Lecture 6_Signal transduction (1)

Signal transduction involves mechanisms by which signals detected at a cell's surface are transmitted into the cell's interior, leading to changes in the cell's behavior or gene expression.

The two types of cell signaling are:

1. Hormonal Signaling: Involves endocrine signals released into the bloodstream to reach target cells.
2. Local Mediator Signaling: Includes autocrine signals (acting on the same cell) and paracrine signals (acting on nearby cells).

Ligands, also known as primary messengers, are molecules that bind to receptors on the cell membrane, initiating the process of cell signaling. They are crucial for transmitting information from the extracellular environment to the inside of the cell.

After a ligand binds to its receptor, it triggers a signaling pathway known as signal transduction. This process can involve second messengers and leads to various cellular responses, including changes in gene expression.

Signaling pathways, initiated by receptor-ligand binding, can lead to changes in gene expression by transmitting signals to the nucleus, where they influence the transcription of specific genes, ultimately altering cellular behavior.

The key outcomes of receptor-ligand interactions include:

1. Conformational changes in the receptor.
2. Clustering of receptors.
3. Combination of different receptor types.
4. Binding affinities measured in K_d (dissociation constant).

An agonist is a synthetic compound that mimics a natural substance, leading to a similar response in cellular activity. It enhances cellular activity when it binds to a receptor site.

An antagonist acts like an inhibitor by blocking the response of a receptor site, thereby preventing normal cellular activity. It effectively reduces or blocks cellular activity when it binds to the receptor.

Receptors can be switched off through the following mechanisms:

1. Desensitization: The receptor is bound to its ligand, but its affinity is reduced, leading to a decreased response.

2. Reducing Signal: The signal from the ligand is diminished, which can lead to a reduced effect even when the receptor is still bound.

3. Reducing Number of Receptors: The total number of receptors available for binding is decreased, which lowers the overall response to the ligand.

Signal amplification allows a single molecule of epinephrine to trigger a cascade of events that results in the activation of millions of molecules, leading to a significant physiological response. This process involves multiple steps where each activated molecule catalyzes the activation of many more molecules, enhancing the overall effect of the initial signal.

1. Reception: Epinephrine binds to a G protein-coupled receptor, activating 1 molecule.

2. Transduction:

   • Inactive G protein is activated to produce 10² molecules.
   • Inactive adenylyl cyclase is activated, producing another 10² molecules.
   • ATP is converted to Cyclic AMP, resulting in 10⁴ molecules.
   • Inactive protein kinase A is activated, producing another 10⁴ molecules.
   • Inactive phosphorylase kinase is activated, resulting in 10⁵ molecules.
   • Inactive glycogen phosphorylase is activated, producing 10⁶ molecules.

3. Response: Glycogen is converted to Glucose-1-phosphate, resulting in 10⁸ molecules.

1. Ligand-gated ion channel
2. G protein-coupled receptor (GPCR)
3. Enzyme-coupled receptor (e.g., receptor tyrosine kinase)
4. Nuclear receptor

Insulin causes liver cells to make glycogen, which helps lower blood glucose levels.

A lower dissociation constant (K) of 10-8 M indicates that the insulin receptors have a higher affinity for insulin compared to normal receptors with a K of 10-7 M, which may lead to increased glycogen synthesis despite low blood glucose levels.

Despite having a higher affinity for insulin, the patient's low blood glucose levels may be due to insufficient insulin secretion or other metabolic issues that prevent effective glucose utilization or storage.

Ligand binding activates a specific G protein, which is a guanine-nucleotide binding protein. This activation leads to the G protein binding to a target enzyme or channel, thereby altering their activity.

Examples of G-protein-coupled receptors include:

1. Olfactory receptors
2. β-adrenergic receptors
3. Some hormone receptors
4. Opioid receptors

The first step is the resting state, where the receptor is not bound to a ligand, and the Ga subunit is bound to GDP and associated with Gβγ.

When a ligand binds to the GPCR, the receptor binds a G protein, causing Ga to release GDP and acquire GTP.

After the ligand binds, the Ga and Gβγ subunits separate, with the alpha subunit now bound to GTP.

The Ga subunit activates or inhibits target proteins, initiating signal transduction events while still bound to GTP.

The Ga subunit hydrolyzes its bound GTP to GDP, becoming inactive, and releases the target protein along with an inorganic phosphate molecule.

The final step is the recombination of the subunits to form an inactive G protein, with the alpha subunit now bound to GDP re-associating with the beta and gamma subunits.

The βγ subunit activates GPR kinases, which are involved in the regulation of G protein-coupled receptor signaling pathways.

The α subunits interact with adenylyl cyclase and phospholipase C, which are key enzymes in the signaling pathways activated by G protein-coupled receptors.

