors PDE, ensuring that cAMP is degraded after transient pulse.
Ensures short, tightly regulated PKA activation.
Prevents diffuse phosphorylation, increasing signalling specificity.
Short-Term (Cytosolic) vs Long-Term (Nuclear) cAMP Effects:
Short-Term:
Rapid cAMP rise, leading to PKA activation and phosphorylation of cytosolic proteins.
E.g. Muscle Cells - PKA phosphorylates glycogen phosphorylase kinase, leading to glycogen breakdown and increased glucose availability for energy production.
E.g. Olfactory Neurones - the odourant stimulates GPCRs to produce Gαolf, which stimulates adenylyl cyclase to produce more cAMP.
cAMP then opens CNG channels, leading to an influx of sodium and calcium ions.
Ca2+-gated Cl- channels lead to an efflux of Cl-.
This leads to a local depolarisation, followed by an action potential. This is a short-term signal.
Long-Term:
PKA catalytic subunits phosphorylate CREB (cAMP response element-binding protein).
The phosphorylation leads to a conformational change, allowing it to bind to CRE (cAMP response element) on DNA.
It recruits CBP (CREB-binding protein), a transcriptional co-activator. It induces transcription of multiple genes.
E.g. Olfactory Neurones - CREB leads to the transcription of neuropilin (adhesion molecules). This ensures axon targeting to the correct glomerulus.
Temporal Dynamics of cAMP Signalling:
cAMP signalling is pulsatile:
Resting: low basal cAMP.
Stimulus: rapid rise in cAMP, activating PKA.
PKA phosphorylates neighbouring PDE, which accelerates cAMP degradation.
Result: short pulse of PKA activity.
Role of Phospholipase C Beta (PLCβ) and Second Messenger Generation:
Activated phospholipase Cβ (PLCβ) is a key enzyme that hydrolyses a specific phospholipid embedded in the inner plasma membrane: PIP2 (phosphatidylinositol 4,5-bisphosphate).
The cleavage of PIP2 yields two crucial second messengers:
Diacylglycerol (DAG): This lipid-soluble molecule remains tethered within the plasma membrane, acting as a crucial activator for downstream proteins.
Inositol 1,4,5-trisphosphate (IP3): This water-soluble molecule rapidly diffuses away from the membrane into the cytosol, targeting intracellular calcium stores.
Action of IP3 and DAG in Cellular Signalling:
IP3 Function:
As IP3 diffuses into the cytosol, it specifically binds to and opens IP3-gated calcium release channels located on the membrane of the endoplasmic reticulum (ER) (or sarcoplasmic reticulum (SR) in muscle cells).
This binding event triggers a rapid and significant release of stored calcium ions from the ER/SR lumen directly into the cytosol, dramatically increasing cytosolic Ca2+ concentrations.
DAG Function:
Diacylglycerol (DAG), found at the plasma membrane, plays a crucial role in activating Protein Kinase C (PKC), an enzyme central to many cellular responses by phosphorylating a variety of target proteins.
Calcium (Ca2+) as a Versatile Second Messenger:
Calcium ions (Ca2+) are tightly regulated in cells, with significantly higher concentrations stored within the endoplasmic/sarcoplasmic reticulum lumen compared to the relatively low basal levels in the cytosol.
The IP3-mediated release of Ca2+ into the cytosol is a rapid and potent signal.
This influx of cytosolic calcium then acts as a secondary messenger, influencing a vast array of cellular functions, from muscle contraction and neurotransmitter release to gene expression and cell division.
Activation of Protein Kinase C (PKC):
For Protein Kinase C (PKC) to activate, these components are needed:
Elevated cytosolic calcium ions (Ca2+), which often contribute to PKC's translocation to the membrane.
Diacylglycerol (DAG), which binds to a regulatory domain on PKC, promoting its association with the plasma membrane.
Phosphatidylserine: a negatively charged phospholipid, present in the inner leaflet of the plasma membrane. It binds to PKC, helping to stabilise its active conformation at the membrane surface.
