TB

Cell Signaling & Secondary-Messenger Pathways

Calcium Control & Secondary Messengers

Fine Control of Calcium

  • The body maintains precise control over intracellular calcium levels, keeping cytosolic concentrations very low ( \approx 10^{-7} M) compared to extracellular levels (\approx 10^{-3} M).

  • Essential for numerous cellular processes, including

    • muscle contraction,

    • nerve impulse transmission,

    • hormone secretion,

    • enzyme activation,

    • gene expression.

  • More calcium is rapidly released into the cytosol from intracellular stores (like the endoplasmic reticulum) or from the extracellular space when needed (e.g., during excitation-contraction coupling in muscle cells).

Phospholipase C Pathway

  • When a first messenger (e.g., a hormone or neurotransmitter) binds to its G protein-coupled receptor (GPCR) on the cell membrane, it activates a specific G protein, typically the Gq subunit.

  • The activated Gq protein then stimulates the enzyme phospholipase C (PLC), specifically PLC-beta isoform.

  • Phospholipase C cleaves the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) into two key secondary messengers: inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG).

IP3

  • IP3 is a water-soluble molecule that diffuses rapidly into the cytosol.

  • It binds to and directly opens specific ligand-gated calcium ion channels, known as IP3 receptors (IP_3R), located on the membrane of the endoplasmic reticulum (ER) and sarcoplasmic reticulum (SR) in muscle cells.

  • This binding causes a rapid efflux of calcium ions from the ER/SR lumen into the cytosol, significantly increasing cytosolic calcium concentration.

DAG

  • DAG is a lipid-soluble molecule that remains embedded within the plasma membrane.

  • Along with calcium ions, DAG activates protein kinase C (PKC), a serine/threonine kinase.

Protein Kinase C (PKC)

  • PKC is a family of enzymes activated by the combined presence of DAG and an increase in cytosolic calcium ions.

  • Once activated, PKC translocates from the cytosol to the plasma membrane and phosphorylates various target proteins, altering their activity and leading to diverse cellular responses such as cell growth, differentiation, and secretion.

PPI vs. Protein Kinase C

  • PKA (Protein Kinase A): Associated with the cyclic AMP (cAMP) pathway, activated by cAMP.

  • PKC (Protein Kinase C): Associated with the IP3/DAG pathway, activated by DAG and Ca^{2+}.

  • Both are types of protein kinases that phosphorylate downstream proteins, leading to diverse cellular responses, but they are activated by different secondary messengers derived from distinct G protein-mediated pathways.

Calcium as a Secondary Messenger

  • Calcium (Ca^{2+}) is a versatile secondary messenger whose precise spatial and temporal concentration changes in the cytosol can trigger distinct cellular responses.

  • Increased cytosolic calcium can directly affect the activity of various signaling proteins by binding to them, often inducing conformational changes.

  • Calcium influx or release can alter the cell's membrane potential, typically by opening or closing ion channels. The resting membrane potential of cells is generally negative inside and positive outside ( \approx -70mV in many cells), and changes in calcium flow can depolarize or hyperpolarize the membrane.

Calcium's Effects

  • Calcium can directly target specific ion channels (e.g., Ca^{2+}-gated K^+ channels) which can lead to further changes in membrane potential or the release of more calcium (calcium-induced calcium release).

  • It can activate protein kinase C, either indirectly through the DAG-PKC pathway or directly by binding to PKC, which regulates its activity.

  • The existence of multiple pathways activated by G proteins (e.g., adenyl cyclase for cAMP and phospholipase C for IP3/DAG) or by different calcium-binding proteins provides chemical specificity and allows for diverse and finely tuned cellular responses from a single first messenger, or distinct responses from different first messengers.

Calcium Channel Activation

  • Plasma membrane calcium channels can open directly when the first messenger binds to a ligand-gated receptor that also functions as an ion channel.