Adenylyl cyclase catalyzes the conversion of ATP to cyclic AMP (cAMP) by removing two phosphate groups from ATP, resulting in the formation of the cyclic structure of cAMP.

Cyclic AMP (cAMP) is broken down into AMP by the enzyme phosphodiesterase, which hydrolyzes the cyclic structure, resulting in the release of a phosphate group and water.

Cyclic AMP (cAMP) acts as a second messenger that transmits signals from hormones and other signaling molecules, leading to various cellular responses such as gene expression, metabolism, and cell growth.

The Gsα subunit activates adenylyl cyclase, which is responsible for converting ATP into cAMP.

cAMP activates protein kinase A (PKA), which then phosphorylates target proteins to elicit cellular responses.

Phosphodiesterase breaks down cAMP into AMP, thus terminating the signaling pathway.

cAMP regulates PKA activity by binding to the regulatory subunits, causing them to change conformation and detach from the catalytic subunits, which activates PKA.

Once activated, Protein Kinase A (PKA) transfers phosphate from ATP to serine or threonine residues on target proteins, thereby phosphorylating them and altering their function.

Protein Kinase A (PKA) is composed of two catalytic subunits and two regulatory subunits. The regulatory subunits inhibit the catalytic subunits in the absence of cAMP.

Epidermal Growth Factor (EGF) binds to a tyrosine kinase receptor complex.

Inositol-1,4,5-trisphosphate (IP3) functions as a second messenger in cell signaling, facilitating various physiological responses by mobilizing calcium ions from the endoplasmic reticulum into the cytoplasm.

The following table summarizes the regulated functions of IP3 and DAG in various target tissues:

IP3 activates the IP3 receptor channel, which is a ligand-gated calcium channel, leading to the release of Ca2+ from the endoplasmic reticulum into the cytosol.

DAG remains in the membrane and activates protein kinase C (PKC), which plays a crucial role in various signaling pathways and cellular responses.

The activation of Gq is initiated by the binding of a ligand to its corresponding G-protein-coupled receptor (GPCR), which leads to the displacement of GDP by GTP.

Calcium ion release is crucial in various signaling processes as it acts as a second messenger, facilitating communication between different cellular components. It influences numerous cellular functions, including muscle contraction, neurotransmitter release, and gene expression.

Receptor tyrosine kinases activate phospholipase C (PLC), which produces inositol trisphosphate (IP3), leading to calcium release from the endoplasmic reticulum. G protein-coupled receptors also activate PLC, contributing to the increase of intracellular calcium levels, thus amplifying the signaling response.

The sodium-calcium exchanger helps regulate intracellular calcium levels by exchanging sodium ions for calcium ions across the plasma membrane. This mechanism is essential for maintaining calcium homeostasis and facilitating calcium signaling in response to various stimuli.

Calcium ATPases in the plasma membrane and endoplasmic reticulum (ER) membrane are responsible for pumping calcium ions out of the cell or back into the ER, respectively. This action is vital for restoring calcium levels after signaling events and preventing excessive calcium accumulation, which can be detrimental to cell function.

Calmodulin mediates calcium-activated processes by binding to four calcium ions, which induces a conformational change in calmodulin. This change allows calmodulin to wrap its two globular 'hands' around a binding site on a target protein, forming an active complex.

Ca2+ is considered a better second messenger than Na+ due to the following reasons:

1. Concentration Gradient:

   • Ca2+ has a much lower concentration in unstimulated cells (about 10^-7 M) compared to Na+ (about 10^-3 M). This steep gradient allows for a significant change in intracellular Ca2+ concentration upon stimulation.

2. Rapid Response:

   • The ability to quickly increase Ca2+ concentration in response to signals allows for rapid cellular responses, which is crucial for processes like muscle contraction and neurotransmitter release.

3. Specificity of Action:

   • Ca2+ can activate specific signaling pathways and proteins, such as calmodulin and various kinases, which are not activated by Na+.

4. Role in Various Cellular Functions:

   • Ca2+ is involved in a wide range of cellular functions, including muscle contraction, neurotransmitter release, and gene expression, making it a versatile second messenger.

ATP binds to the catalytic domain of a protein kinase, which then hydrolyzes ATP into ADP and inorganic phosphate (P). This phosphate group is transferred to specific amino acid residues, such as serine, leading to the activation or inactivation of the substrate through phosphorylation.

Interleukin-2 (IL-2) targets cytotoxic T lymphocytes and uses a receptor complex associated with foreign antigens.

Transforming Growth Factor ẞ (TGF ẞ) targets fibroblastic cells and uses an acetylcholine receptor complex.

The growth factors that bind to tyrosine kinase receptor complexes include:

1. Epidermal growth factor (EGF)
2. Transforming growth factor a (TGF a)
3. Platelet-derived growth factor (PDGF)
4. Fibroblast growth factor (FGF)
5. Colony-stimulating factor-1 (CSF-1)

Wnts bind to a frizzled (seven-pass protein) receptor complex, while Hedgehogs bind to a patched (seven-pass protein) receptor complex.

