Lecture+14_Signaling+Lecture+II

Signal Transduction - Lecture II

Receptors Relay Signals Via Intracellular Signaling Molecules

• Protein signaling molecules relay signals by activating the next protein in the pathway or by generating second messengers.
• Many behave as molecular switches with "active" and "inactive" states (e.g., ATP, GTP).
• Small chemical signaling molecules (second messengers) are generated in large amounts in response to receptor activation.
• They diffuse away from the source to spread the signal throughout the cell.
  • Water-soluble molecules spread through the cytosol (e.g., cyclic AMP, $Ca^{2+}$).
  • Lipid-soluble molecules spread along the plasma membrane (e.g., diacylglycerol).
• They bind to and alter the behavior of specific proteins, amplifying signals.

Signaling Via Proteins That Act as Molecular Switches

• Use of ATP in signaling:
  • Humans have approximately 520 protein kinases and 150 protein phosphatases.
  • 30-50% of human proteins can be phosphorylated.

Kinase Cascade

• Protein Kinase 1 phosphorylates Protein Kinase 2, which phosphorylates Protein Kinase 3, relaying the signal forward.
• Cascades can amplify signals and interact with other pathways.
• Protein Kinases can phosphorylate downstream target proteins (a, b, c).

GEF and GAP

• Small GTPases are used in signaling.
• GEF (guanine nucleotide exchange factor) activates the GTPase by promoting GTP binding.
• GAP (GTPase activating protein) inactivates the GTPase by stimulating GTP hydrolysis.

Signaling Complexes

• Two main functions:
  • Specificity: Ensures proteins in a signaling pathway only interact with each other, avoiding undesirable cross-talk with other signaling pathways.
  • Concentration: Generates high local concentrations of signaling proteins, enabling sequential and rapid activation of proteins in a signaling pathway and allowing some signaling proteins to be used in different pathways.

Intracellular Signaling Complexes Form at Activated Receptors

• Preformed Signaling Complex on a Scaffold Protein
  • An inactive receptor is bound to a scaffold protein, which also binds intracellular signaling proteins.
  • Upon activation by a signal molecule, the receptor undergoes a conformational change, activating the intracellular signaling proteins, which then transmit downstream signals.
• Assembly of Signaling Complex on an Activated Receptor
  • An inactive receptor, upon activation by a signal molecule, recruits intracellular signaling proteins.
  • The receptor phosphorylates these proteins, leading to the assembly of a signaling complex that transmits downstream signals.
• Assembly of Signaling Complex on Phosphoinositide Docking Sites
  • Upon activation by a signal molecule, the receptor causes hyperphosphorylation of specific phospholipid molecules (phosphoinositides) in the plasma membrane.
  • These hyperphosphorylated phosphoinositides serve as docking sites for inactive intracellular signaling proteins, leading to their activation and transmission of downstream signals.

Signaling Proteins Interact Via Modular Interaction Domains

• PTB (phosphotyrosine-binding) and SH2 (src homology 2) These domains bind phosphorylated tyrosine residues.
• PH (pleckstrin homology) These domains bind specific phosphoinositides in the plasma membrane.
• SH3 (src homology 3) These domains bind proline-rich regions.

Positive and Negative Feedback

• Feedback loop: The output of a process feeds back to regulate that same process.
• Positive Feedback
  • A transient signal induces a long-term change (cellular memory).
• Negative Feedback
  • Limits response to a signal and maintains homeostasis.
  • Loss of negative feedback can result in several diseases (e.g., cancer).

Positive Feedback Can Generate an All-or-None Response

• Bistable system: Either on or off.
• A transient stimulus switches it between states, and this switch can be permanent (e.g., cell fate).

Negative Feedback Can Generate Different Types of Responses

• Short delay: Strong, brief response.
• Long delay: Response that oscillates.

Cells Can Adjust Their Sensitivity to a Signal Via Desensitization

• Desensitization/Adaptation: Prolonged exposure to a stimulus decreases the response to that level of stimulus.
• Allows cells to respond to changes in a signal rather than the absolute level of the signal over a wide range of concentrations.
• Mechanism: Negative feedback with a short delay.
• A strong response modifies the signaling machinery to become less responsive to the signal.

G-Protein-Coupled Receptors (GPCRs)

• These receptors have 7 transmembrane domains; the ligand binds in the center.
• They use G proteins to relay signals into the cell.
• The GPCRs are the largest family of receptors (>800 in humans).
• They mediate most responses to signals from the environment and other cells, including sight, smell, and taste.
• Ligands: Proteins, small molecules (amino acids, fatty acids, odorants, tastants), and light.
• One ligand can often activate several GPCRs.
  • Example: Adrenaline activates at least 9 distinct GPCRs, eliciting different responses.
• The GPCRs are the most common drug target (half of FDA-approved drugs) (e.g., Wegovy).

G-Protein-Coupled Receptors (GPCRs) Ligand Binding Site:

• Binding due to interactions with specific receptor amino acids
• Example epinephrine bound to beta-adrenergic receptor

Trimeric G Proteins Relay Signals from GPCRs

• α subunit: GTPase switch.
• α and γ subunits: Anchored to the cell membrane via covalently attached lipids.

Trimeric G Proteins Relay Signals from GPCRs Process

• Inactive GPCR is bound to an inactive G protein (α, β, γ subunits) with GDP.
• Activated GPCR causes the α subunit to exchange GDP for GTP, activating both the α subunit and the βγ complex.
• The activated α subunit and βγ complex then activate downstream effector proteins.

Signaling Downstream of GPCRs

1. Cyclic AMP (e.g., β adrenergic receptor).
2. Phospholipids (e.g., α adrenergic receptor).
3. Ion Channels (e.g., muscarinic acetylcholine receptor).