  • Alternatively, receptors may activate a G protein, which then, via secondary messengers like IP3, can indirectly open calcium channels located on intracellular stores (ER/SR) or sometimes on the plasma membrane.

IP3 and Endoplasmic Reticulum

  • IP3 serves as the crucial signal that triggers calcium release from the endoplasmic reticulum, the primary intracellular calcium store.

Active Calcium Transport

  • Following calcium release, active calcium transport pumps (e.g., SERCA pumps on the ER membrane, and plasma membrane Ca^{2+}-ATPases) work to pump calcium back into the ER or out of the cell, respectively.

  • This re-uptake of calcium is essential for terminating the calcium signal and refilling intracellular stores. Sometimes, these pumps can be inhibited by secondary messengers or their downstream effects to selectively retain calcium within the cell for prolonged signaling or specific processes.

Calmodulin

  • Calmodulin (CaM) is a ubiquitous calcium-binding 'sensor' protein in eukaryotic cells. It has four EF-hand motifs, each capable of binding a calcium ion.

  • When calcium binds to calmodulin, it undergoes a significant conformational change, exposing hydrophobic regions.

  • This altered calmodulin then acts as a regulatory protein that can bind to and activate or inhibit a wide array of target enzymes and proteins, including various protein kinases (e.g., CaMKs), phosphodiesterases, and ion channels.

  • Calcium also affects other calcium-binding proteins, such as troponin in muscle contraction

  • Fine control over intracellular calcium concentration is crucial to prevent unwanted and potentially damaging reactions like excitotoxicity or apoptosis.

Calcium-Calmodulin-Dependent Protein Kinase System

  • This system typically functions downstream of the G-coupled receptor response that leads to increased cytosolic calcium.

  • Increased calcium concentration activates calmodulin by binding to it.

  • Activated calmodulin then binds to and activates a family of serine/threonine protein kinases known as calmodulin-dependent protein kinases (CaMKs), such as CaMKII.

  • These protein kinases utilize ATP as an energy source to phosphorylate other proteins, thereby propagating and furthering the cellular response in a calcium-dependent manner.

Importance of Calcium Release

  • Rapid and controlled calcium release is fundamentally necessary for triggering essential physiological processes such as muscle contraction (where Ca^{2+} binds to troponin C), stimulus-secretion coupling (e.g., neurotransmitter release at synapses, insulin secretion from pancreatic beta cells), and initiation of nerve action potentials in excitable cells.

  • The IP3 pathway is a particularly key and rapid pathway in initiating this crucial calcium release from intracellular stores.

Key Secondary Messengers

  • Adenylyl cyclase activity producing cyclic AMP (cAMP) and the downstream activation of protein kinase A (PKA) represents a major G-coupled response pathway.

  • Similarly, guanylyl cyclase producing cyclic GMP (cGMP) and activating protein kinase G (PKG) is another important pathway.

  • These secondary messengers cannot cross the plasma membrane themselves due to their hydrophilic nature, thus necessitating an intracellular cascade effect to amplify and propagate the signal from the cell surface to the interior.

Cyclic AMP Pathway

  • The binding of a first messenger (e.g., epinephrine, glucagon) to its GPCR activates a Gs protein, which then stimulates adenylyl cyclase.

  • Adenylyl cyclase catalyzes the conversion of ATP to cyclic AMP (cAMP).

  • Cyclic AMP then primarily activates protein kinase A (PKA) by binding to its regulatory subunits, releasing and activating its catalytic subunits.

  • PKA, in turn, phosphorylates a diverse range of target proteins (enzymes, ion channels, transcription factors), leading to downstream responses such as glycogenolysis in liver cells, lipolysis in adipose tissue, and altered heart rate and contractility (e.g., in fight-or-flight responses).

IP3 Pathway

  • As previously detailed, phospholipase C, activated by Gq, splits PIP2 into IP3 and DAG.

  • IP3 immediately triggers the release of calcium from the endoplasmic reticulum.