EGF binds to the extracellular domains of two receptor monomers, causing them to dimerize, which activates the kinase domains and leads to phosphorylation of tyrosine residues on the cytoplasmic tails of the receptors.

An inactive EGFR consists of an EGF-binding site located outside the cell, a plasma membrane, a cytosolic region containing a tyrosine kinase, and a cytosolic tail.

Upon activation, the kinase domains phosphorylate the tyrosine residues in the cytoplasmic tail, converting them to phosphorylated tyrosine (P-Tyr).

Receptor dimerization is crucial for the activation of receptor tyrosine kinases as it brings the kinase domains of the receptors into proximity, allowing them to activate each other and initiate downstream signaling pathways.

EGF (Epidermal Growth Factor) acts as a ligand that binds to the Receptor Tyrosine Kinase (RTK), leading to the activation of the receptor and subsequent downstream signaling events.

Ras-GTP, which is formed after the activation of the RTK, binds to and activates Raf, initiating a downstream signaling cascade that ultimately leads to the phosphorylation of transcription factors and activation of response genes.

GAP (GTPase Activating Protein) converts Ras-GTP back to Ras-GDP, effectively terminating the signaling pathway by shutting off the signal once the response has been achieved.

1. EGF binds to the RTK, activating its tyrosine kinase domain.
2. This leads to the phosphorylation of the receptor and the formation of Ras-GTP.
3. Ras-GTP activates Raf, which phosphorylates MEK.
4. MEK then activates MAPK, which enters the nucleus to phosphorylate transcription factors Ets and Jun, activating response genes.

In the absence of TGFB, the type I and type II receptors are not clustered or phosphorylated, and R-Smads and Smad4 remain in the cytosol.

The binding of TGFB results in the clustering of type I and type II receptors, followed by the phosphorylation of type I receptors by type II receptors.

The activated type I receptors bind a complex of an anchoring protein and an R-Smad, resulting in the phosphorylation of R-Smad.

The phosphorylated R-Smad binds Smad4, and the complex enters the nucleus to activate or repress gene expression. Eventually, the R-Smad is degraded or leaves the nucleus, and Smad4 returns to the cytosol, terminating the signal.

Normal cells are expected to show an increase in cell division rate in response to EGF, while cells with knocked down GAP for Ras are likely to show a reduced or altered response due to impaired Ras signaling.

Scaffolding complexes facilitate the interaction between various signaling molecules, enhancing the efficiency and specificity of signal transduction. They organize the signaling components, such as Ras-GTP, KSR, and protein kinases (Raf, MEK, MAPK), allowing for coordinated phosphorylation events and downstream signaling to the nucleus.

The interaction of 14-3-3 with KSR through phosphorylation at specific sites (S297 and S392) modulates the activity of KSR, which is crucial for the assembly and function of the signaling complex, thereby impacting the downstream signaling cascade involving MAPK.

Phosphorylation of MAPK is a critical step in the signaling cascade that leads to the activation of transcription factors in the nucleus, ultimately resulting in changes in gene expression and cellular responses. This highlights the importance of MAPK as a key mediator in signal integration from RTK and GPCR pathways.

The main types of receptors involved are:

1. Ion Channel - triggers depolarization.
2. G-Protein Coupled Receptor (GPCR) - stimulates Adenylyl cyclase to convert ATP to cAMP and activates Phospholipase C.
3. Receptor Tyrosine Kinase (RTK) - undergoes clustering and autophosphorylation, activating the GRB2/Sos complex.

The GPCR receptor influences intracellular signaling by:

1. Stimulating Adenylyl cyclase to convert ATP to cAMP, activating PKA.
2. Activating Phospholipase C through Gα-GTP, leading to the conversion of PIP2 to IP3 and DAG.
3. IP3 binds to the IP3 receptor on the ER, while DAG activates PKC.

The RTK receptor plays a crucial role by:

1. Undergoing clustering and autophosphorylation.
2. Activating the GRB2/Sos complex, which leads to the activation of Ras-GTP.
3. This activation contributes to downstream signaling that influences gene expression.

cAMP is significant in the signaling pathway because it:

1. Activates PKA (Protein Kinase A), which phosphorylates target proteins.
2. Interacts with the endoplasmic reticulum (ER) to influence various cellular responses.
3. Plays a key role in the convergence of signals that ultimately influence gene expression in the nucleus.

IP3 and DAG contribute to the signaling process by:

1. IP3 binding to its receptor on the ER, leading to the release of Ca2+ ions into the cytoplasm.
2. DAG activating PKC (Protein Kinase C), which further propagates the signaling cascade.
3. Together, they enhance the cellular response and influence various downstream effects, including gene expression.