Cell Signaling Notes

Cell Signaling

Trimeric G Proteins

• Four major families of trimeric G proteins are categorized by the amino acid sequence relatedness of their α subunits.
• Humans have approximately 20 α subunits, at least 6 β subunits, and 11 γ subunits.
• Examples of G protein families and their functions:
  • Gs: Activates adenylyl cyclase and Ca2+ channels.
  • Golf: Activates adenylyl cyclase in olfactory sensory neurons.
  • Gi: Inhibits adenylyl cyclase; activates K+ channels; inactivates Ca2+ channels.
  • Gq: Activates phospholipase C-β.
  • Gt (transducin): Activates cyclic GMP phosphodiesterase in vertebrate rod photoreceptors.
  • G12/13: Activates Rho family monomeric GTPases (via Rho-GEF) to regulate the actin cytoskeleton.

Cell Signaling Through Second Messengers

• Overview:
  • A signal molecule binds to a receptor (e.g., GPCR, RTK) on the plasma membrane.
  • This activates intracellular signaling pathways involving G proteins, adenylyl cyclase, phospholipase C, Ras-GEF, PI 3-kinase, etc.
  • Second messengers (e.g., cyclic AMP, Ca2+, diacylglycerol, IP3) are produced.
  • Protein kinases (e.g., PKA, CaM-kinase, PKC, MAP kinase, Akt kinase) are activated.
  • Transcription regulators and other target proteins are phosphorylated, leading to cellular responses.

Cyclic AMP (cAMP)

• cAMP concentrations can change rapidly in response to stimuli such as serotonin.
• Second Messenger Role:
  • G proteins are membrane-bound and activate membrane-bound enzymes.
  • These enzymes produce small molecules referred to as second messengers.
  • Second messengers diffuse through the cytoplasm to spread the signal.
  • Cyclic AMP is a common second messenger of GPCRs.
  • The activated α subunit of a G protein activates adenylyl cyclase.
  • Adenylyl cyclase generates cAMP from ATP by catalyzing the removal of two phosphates.
  • cAMP phosphodiesterase linearizes cAMP, terminating the signal. Ordinary AMP does not continue to signal in the cAMP pathway.

Hormone-Induced Cell Responses Mediated by Cyclic AMP

• Examples:
  • Thyroid gland (TSH): Thyroid hormone synthesis and secretion.
  • Adrenal cortex (ACTH): Cortisol secretion.
  • Ovary (Luteinizing hormone): Progesterone Secretion.
  • Muscle (Adrenaline): Glycogen breakdown.
  • Bone (Parathormone): Bone resorption.
  • Heart (Adrenaline): Increase in heart rate and force of contraction.
  • Liver (Glucagon): Glycogen breakdown.
  • Kidney (Vasopressin): Water resorption.
  • Fat (Adrenaline, ACTH, glucagon, TSH): Triglyceride breakdown.

Cyclic AMP-Dependent Kinase (PKA)

• PKA is the primary target of cAMP.
• Structure:
  • Consists of four subunits: two regulatory and two catalytic.
  • cAMP binds to the regulatory subunits, causing the release and activation of the catalytic subunits.
• Function:
  • PKA activates effector proteins to trigger a cellular response.
  • PKA can phosphorylate and activate many proteins, such as phosphorylase kinase.
  • Phosphorylase kinase can phosphorylate and activate different effector proteins (e.g., glycogen phosphorylase, which catalyzes glycogen breakdown).
  • PKA is a serine/threonine kinase.
  • Activated PKA can enter the nucleus through the nuclear pore complex.
  • PKA phosphorylates and activates transcription factors in the nucleus.
  • Transcription factors activate target gene transcription.