  • DAG, in conjunction with calcium, activates protein kinase C, which phosphorylates its target proteins. The activation of PKC can also be influenced indirectly by calcium via its role in DAG-mediated translocation and activity modulation.

Secondary Messengers Summary

  • Calcium: Increase in cytosolic Ca^{2+} through plasma membrane channels or release from ER (via IP3 or ryanodine receptors). It activates protein kinase C (indirectly with DAG), calmodulin, and other Ca^{2+}-binding proteins (e.g., troponin, synaptotagmin).

  • Cyclic AMP (cAMP) and Cyclic GMP (cGMP): A G protein activates adenylyl cyclase (for cAMP synthesis from ATP) or guanylyl cyclase (for cGMP synthesis from GTP). These cyclic nucleotides then activate protein kinase A or G, respectively, initiating phosphorylation cascades.

  • DAG and IP3: Phospholipase C catalyzes the breakdown of the membrane lipid PIP2. DAG (diacylglycerol) remains in the membrane and directly activates protein kinase C (in the presence of Ca^{2+}). IP3 (inositol trisphosphate) diffuses into the cytosol and triggers the rapid release of Ca^{2+} from intracellular stores (ER), which can then further activate protein kinase C or calmodulin.

Eicosanoids

  • Eicosanoids are a family of lipid molecules derived from arachidonic acid, a 20-carbon polyunsaturated fatty acid (e.g., C_{20:4}).

  • They act as short-range signaling molecules (paracrine or autocrine mediators) in various tissues.

  • Examples include cyclic endoperoxides, prostaglandins, thromboxanes, and leukotrienes.

  • They are of significant importance in immune research, inflammation, pain, fever, and blood coagulation.

Reactions

  • Eicosanoid synthesis begins with the activation of membrane-bound phospholipase A2 (PLA2) by a first messenger binding to its receptor.

  • PLA2 cleaves arachidonic acid from membrane phospholipids.

  • Arachidonic acid then enters different enzymatic pathways:

    • Cyclooxygenase (COX) pathway: Leads to the production of cyclic endoperoxides (like PGH_2), which are then converted into prostaglandins and thromboxanes.

      • Prostaglandins: Diverse roles, including aiding in local vascular action (vasodilation/constriction), mediating inflammation, pain, fever, and decreasing gastric acid secretion. Anti-inflammatory drugs like NSAIDs inhibit COX enzymes.

      • Thromboxanes: Primarily important for blood clotting (platelet aggregation) and vasoconstriction.

    • Lipoxygenase (LOX) pathway: Leads to the synthesis of leukotrienes.

      • Leukotrienes: Potent mediators of allergic and inflammatory reactions, particularly in the respiratory tract (e.g., bronchoconstriction in asthma).

Stopping the Responses

  • The duration of the cellular response is tightly regulated.

  • Messenger binding to the receptor is typically brief and reversible; the response subsides rapidly when the first messenger is removed or its concentration decreases.

  • Intracellular concentrations of secondary messengers are quickly decreased by specific enzymes, re-uptake pumps, or sequestration mechanisms to prevent chronic overstimulation and ensure the cell can respond to new signals.

IP3 Pathway Shutdown

  • Decrease in IP3: IP3 is quickly dephosphorylated by IP3 phosphatases or phosphorylated by IP3 kinases, converting it into inactive forms.

  • Decrease in DAG: DAG is either phosphorylated by DAG kinase to form phosphatidic acid or metabolized by DAG lipase, preventing active protein kinase C signaling.

  • Decreased activated protein kinase C: As DAG and Ca^{2+} levels fall, PKC dissociates from the membrane and becomes inactive, or is degraded.

  • Decreased calcium: Cytosolic Ca^{2+} is rapidly pumped back into the ER/SR by SERCA pumps (Ca^{2+}-ATPases) or expelled from the cell by plasma membrane Ca^{2+}-ATPases and Na^+/Ca^{2+} exchangers, preventing sustained calmodulin activation and subsequent protein kinase activation.

