15.3 Signal transduction pathways and second messengers

Signal transduction pathways are cellular communication systems that convert external signals into internal responses. These pathways use receptors, second messengers, and protein kinases to relay and amplify information, allowing cells to adapt to their environment and coordinate activities like metabolism, growth, and gene expression.

Receptor Types

G Protein-Coupled Receptors (GPCRs)

GPCRs are the largest family of cell surface receptors. They have seven transmembrane-spanning alpha helices, an extracellular ligand-binding domain, and an intracellular domain that interacts with heterotrimeric G proteins.

Here's how GPCR signaling works:

1. A ligand (hormone, neurotransmitter, etc.) binds the extracellular domain.

2. Ligand binding induces a conformational change in the receptor, exposing a binding site on the intracellular face.

3. The receptor acts as a guanine nucleotide exchange factor (GEF), catalyzing the exchange of GDP for GTP on the G$\alpha$ subunit of the associated G protein.

4. The GTP-bound G$\alpha$ subunit dissociates from the G$\beta\gamma$ dimer, and both can now activate downstream effectors.

5. Depending on the G protein subtype, different effectors are engaged:

   • G$_s$ stimulates adenylyl cyclase → increases cAMP
   • G$_i$ inhibits adenylyl cyclase → decreases cAMP
   • G$_q$ activates phospholipase C (PLC) → generates IP$_3$ and DAG

6. The signal terminates when the intrinsic GTPase activity of G$\alpha$ hydrolyzes GTP back to GDP, causing reassociation with G$\beta\gamma$.

GPCRs respond to a wide variety of stimuli including hormones (epinephrine, glucagon), neurotransmitters (dopamine, serotonin), and sensory signals (light via rhodopsin, odorant molecules).

Receptor Tyrosine Kinases (RTKs)

RTKs are cell surface receptors with an extracellular ligand-binding domain, a single transmembrane helix, and an intracellular domain that possesses intrinsic tyrosine kinase activity.

Activation follows a distinct sequence:

1. Ligand binding induces receptor dimerization, bringing two intracellular kinase domains into close proximity.
2. The kinase domains trans-autophosphorylate each other on specific tyrosine residues.
3. These phosphotyrosine residues serve as docking sites for downstream signaling proteins that contain SH2 domains (Src homology 2) or PTB domains (phosphotyrosine-binding).
4. Recruitment of adaptor and effector proteins to the activated receptor initiates branching signaling cascades, most notably:
   • The Ras → MAPK pathway (cell proliferation and differentiation)
   • The PI3K → Akt pathway (cell survival and metabolic regulation)

RTKs respond to growth factors such as insulin, epidermal growth factor (EGF), and platelet-derived growth factor (PDGF). Mutations that constitutively activate RTKs are commonly found in cancers, which is why these receptors are major drug targets.

Nuclear Receptors

Unlike GPCRs and RTKs, nuclear receptors are intracellular receptors that function as ligand-activated transcription factors. Their ligands are lipophilic molecules that can cross the plasma membrane on their own.

Nuclear receptors contain three key domains:

• A ligand-binding domain (LBD)
• A DNA-binding domain (DBD) with zinc finger motifs
• One or more transcriptional activation domains

Without ligand, many nuclear receptors are bound to corepressor complexes (often including histone deacetylases) that keep target genes silenced. When a lipophilic ligand binds, the receptor undergoes a conformational change, releases corepressors, and recruits coactivator proteins that promote transcription.

Examples include receptors for steroid hormones (glucocorticoids, estrogen), thyroid hormones, retinoic acid, and vitamin D. Because they directly regulate transcription, nuclear receptor responses are typically slower (hours) than GPCR or RTK responses (seconds to minutes), but they produce longer-lasting changes in cell behavior.

Second Messengers

Second messengers are small, rapidly produced intracellular molecules that amplify the signal initiated at the receptor. A single activated receptor can trigger the production of thousands of second messenger molecules, which is a major source of signal amplification.

Cyclic Nucleotides (cAMP and cGMP)

Cyclic AMP (cAMP) is synthesized from ATP by adenylyl cyclase, which is activated downstream of G$_s$-coupled receptors. cAMP activates protein kinase A (PKA) by binding to its regulatory subunits, releasing the catalytic subunits to phosphorylate targets. Key PKA substrates include:

• Glycogen phosphorylase kinase (activates glycogen breakdown)
• CREB (cAMP response element-binding protein), a transcription factor that drives expression of cAMP-responsive genes

Cyclic GMP (cGMP) is synthesized by guanylyl cyclase, which can be activated by nitric oxide (NO). cGMP activates protein kinase G (PKG), which promotes smooth muscle relaxation and inhibits platelet aggregation. This is the mechanism behind the vasodilatory effects of nitroglycerin.