Once activated, PKC phosphorylates specific serine and threonine residues on various target proteins. This phosphorylation event modifies the activity, localisation, or interaction of these substrate proteins, leading to diverse cellular responses.
Diverse Cellular Responses Initiated by PKC Activation:
The activation of PKC leads to a wide range of specific effects:
Liver: PKC activation promotes glycogenolysis, releasing glucose into the bloodstream as part of metabolic regulation.
Pancreas: In pancreatic acinar cells, PKC stimulates amylase secretion, aiding in digestion.
Smooth Muscle: In smooth muscle cells, it can induce muscle contraction by influencing pathways involved in actin-myosin interactions.
Blood Platelets: PKC activation is a key step in platelet aggregation, a crucial process for blood clotting and wound healing.
Functions of Calcium in Cells:
Calcium ions exert diverse effects across various cell types, influencing both rapid and sustained cellular responses.
Rapid Effects (milliseconds): Include neurotransmitter release via exocytosis in nerve cells, muscle contraction, and hormone secretion.
Delayed Effects (hours to days): Involve modulation of gene transcription, leading to long-term changes in cell behaviour, differentiation, and proliferation.
Calcium Signalling Toolkit:
The "calcium signalling toolkit" refers to the conserved set of molecular components that govern calcium dynamics within a cell:
Calcium Channels: Transmembrane proteins that facilitate the influx of Ca2+ into the cytosol from the extracellular space or intracellular stores (e.g., ER/SR), thereby increasing cytosolic calcium concentration. They are separated into:
Voltage-Operated Channels (VOCs): respond to changes in membrane potential (e.g. L-type, N-type and T-type channels).
Receptor-Operated Channels (ROCs): activated directly by extracellular ligand binding (e.g. NMDA receptors).
Second Messenger-Operated Channels (SMOCs): Triggered by intracellular secondary messengers like IP3.
Pumps and Exchangers: Active transporters responsible for extruding Ca2+ from the cytosol (e.g., plasma membrane Ca2+ ATPases) or pumping it back into intracellular stores (e.g., sarcoplasmic/endoplasmic reticulum Ca2+ATPases), thus reducing cytosolic calcium concentration.
Buffers: Proteins or small molecules that bind reversibly to Ca2+ ions, sequestering them to prevent inappropriate interactions with calcium sensors and to regulate Ca2+ concentration.
Calcium Sensors: Proteins that detect changes in cytosolic Ca2+ levels, undergo conformational changes upon binding Ca2+, and transduce these signals to downstream effectors, thereby modifying cellular behaviour. They are separated into:
C2 Domain Family - interacts with cell membranes in a Ca2+-dependent manner (e.g. Protein Kinase C).
EF-Hand Family - calcium sensors that feature a conserved helix-loop-helix motif that directly binds Ca2+ ions. This binding induces conformational changes, allowing them to transduce signals.
Mechanisms of Calcium Signalling:
Low Basal Calcium Levels: Resting cells maintain low cytosolic free calcium concentrations (around 100nM), which ensures a steep electrochemical gradient is available for Ca2+ influx.
Calcium Store: The endoplasmic reticulum/sarcoplasmic reticulum serve as the primary intracellular reservoir, storing high concentrations of Ca2+ (millimolar range) that can be rapidly released upon specific stimuli.
Signal Induction: External signals, such as hormones, neurotransmitters, or growth factors, activate specific receptors.
This activation can lead to the opening of calcium channels on the plasma membrane or the ER/SR, causing a rapid and substantial increase in cytosolic calcium levels, often by 10-20 fold (to 500 nM - a few μM).
Channel Types and Responses:
The diversity of extracellular stimuli necessitates various receptor-channel coupling mechanisms for calcium influx:
Voltage-operated Channels (VOCs): found in excitable cells (e.g., neurons, muscle cells), these channels are sensitive to changes in membrane potential.
Examples include L-type, N-type, and T-type Ca2+ channels.
