Cell Signaling II Notes

Announcements

• Wiley Plus assignment (chapter 15) due this Friday.
• Start reviewing materials for final exams.

Chapter 15: Cell Signaling II

• Topics Covered:
  • GPCR signal desensitization and termination
  • Phosphatidylinositol-derived signaling
  • RTK signaling and insulin
  • Ca2+ signaling
  • Cross talk between signaling pathways
  • Steroid signaling

Learning Objectives

• Understand how a signaling pathway can be modulated, desensitized, and terminated.
• Compare and contrast cell signaling messengers cAMP, IP3, DAG, Ca2+, and some major pathways in which they act.
• Explain kinase cascade activation.
• Explain the mechanism of RTK activation and signaling pathways.
• Understand how several pathways are regulated by insulin and key molecular components.
• Describe Ca2+ signaling and its important role in cellular activity.
• Understand signal convergence and crosstalk.
• Explain the unique features of steroid hormones.

GPCR Signaling Modulation and Termination

• Analogy: Smelling apple pie and the eventual reduction in smell due to receptor desensitization.
• Ending the signaling is important.
• Proteins involved in signal termination include:
  • G-protein coupled receptor kinase (GRKs)
  • Arrestins
  • Regulator of G protein signaling (RGS) proteins
  • Phosphodiesterases
• Negative feedback reduces the output of the pathway.
• DESENSITIZATION/ADAPTATION: Reduced outcome in response to a prolonged stimulation.
  • Examples: adenylyl cyclase, GRK – GPCR kinase
  • RGS – regulators of G-protein signaling (internalization)

Termination of G-protein-coupled Signaling by Internalization of Receptors

• G protein-coupled receptor kinase (GRK) phosphorylates GPCRs.
• Arrestins bind to the phosphorylated GPCR.
• Receptor-mediated endocytosis.
• Internalizing GPCR to terminate the signal.
• GPCR can be recycled.
• Arrestin-mediated internalization of GPCRs

Feedback Regulation

• Feedback regulation adjusts the response to a signal.
• Positive feedback: Y enhances the production of X.
• Negative feedback: N inhibits the production of X.

Signaling Molecules as Switches

• Small GTP binding proteins are turned on by association with GTP, which is mediated by exchange factors.
• GTP hydrolysis shuts signaling off (can be mediated by proteins).
• Some signaling molecules act as switches.

Other Second Messengers

• cAMP
  • Formed from ATP by adenylyl cyclase.
  • Broken down by cyclic AMP phosphodiesterase to AMP.
• DAG (diacylglycerol), IP3 (inositol 1,4,5-trisphosphate), and Ca2+
  • Generated from inositol phospholipid.
  • IP3 opens Ca2+ channels in the ER lumen.
  • DAG activates PKC (protein kinase C).

Phosphatidylinositol-derived Signaling

• A major class of signaling molecules is phosphoinositides generated by kinases.
• Phosphoinositide can bind to the Pleckstrin homology (PH) domain of a protein to relay the signal.
• Phosphoinositides: tiny lipids with a significant role in cell signaling and regulation.
• Chemoattractant stimulated generation of PIP3 (visualized with an anti-PIP3 antibody) at the cell leading edge

Second Messengers Produced by PLCβ

• PIP2 is cleaved by phosphatidylinositol-specific phospholipase C-β (PLCβ) to generate inositol 1,4,5 triphosphate (IP3) and diacylglycerol (DAG).
• Note the cross-talk between the G-protein and phosphoinositide signaling pathway.
• IP3 leads to $Ca^{2+}$ release from the smooth endoplasmic reticulum (sER).
• Protein kinase C activation is Ca2+ dependent.

Changes in Cytosolic $Ca^{2+}$

• Influx of $Ca^{2+}$ through open channels in the plasma membrane.
• Release of $Ca^{2+}$ from internal $Ca^{2+}$ stores, such as from IP3 receptors.
• Returning to resting level by $Na^+/Ca^{2+}$ exchangers or $Ca^{2+}$ pumps.

Important Factors About $Ca^{2+}$ Signaling

1. Universal, regulating almost every cellular activity.
2. Changes in $Ca^{2+}$ level are transient, often regional, either influx from extracellularly through ion channels, or released internally from $Ca^{2+}$ stores.
3. Cytosol $Ca^{2+}$ tightly regulated, low at rest.
4. Many $Ca^{2+}$ binding proteins.
5. Can be monitored using $Ca^{2+}$ sensitive dye (Fura 2 AM) or proteins (GCamP).

Visualizing Cytoplasmic $Ca^{2+}$ Concentration

• Intracellular $Ca^{2+}$ increase induced by the sperm’s contact with the plasma membrane of the egg.
• The $Ca^{2+}$ rise following fertilization triggers a number of events, driving the zygote toward its first mitotic division.
• Calcium wave in a starfish egg due to fertilization.
• Pseudocolored: blue- low $[Ca^{++}]$ , red- high $[Ca^{++}]$

$Ca^{2+}$ Binding Proteins

• Calcium can:
  • Affect a number of different types of cellular effectors, including protein kinases.
  • Activate or inhibit various enzyme and transport systems.
  • Change the ionic permeability of membranes.
  • Induce membrane fusion.
  • Alter cytoskeletal structure and function.
• Calcium acts in conjunction with a number of calcium‐binding proteins to alter cytoskeletal structure and function, such as tropomyosin and calmodulin.