Cyclic AMP (cAMP) Signaling Downstream of GPCRs

• Adenylyl cyclase converts ATP into cAMP (second messenger).
• cAMP is rapidly degraded by cAMP phosphodiesterase.

GPCRs Can Activate and Inhibit Adenylyl Cyclase

• $G \alpha_s$ = stimulatory G protein
• $G \alpha_i$ = inhibitory G protein

Different G Proteins Act In Different Signaling Pathways

• Four Major Families of Trimeric G Proteins
  • Family I:
  • Some members: $G<em>s, G</em>{olf}$
  • Subunits that mediate action: α
  • Some functions: Activates adenylyl cyclase, activates $Ca^{2+}$ channels
  • Family II:
  • Some members: $G_i$
  • Subunits that mediate action: α and βγ
  • Some functions: Inhibits adenylyl cyclase, activates $K^+$ channels, inactivates $Ca^{2+}$ channels
  • Family III:
  • Some members: $G<em>q, G</em>{12/13}$
  • Subunits that mediate action: α
  • Some functions: Activates phospholipase C-β, activates Rho family monomeric GTPases (via Rho-GEF) to regulate the actin cytoskeleton

cAMP Acts Via cAMP Dependent Protein Kinase A (PKA)

• cAMP binds to the regulatory subunits of inactive PKA, causing them to dissociate from the catalytic subunits.
• This dissociation activates the catalytic subunits.

Sharpness of Enzyme Activation Response Increases With The Number of Molecules That Must Bind To Activate The Enzyme

Advantages:

1. Prevents responses to background noise
2. Respond with high sensitivity when stimulus crosses a threshold level – acts like on/off switch

cAMP-Mediated Signaling Cascade: Signal Amplification

• Example: Epinephrine (at $10^{-10}$ M) activates a GPCR, which activates adenylyl cyclase.
• Adenylyl cyclase produces cAMP (at $10^{-6}$ M), which activates protein kinase A.
• Protein kinase A phosphorylates target proteins.

cAMP-Induced Responses Can Be Slow and Long-Lasting By Inducing Gene Expression

1. Activated PKA catalytic subunits enter the nucleus.
2. PKA phosphorylates CRE-binding (CREB) protein.
3. Phosphorylated CREB binds CREB-binding protein (CBP).
4. CREB/CBP binds cAMP response element (CRE) – DNA enhancer that stimulates expression of genes (e.g., learning and memory).

Phospholipid Signaling Downstream of GPCRs

• Activated GPCR activates phospholipase C-β.
• Phospholipase C-β hydrolyzes PI(4,5)P2 (PIP2) into diacylglycerol and inositol 1,4,5-trisphosphate (IP3).
• Diacylglycerol activates protein kinase C.
• IP3 releases $Ca^{2+}$ from the endoplasmic reticulum.

Phospholipid Signaling Downstream of GPCRs Process

• Signal molecule activates GPCR.
• Activated GPCR activates phospholipase C-β.
• Phospholipase C-β hydrolyzes PI(4,5)P2 (PIP2) into diacylglycerol and inositol 1,4,5-trisphosphate (IP3).
• Diacylglycerol activates protein kinase C.
• IP3 releases $Ca^{2+}$ from the endoplasmic reticulum.

Calcium Signaling Downstream of GPCRs

• $Ca^{2+}$ concentration is low in the cytosol ($10^{-7}$ M) and high in the extracellular fluid and ER lumen ($10^{-3}$ M).
• IP3 activates IP3 receptors in the ER membrane, releasing $Ca^{2+}$ into the cytosol.
• Local $Ca^{2+}$ release promotes the opening of nearby IP3 and ryanodine receptors, causing more $Ca^{2+}$ release (positive feedback).
• This positive feedback produces a $Ca^{2+}$ wave that rapidly spreads across the cell.
• High $Ca^{2+}$ levels inactivate IP3 and ryanodine receptors, blocking $Ca^{2+}$ release (negative feedback).
• $Ca^{2+}$ pumps reduce cytosolic $Ca^{2+}$ back to normal level.

Example of Calcium Wave: Egg Fertilization

• After sperm entry, you can observe waves of calcium concentration fluctuation across the egg.

Calmodulin: A Multipurpose Intracellular $Ca^{2+}$ Receptor

• Calmodulin binds $Ca^{2+}$ and then binds to target proteins to activate them.

Gaseous Signaling Between Cells: Nitric Oxide

• Nitric oxide (NO) causes the dilation of blood vessels.
• NO acts locally because its half-life in the extracellular space is only 5-10 seconds, as $O<em>2$ and $H</em>2O$ convert it into nitrates and nitrites.

GPCRs Can Directly Regulate Ion Channels

• Example: In neurons, the rapid opening and closing of ion channels causes changes in membrane potential that transmit nerve impulses.
• Acetylcholine released from the vagus nerve reduces heart rate.
• Activated GPCR activates $G \alpha_i$.

GPCRs Can Indirectly Regulate Ion Channels: Olfaction

• Humans have 350 olfactory receptors, each recognizing a different set of odorants.
• Each olfactory sensory neuron expresses only one type of olfactory receptor.
• Olfactory receptors are GPCRs present on cilia that extend from each olfactory sensory neuron.

GPCRs Can Indirectly Regulate Ion Channels: Vision

• Visual transduction is the fastest G-protein mediated response in vertebrates.
• The signal is light.
• Rod photoreceptor: non-color vision in dim light.
• The outer segment has a stack of discs, each formed by a sac of membrane containing rhodopsin (GPCR).
• Rhodopsin has a covalently attached chromophore: 11-cis-retinal.
• Absorption of a photon induces isomerization to 1