Receptor-operated Channels (ROCs): Activated directly by the binding of specific extracellular ligands (e.g., neurotransmitter-gated ion channels like NMDA receptors, which are also voltage-sensitive).
Second Messenger-operated Channels (SMOCs): Respond to intracellular secondary messengers (e.g., IP3, cyclic nucleotides), which are generated in response to receptor activation.
IP3 Signalling Pathway:
Activation of G protein-coupled receptors (GPCRs) often leads to the activation of Phospholipase C-beta (PLCβ).
PLCβ cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3).
IP3, acting as a second messenger, binds to specific IP3-gated Ca2+ channels (IP3Rs) located on the ER/SR membrane.
This binding triggers the efflux of stored Ca2+ from the ER/SR into the cytosol.
Ryanodine Receptors (RyRs): a crucial class of Ca2+ release channels on the ER/SR.
RyRs are particularly sensitive to elevated cytosolic calcium, leading to Calcium-Induced Calcium Release (CICR), which further amplifies the initial calcium signal in a positive feedback loop.
Feedback Mechanisms:
Positive Feedback Loop:
The initial IP3-induced Ca2+ release can stimulate Ryanodine Receptors (RyRs) via CICR, leading to further Ca2+ release. This amplification can generate regenerative calcium waves.
Negative Feedback Loop:
As cytosolic Ca2+ levels rise, they can also paradoxically inhibit both IP3Rs and RyRs at higher concentrations, providing a mechanism to eventually halt or limit further release and contribute to the oscillating nature of calcium signals.
❗This dynamic interplay of activation and inhibition often generates intricate calcium waves or oscillations, which encode information that dictates how cells respond to continuous signalling.
Calcium Ion Regulation:
The calcium signalling toolkit efficiently restores basal calcium levels after a signal, critical for maintaining cellular excitability and responsiveness:
Pumps and Exchangers on Plasma Membrane:
Plasma Membrane Calcium ATPase (PMCA): An ATP-dependent pump that actively extrudes Ca2+ from the cytosol to the extracellular space.
Sodium-Calcium Exchanger (NCX): Utilizes the electrochemical gradient of sodium ions (3 Na+ in for 1 Ca2+ out) to remove Ca2+ from the cytosol. This is an electrogenic exchanger.
SerCA (Sarco/Endoplasmic Reticulum Calcium ATPase): An ATP-dependent pump that actively transports Ca2+ from the cytosol back into the ER/SR lumen.
Mitochondrial Role: Mitochondria can act as temporary, lower-affinity, but high-capacity calcium buffers, particularly in microdomains of high Ca2+ concentration near open channels.
They take up Ca2+ via the mitochondrial calcium uniporter (MCU) and release it via other carriers, aiding in swift restoration of cytosolic calcium levels and regulating mitochondrial function.
Calcium Buffers and Sensors:
Buffers:
These ubiquitous proteins and small molecules bind to Ca2+ ions, moderating their diffusion and interaction with downstream effectors.
Calcium Sensors:
These are the proteins that translate the Ca2+ signal into specific cellular responses.
Two primary structural families of calcium sensors are recognized:
C2 Domain Family: domains that typically interact with cell membranes in a Ca2+-dependent manner, influencing protein localisation or enzymatic activity (e.g., found in protein kinase C).
EF-Hand Family: characterised by a conserved helix-loop-helix motif that directly binds Ca2+ ions. Binding of Ca2+ to the loop induces conformational changes in the helices.
Major Calcium Sensor: Calmodulin (CaM):
A ubiquitous and highly conserved EF-hand protein, containing four EF-hand motifs, each capable of binding one Ca2+ ion.
Upon binding four Ca2+ ions, Calmodulin undergoes a significant conformational change, exposing hydrophobic surfaces.
This activated Calmodulin then binds to and activates various cellular targets, including protein kinases (e.g., CaM kinases), phosphatases (e.g., calcineurin), and other enzymes or ion channels.