Classes of Receptors

• Response to a signal depends on the type of receptor to which it binds.
• Classes:
  • G-protein coupled receptors (GPCR)
  • Ligand-gated channels (ionotropic receptors)
  • Receptor protein tyrosine kinases (RTK)
  • Steroid hormone receptors (nuclear receptors)
  • Others (T-cell receptor, integrins)

Protein-Tyrosine Phosphorylation as a Mechanism for Signal Transduction

• Protein-tyrosine kinases phosphorylate tyrosine residues on target proteins.
  • Receptor protein‐tyrosine kinases (RTKs), integral membrane proteins (a single transmembrane helix and an extracellular ligand binding domain).
  • Non‐receptor (or cytoplasmic) protein‐tyrosine kinases.
• RTKs’ ligands primarily are growth factors and hormones.

RTK Dimerization and Activation

• In a non-activated state, the receptors are present in the membrane as monomers.
• Ligand binding leads directly to dimerization of the receptor and activation of its kinase activity.

Activation of Downstream Signaling Pathway

• Activation of downstream signaling pathway through SH2 and PTB:
  • Phosphorylated tyrosine residues bind to effector proteins that have either a Src-homology 2 (SH2) domain or a phosphotyrosine-binding (PTB) domain.
  • SH2 and PTB domains contain a conserved binding-pocket for the phosphorylated tyrosine residue.

SH2 and PTB Domain Proteins

• Cells contain numerous proteins with SH2 or PTB domains adaptor docking protein enzymes transcription factor
• SH2 and PTB domain proteins include:
  1. Adaptor proteins that bind other proteins.
  2. Docking proteins that supply receptors with other tyrosine phosphorylation sites.
  3. Signaling enzymes (kinases) that lead to changes in cell.
  4. Transcription factors
• Example: GrB2 (Growth factor receptor-bound protein).

Insulin Signaling

• Binding of insulin to its receptor promotes glucose uptake.

Insulin Signaling Pathway

• Activated insulin receptor recruits insulin receptor substrate proteins (IRSs).
• Through IRSs, a variety of signaling pathways can be activated.
• PH (Pleckstrin homology) domain enables cytosolic proteins to interact with membrane-bound protein or lipids.
• IRS
• PDK1: Phosphoinositide-dependent kinase-1
• PKB: protein kinase B, also called AKT, is a serine/threonine kinase.

Regulation of Glucose Uptake by Insulin

• Regulation of glucose uptake in muscle and fat cells by insulin
• Diabetes mellitus:
  • Type 1: Caused by defects in insulin production.
  • Type 2: Caused by gradual insensitivity to insulin.

IRS Activation

• Tyrosine-phosphorylated IRS activates a variety of signaling pathways
• IRS Insulin receptor-substrate-1 (IRS-1) also recruits adaptors, one of which is GrB2 (Growth factor receptor- bound protein), which recruits Sos (guanine nucleotide exchange factor).
• Sos activates Ras binding site.

The Ras Family

• ras mutations are found in ~25% of human cancers; often constitutively activated.
• Has GTPase activity – active when bound to GTP (inactive when bound to GDP).
• Associated with the membrane by lipids.
• H-Ras monomeric GTPase!

MAP Kinase Cascade

• Mitogen-Activated Protein (MAP) kinase cascade:
  1. Growth factor binds receptor.
  2. Receptor PTK (protein tyrosine kinase) activated.
  3. Grb2 and Sos recruited.
  4. Ras-GDP converted to Ras-GTP.
  5. Soluble Raf recruited to membrane-bound Raf.
  6. Raf (MAPKKK) phosphorylates MEK (MAPKK).
  7. MEK phosphorylates ERK (MAPK).
  8. ERK phosphorylates transcription factors (TF).
  9. Transcription factors activate gene transcription.
  10. MKP-1 (MAP kinase phosphatase) deactivates MAPK.
• Results in activation of transcription factors leading to cell growth and proliferation.

Convergence, Divergence, and Cross-Talk

• Signals from unrelated receptors can converge to activate a common effector.
• Identical signals can diverge to activate a variety of effectors.
• Signals can be passed back and forth between pathways from crosstalk.

Convergence Among Different Signaling Pathways

• Signals transmitted from three sources all converge on Ras and are then transmitted along the MAP kinase cascade.
• Examples:
  • GrB2: Growth factor receptor-bound protein 2
  • Sos: guanine nucleotide exchange factor
• Convergence: Signals from unrelated receptors lead to activation of a common effector.

Cross-Talk Among Different Signaling Pathways

• Crosstalk: more and more crosstalk is found between signaling pathways.
• cAMP can block signals transmitted through the MAP kinase cascade.
• $Ca^{2+}$ and cAMP can influence each other’s pathways.

Steroid Hormones

• Steroid hormones bind to intracellular receptors to influence transcription directly.
• Ligands that bind to intracellular receptors include small and lipophilic estrogen, testosterone, retinoic acid, vitamin D etc. (chapter 12).