  • Ultimately, the splitting of ATP by kinases stops as the activating signals (secondary messengers) are removed, turning off the cellular response.

Analogy (LEGO Set)

  • Continuous delivery of LEGO pieces = First messenger continuously binding to the receptor.

  • Building intermediate steps (e.g., assembling sub-sections of the LEGO model) = Secondary messengers being produced and activating downstream components.

  • Finishing the LEGO set (the completed model) = The final cellular response.

  • No more LEGO pieces = No more first messenger, leading to the rapid disassembly of intermediate steps and termination of the building process (response).

More Reaction Needs

  • New first messengers must bind to receptors to initiate a fresh cycle of reaction.

  • The cell can increase the affinity of the first messenger binding to its receptor to prolong the signal, or increase the number of receptors on the surface.

  • The cascade effect, where one activated component can activate many downstream components (e.g., one receptor activates many G proteins, one adenylyl cyclase produces many cAMP molecules, one PKA phosphorylates many targets), helps amplify the initial signal, allowing a small amount of first messenger to elicit a robust cellular response.

Interactions Between Pathways (Crosstalk)

  • Cellular signaling pathways rarely operate in isolation; they exhibit extensive crosstalk, meaning they can influence each other.

  • A single first messenger can indeed trigger changes in multiple distinct pathways (e.g., through different G protein subtypes or by activating different receptor types).

  • Conversely, different first messengers can stimulate different influences on the same pathway or activate entirely distinct pathways, leading to integrated cellular outcomes.

  • Secondary messengers from different pathways can synergize, antagonize, or modify each other's effects, allowing for highly complex and specific regulation of cellular processes.

IP3 and Cyclic AMP Combination

  • In some cells, a single receptor, upon binding a first messenger, can activate different G protein complexes (e.g., Gs and Gq or G_i), leading to diverse and sometimes opposing outcomes.

  • For example, one G protein complex might activate the IP3 pathway (leading to calcium release), while another G protein complex might simultaneously inhibit the cyclic AMP pathway (via G_i). This allows for a refined balance of cellular activity.

  • This multi-faceted activation from a single receptor highlights the intricate 'personalities' or specific functional outcomes that G proteins can mediate.

  • Hundreds of thousands of receptors can be active simultaneously in a single cell membrane, integrating vast amounts of information.

  • Fine Control: This extensive crosstalk and parallel activation allow for sophisticated, fine control over cellular reactions, ensuring chemical specificity and enabling simultaneous and coordinated cellular responses to complex extracellular cues.

Nervous System Reactions

  • Cell signaling mechanisms are fundamental to the nervous system. A signal generated within one neuron often needs to travel and be relayed to stimulate the next cell (another neuron, muscle cell, or gland cell) across a synapse.

  • The nervous system utilizes both rapid ion channel-mediated reactions (ligand-gated and voltage-gated channels) and slower, but often more sustained, G protein-coupled receptor reactions (neuromodulation).

Ligand vs. Voltage Gated Ion Channels

  • Ligand-gated ion channels: These transmembrane proteins open or close their ion pores in direct response to the binding of a specific extracellular signaling molecule (a ligand, e.g., a neurotransmitter like acetylcholine or GABA). The ligand acts as the 'first messenger' directly causing the channel to open, allowing ions to flow across the membrane and change the membrane potential.

  • Voltage-gated ion channels: These channels differ significantly in their activation mechanism. Instead of ligand binding, their opening and closing are regulated by changes in the electrical potential (voltage) across the cell membrane. They possess charged amino acid segments (voltage sensors) that detect changes in the membrane potential.

  • The inside of most resting cells is typically negatively charged relative to the outside ( \approx -70mV resting potential). When a change in membrane potential (e.g., from a ligand-gated channel opening) reaches a certain threshold, it 'flips' or alters the conformation of the voltage sensors, causing the voltage-gated channel to open rapidly. This is crucial for the propagation of action potentials along neurons and muscle cells.