Cell Signaling Pathways

Signaling Pathways Overview

• Scaffolds, adaptors, and compartments like primary cilia bring signaling molecules together.
• Protein domains include interaction domains (SH2, SH3, PBD, PH) and catalytic domains (Kinase, Phosphatase, GTPase).
• Protein-protein interactions are experimentally detected via Co-immunoprecipitation (Co-IP), Yeast two-hybrid (Y2H), FRET, BiFC, and BioID.
• Signal-response relationships involve response timing, signal processing, response duration, and sensitivity.
• Signal processing can be hyperbolic, sigmoidal, or all-or-none.

Response Behavior & Signal Processing

• Response timing can be fast or slow.
• Possible signal processing behaviors: hyperbolic, sigmoidal, or all-or-none.
• Positive and negative feedback affect signal processing.
• Response duration can be sustained or transient.
• Positive feedback generates an all-or-none response.

Positive & Negative Feedback Loops

• Positive feedback can generate an all-or-none response.
• Positive feedback mechanisms can create a sustained response.
• Negative feedback mechanisms generate transient responses.
• Negative feedback can generate transient or oscillatory responses.

Sensitivity in Signaling

• Sensitivity refers to the amount of signal required to elicit a response.
• Hormones act at low concentrations, representing a highly sensitive system.
• Neurotransmitters act at high concentrations, representing a less sensitive system.
• Sensitivity is determined by the number and affinity of receptors, and the activity/availability of intracellular signaling molecules.
• Cells can adjust sensitivity through negative feedback loops, receptor sequestration, or receptor degradation.

Adaptation and Desensitization

• Adaptation (desensitization) results from pathway inactivation through negative feedback loops, receptor sequestration, or degradation.
• Insulin receptor desensitization relates to Type 2 diabetes.
• Drug addiction and tolerance can be related to receptor desensitization and internalization
• Drug dependence can result from receptor degradation

Sensitivity Experiment

• Progesterone promotes frog oocyte maturation through the MAP kinase signaling module.
• In pooled oocytes, the % active MAP kinase varied with varying progesterone concentrations.
• Individual oocytes showed an all-or-none response to progesterone, either phosphorylated or not at certain concentrations.

G-Protein Coupled Receptors (GPCRs)

• GPCR introduction highlights that there are >800 GPCRs in humans.
• Mice have ~1000 GPCRs, mostly related to smell.
• GPCR targets comprise 12% of genes targeted by approved drugs.
• Ligands include light (rhodopsin receptor), smell, taste (amino acids, small molecules), hormones, and neurotransmitters.
• A single ligand can activate multiple GPCRs: Adrenalin (9), Acetylcholine (5), Serotonin (14).
• GPCR relays trimeric G-proteins by binding GTP and hydrolyzing it to GDP, active when bound to GTP.
• GAP inactivates G-proteins by accelerating GTP hydrolysis rates.
• GEF activates G-proteins and exchanges GDP with GTP.
• G protein activation needs GPCR conformational change upon ligand binding
• GPCR acts as a GEF for G-protein
• Trimeric G-protein is activated when: GTP binds α-subunit and Separated β/ɣ-subunit.

G-Protein Inactivation and Regulation

• GTP hydrolysis by the α-subunit inactivates G-proteins.
• RGS (Regulator of G-protein Signaling) acts as GAP for α-subunit.
• Following prolonged stimulation, GPCR desensitization occurs which depends on receptor phosphorylation.
• Downstream signaling proteins activated by trimeric G-proteins: adenylyl cyclase → cAMP → PKA and PLC→IP3+DAG→Ca2+→PKC
• Adenylyl cyclase activation leads to cAMP production, a second messenger.
• Gs is stimulatory, while Gi is inhibitory
• Some olfactory receptors are GPCRs coupled to Gs, increasing cAMP and opening Na+ channels to start nerve impulses.
• Some GPCRs coupled to G proteins suppress cGMP production, using different cyclic nucleotide monophosphates and cGMP-gated cation channels.
• Certain bacterial toxins like cholera and pertussis target Gs and Gi proteins respectively.