Both cAMP and cGMP are degraded by phosphodiesterases (PDEs), which hydrolyze them to AMP and GMP, respectively. PDEs are themselves drug targets: caffeine inhibits certain PDEs, prolonging cAMP signaling.

Lipid-Derived Second Messengers (IP$_3$ and DAG)

When G$_q$-coupled receptors activate phospholipase C (PLC), PLC cleaves the membrane phospholipid PIP$_2$ (phosphatidylinositol 4,5-bisphosphate) into two second messengers:

• IP$_3$ (inositol 1,4,5-trisphosphate): a soluble molecule that diffuses through the cytosol and binds IP$_3$ receptors on the ER membrane, triggering release of stored $Ca^{2+}$ into the cytosol.
• DAG (diacylglycerol): remains embedded in the plasma membrane and activates protein kinase C (PKC).

These two messengers work cooperatively. PKC requires both DAG and $Ca^{2+}$ (released by IP$_3$) for full activation. Once active, PKC phosphorylates a range of targets including ion channels and transcription factors.

Calcium Signaling

$Ca^{2+}$ is one of the most versatile second messengers, involved in muscle contraction, neurotransmitter release, cell proliferation, and apoptosis.

Resting cytosolic $Ca^{2+}$ is kept very low (around 100 nM), roughly 10,000-fold lower than extracellular concentrations. This steep gradient is maintained by:

• SERCA pumps that move $Ca^{2+}$ back into the ER
• Plasma membrane $Ca^{2+}$-ATPases and $Na^+/Ca^{2+}$ exchangers that export $Ca^{2+}$ from the cell

$Ca^{2+}$ can enter the cytosol through several routes:

• Voltage-gated $Ca^{2+}$ channels in excitable cells (neurons, muscle)
• Ligand-gated channels at synapses
• Store-operated $Ca^{2+}$ entry (SOCE) channels, activated when ER stores are depleted
• IP$_3$ receptors and ryanodine receptors on the ER membrane

Once in the cytosol, $Ca^{2+}$ exerts its effects by binding to calcium-sensing proteins. The most important is calmodulin (CaM), which upon binding four $Ca^{2+}$ ions undergoes a conformational change that allows it to activate CaM-dependent kinases (CaMKs), phosphatases (calcineurin), and other effectors. In muscle, $Ca^{2+}$ binds troponin C to initiate contraction.

Signal Transduction

Protein Kinases

Protein kinases catalyze the transfer of the $\gamma$-phosphate group from ATP to the hydroxyl group of specific amino acid residues on target proteins. This is one of the most common post-translational modifications in the cell.

Phosphorylation can alter a target protein's function in several ways:

• Conformational change that activates or inhibits enzymatic activity
• Creation of docking sites for other signaling proteins (as with phosphotyrosines on RTKs)
• Altered subcellular localization (e.g., nuclear import/export)

Kinases are classified by the residues they phosphorylate:

• Serine/threonine kinases: PKA, PKC, Akt, RAF
• Tyrosine kinases: Src, Abl, the intracellular domains of RTKs
• Dual-specificity kinases: MEK (phosphorylates both threonine and tyrosine on MAPK)

Protein phosphatases reverse kinase activity by hydrolyzing the phosphoester bond, removing the phosphate group. The balance between kinase and phosphatase activity determines the phosphorylation state of any given protein, and therefore the strength and duration of a signal.

Phosphorylation Cascades

Phosphorylation cascades are sequential chains in which one kinase phosphorylates and activates the next kinase downstream. They serve three critical functions:

1. Signal amplification: Each activated kinase can phosphorylate many copies of the next kinase, so the number of activated molecules increases at each tier.
2. Signal integration: Each step in the cascade can receive inputs from multiple pathways, allowing the cell to combine information from different signals.
3. Signal modulation: Scaffolding proteins, phosphatases, and feedback loops at each step provide fine-tuned control over signal strength and duration.

The MAPK cascade is the classic example. It proceeds through three sequential kinases:

1. MAPKKK (e.g., RAF): activated by Ras-GTP at the membrane
2. MAPKK (e.g., MEK): phosphorylated and activated by RAF
3. MAPK (e.g., ERK): phosphorylated on both a threonine and a tyrosine by MEK (dual-specificity)

Activated ERK then enters the nucleus and phosphorylates transcription factors that drive expression of genes involved in cell proliferation and differentiation. This cascade is activated by growth factors binding RTKs and is one of the most frequently mutated pathways in human cancers (especially at the level of Ras and RAF).