Health Focus: GPCR Signaling Gone Bad

• Cholera Toxin:
  • Vibrio cholerae produces cholera toxin.
  • The A subunit of the toxin modifies a G protein, causing it to remain constitutively active.
  • This leads to continuous activation of adenylyl cyclase, increasing cAMP levels.
  • The increased cAMP causes epithelial cells to secrete excessive amounts of Cl- ions, leading to water loss and diarrhea.
• Pertussis Toxin:
  • Pertussis toxin inhibits the activity of adenylyl cyclase, decreasing cAMP levels.

Phospholipase C Pathway

• Another GPCR-activated second messenger pathway involves activation of phospholipase C (PLC).
• Phospholipase C (PLC) is a membrane-bound enzyme.
• Activated α or βγ subunits of G proteins activate phospholipase C.

Phosphatidyl Inositol Bisphosphate

• Phospholipase C acts on its substrate, phosphatidyl inositol bisphosphate (PI(4,5)P2).
• Phosphatidyl inositol (PI):
  • A membrane phospholipid comprising a small percentage of membrane lipids.
• PLC Cleavage:
  • PLC cleaves PI(4,5)P2 into two molecules:
    • Membrane-bound diacylglycerol (DAG).
    • Inositol triphosphate (IP3).
  • Both DAG and IP3 are second messengers.

IP3 and DAG Signaling

• DAG:
  • Recruits protein kinase C (PKC) to the cell membrane and activates it.
  • PKC also needs Ca2+ to be activated.
  • PKC activates effector proteins through phosphorylation.
• IP3:
  • Binds to a Ca2+ channel in the ER membrane.
  • Ca2+ is released as another second messenger.

Calcium Signaling

• A wave of Ca2+ release can be observed following fertilization of an egg.

Calmodulin

• Calmodulin is a critical Ca2+-dependent effector protein.
• Activation:
  • Calmodulin binds four Ca2+ ions.
  • Ca2+ induces a conformational change in Calmodulin.
  • The conformational change allows calmodulin to bind to target effector proteins.

CaM Kinases

*CaM kinases are Ca²+/calmodulin effector proteins.

CaM-Kinase II

• Structure:
  • Twelve identical subunits, each with kinase activity and the ability to bind Ca2+/calmodulin.
• Properties:
  • Has multiple states of partial activation.
  • Exhibits a form of “memory”.

Biochemical Techniques in Cell Signaling

• Determining Signaling Pathway Order:
  • Example: A signaling pathway involves three proteins: Ras, protein X, and protein Y.
  • Using cells with mutant proteins, the order can be deduced by introducing an overactive form of one protein and observing whether signaling is restored.
  • If introducing overactive Ras restores signaling in a cell with mutant protein X, but not in a cell with mutant protein Y, this indicates that X is upstream of Ras, and Y is downstream.

Designing Experiments in Cell Signaling

• Manipulating the System:
  • Experiments often perturb the “normal” state to unravel a signaling pathway.
  • Techniques rely heavily on genetic manipulation and/or drug treatments.
• Defining the Readout:
  • Must have an idea of the expected outcome.
  • Can examine the cell or organism as a whole, or can be more targeted and specific.

Methods for Manipulating the System

• Express Mutant Versions of Proteins:
  • Overactive/Constitutively Active: Can’t deactivate.
  • Can’t activate.
  • Mimic a disease mutation.
  • Remove a phosphorylation site.
  • Remove an NLS (Nuclear Localization Signal).
• Manipulate Gene Expression:
  • Overexpress a gene of interest.
  • Knock down gene expression.
  • Knock out a gene.
• Drugs/Toxins:
  • Agonists and antagonists: Activate or block a known component of a pathway.
  • Could also be the known extracellular signaling molecule.
• Disease State:
  • Take cells from a patient with a disease.
  • Create an animal model that gets the disease.

Removing Phosphorylation Sites

• Removing phosphorylation sites can reveal how proteins bind to one another.
• By mutating tyrosine residues (Y) to alanine, phosphorylation at those sites is prevented.
• If a protein fails to bind to a receptor when a specific tyrosine residue is mutated, it indicates that phosphorylation at that site is necessary for binding.