cAMP and PKA Pathway

• Different cell types respond differently to increased cAMP.
• Cyclic AMP sensor changes its fluorescence when bound to cAMP
• Cyclic-AMP-Dependent Protein Kinase (PKA) mediates most effects of cAMP.
• A-kinase anchoring proteins (AKAP): localizes the kinase to certain locations Consensus sequence for PKA phosphorylation: RRxS/Ty x: any a.a., y: any large or hydrophobic a.a.
• Stimulation of adenylyl cyclase leads to Cytoplasmic target proteins and CREB: CRE Binding proteins (transcription factors)

Phospholipids and Calcium Signaling

• Some G proteins signal via phospholipids.
• PLC produces diacylglycerol, and inositol to stimulate Protein Kinase C (PKC) from the ER
• A table highlights cell responses where GPCRs activate PLCβ.
• Calcium (Ca2+) acts as a second messenger, regulating muscle contraction, neuronal transmission, fertilization, cell division, and secretion.
• calcium regulates enzyme and ion pumps Feedback Generates Ca2+ Waves and Oscillations
• Feedback Generates Ca2+ Waves and Oscillations

Calcium and Muscle Contraction

• Ca+2 binds to troponin, exposing myosin-binding sites on actin
• Striated muscle has tropomyosin that covers myosin-binding sites in the relaxed state.
• Calcium binding to troponin C induces a conformational change and reveals the myosin-binding sites
• Myosin can now bind to actin.

Calcium in Fertilization

• There are three main steps: release, regulation, and restoration.
• Sperm brings PLC and triggers Ca2+ release from internal stores
• Ca2+ release in egg:
  • alters egg surface properties to prevent polyspermy.
  • induces egg activation and starts embryonic development.
• Calmodulin is a multipurpose intracellular calcium receptor.
• Calmodulin opens up upon calcium binding, interacts with its targets, and undergoes conformational change

CAM Kinases and Signaling Summary

• Ca2+/Calmodulin-Dependent Protein Kinases (CAMK) mediate the effects of Signals. These have intermediate kinase levels.
• Once phosphorylated, kinase stays active even after calcium signal decays, forming a molecular memory of Ca2+ levels in cells!
• Major points of the summary:
• Adenylyl cyclase → cAMP → PKA→ target proteins
• DAG + IP3 → Ca2+ and other processes. -PLC-beta
• GPRC -> target processes through ion channels and calcium -CAM Kinases are involved with smooth muscle. Nitric Oxide Is a Gaseous Signaling Mediator

Nitric Oxide Is a Gaseous Signaling Mediator

• Nitric Oxide Is a Gaseous Signaling Mediator That Passes Between Cells and increases blood flow by dilating blood vessels

Viagra and Heart Disease

• Viagra (the potency pill) was discovered accidently – designed against cardiovascular diseases.
• Nitroglycerin against heart failure and is metabolized to yield nitric oxide.
• Viagra was designed to block it, increasing flow to the heart
• But was found to make an erect penis -Is Now prescribed commonly there as well

Receptor Tyrosine Kinases (RTKs)

• RTKs are tyrosine kinases involved in diverse cellular processes consisting of 60 of them
• Ligand binding promotes dimerization
• Activation of RTKs
• Dimerization à Activates kinase activity via conformational changes
• Trans-autophosphorylation à Fully activation
• Dimerization activates via conformational changes. Autophosphorylation creates docking sites for signaling proteins.
• Act in this format

Additional Info about RTKs

• Exception: Insulin, Insulin receptor is always a dimer.
• Exception: Kinase is a type, a process
• Proteins bind with SH2
• SH2 means binds phosphorylation of type
• Can transfer this

More Downstream RTK facts

• GTPasw Ras mediates most RTKSs. Ras has SH3, SH3
• PIP2 IS used here -P13 = bad

###Cytokines

• Cytokine Receptors Activate the JAK–STAT Signaling Pathway

TGFB receptors

Signal Proteins of the TGFβ Superfamily Act Through Receptor Serine/Threonine Kinases and Smads.

• Smads type process of transciption factor

Receptors of Latent Transcription

Protein & WNT receptors