Chapter 15: Signaling at the Cell Surface Notes

Signal Transduction

  • Introduction to signal transduction, focusing on signaling within multicellular animals.
  • Key components:
    • Signaling molecule (ligand): synthesized by one cell type.
    • Target cell: responds to the ligand via a specific receptor.
  • Signaling molecule (ligand):
    • Examples: amino acids and derivatives, acetylcholine, small peptides, full proteins.

Signal Transduction Overview

  • Signaling cell releases a ligand.
    • The secretory pathway is involved in ligand release.
  • Responding cell:
    • Cell surface receptor binds the ligand, causing a conformational change.
    • Activates a signal transduction cascade, often elevating second messenger levels.
    • Effector proteins are activated.
  • Responses:
    • Vary, including changes in cellular metabolism, cell division, differentiation, morphology, or mobility.
  • The process involves:
    • Inactive cell-surface receptor becoming active upon ligand binding.
    • Signal transduction proteins and second messengers mediating the signal.
    • Activation of effector proteins, leading to modification of cellular processes.
    • Potential modification of gene expression in the nucleus, affecting development.

Nuclear Receptor Super-Family

  • Transcription factors that bind lipophilic hormones.
  • Lipophilic hormones diffuse through the plasma membrane and act as intracellular ligands.
  • Examples:
    • Progesterone, cortisol, estradiol, testosterone, thyroxine, retinoic acid.
  • Hydrophobic signals:
    • Steroids, retinoids, thyroxine.
  • Heterodimeric Nuclear Receptors:
    • Always in the nucleus, bound to DNA.
    • Bind HAT (histone acetylase).
  • Homodimeric Nuclear Receptors:
    • Binds HDAC (histone deacetylase).

Cell Surface Receptors Overview

  • Receptor-associated kinase.
  • Cytosolic kinase.
  • Protein subunit dissociation.
  • Protein cleavage.
  • Activation or repression of genes in the nucleus.
  • Representative receptors and pathways:
    • RTKs (Receptor Tyrosine Kinases).
    • TGF-β receptors.
    • Cytokine receptors (JAK-STAT).
    • Ras/MAP Kinase.
    • GPCRs (G Protein-Coupled Receptors).
    • cAMP/PKA/CREB.
    • Wnt.
    • Notch/Delta.
    • Hedgehog.
    • NF-κB.

Types of Signaling

  • Endocrine signaling:
    • Hormone secretion into the blood by an endocrine gland.
    • Hormones act on distant target cells.
    • Example: Pancreas cells secreting insulin.
  • Paracrine signaling:
    • Secretory cell affects adjacent target cells.
    • Involves neurotransmitters or growth factors.
    • Example: Nerve cell secreting a neurotransmitter.
  • Autocrine signaling:
    • Target sites on the same cell.
    • Involves growth factors.
    • Secreted growth factors act back on the same cell; often used by cancer cells.
  • Signaling by plasma-membrane-attached proteins:
    • Signal cannot diffuse away from the source cell.
    • Adjacent target cell is required.
  • Combinations:
    • Signaling molecules can act locally or at a distance.
    • Epinephrine: paracrine and endocrine signaling.
    • EGF: plasma membrane-attached signaling, and once cleaved, autocrine or paracrine.

EGF (Epidermal Growth Factor)

  • EGF is a hormone.
  • Plasma membrane-attached EGF mediates signaling to adjacent cells.
  • Cleavage by an extracellular protease releases diffusible EGF that can function in paracrine or autocrine signaling.
  • Neu as a transmembrane protein.

Ligands and Receptors

  • Ligand: a signaling molecule or protein.
  • Binds to a receptor on the cell surface.
  • Receptor changes its conformation to initiate a cell response.
  • Different receptors for the same ligand can initiate different responses (usually in different cell types).
  • The same receptor in different cells may cause different responses (depending on the cell type).
  • Example of different responses triggered by acetylcholine:
    • Causes striated muscles to contract (receptor: acetylcholine-gated ion channel).
    • Causes smooth heart muscle to slow its rate of contraction (receptor: GPCR).
    • Causes pancreatic acinar cells to rapidly secrete enzymes (receptor: a different GPCR).

Ligands (continued)

  • A particular cell response could be triggered by different ligands and receptors.
    • Epinephrine and glucagon both cause glycogen breakdown in liver cells.
    • Both cause production of cAMP in the cytoplasm, which then serves as a second messenger.
  • Only apparent function of the ligands is to bind receptors to initiate signal transduction.
  • Ligands are not used for anything else in signaling pathways.
  • Some ligands/receptors are degraded after binding.
  • Receptor-mediated endocytosis occurs.

Cell Response

  • A particular cell response depends on:
    • Signaling pathway that is activated.
    • Proteins present in the cell that can respond to that pathway.
    • Intracellular proteins determine the response.

Cell Surface Receptor Ligands (Hydrophilic)

  • Cannot diffuse through the plasma membrane.
  • Plasma membrane receptors.
    • Peptide hormones: Insulin, growth factors (EGF, PDGF, etc.), glucagon.
    • Small charged molecules: Epinephrine (adrenaline), histamine.
    • Most derived from amino acids and function as hormones and neurotransmitters.

Small Charged Molecules

  • Examples:
    • Acetylcholine, Dopamine, Glycine, Glutamate, Serotonin, Norepinephrine, Histamine, Epinephrine, y-Aminobutyric acid (GABA).

Lipophilic Ligands

  • Ligands include the prostaglandins (PDs).
  • ~16 total prostaglandins divided into 9 classes: PDA, B, C, … I.
  • Modulate responses of other hormones in paracrine and autocrine signaling.
  • Some PDs participate in pain and inflammatory responses.
  • Most anti-inflammatory drugs like aspirin, ibuprofen, and cortisone act in part to inhibit synthesis of these prostaglandins.
  • Other PDs affect smooth muscle cells (i.e., uterus during childbirth).

Molecular Complementarity

  • Ligands bind through molecular complementarity.
  • Small patch of amino acids are essential for tight binding with receptor.
  • Interactions:
    • H-bonds and ionic interactions.
    • van der Waals interactions.
    • Hydrophobic interactions.

Binding Specificity

  • Function of molecular complementarity.
  • Binding specificity by the receptor: only one kind of ligand can bind.
  • Effector specificity: Receptor-ligand complex initiates a particular cellular response depending on responder proteins present in the cell.
  • A second receptor could bind to form a receptor dimer.

Second Messengers

  • Common second messengers in signal transduction:
    • cAMP: Activates protein kinase A (PKA).
    • cGMP: Activates protein kinase G (PKG) and opens cation channels in rod cells.
    • DAG: Activates protein kinase C (PKC) (in combination with Ca^{++}
    • IP_3: Opens Ca^{2+} channels in the endoplasmic reticulum.
    • Ca^{++}: Muscle contraction, regulated secretion, Calmodulin activation.

Intracellular Proteins

  • Protein kinases and phosphatases are employed in virtually all signaling pathways.
  • Protein Kinases:
    • Tyrosine phosphorylation.
    • Serine/Threonine phosphorylation.
  • Phosphatase activity opposes kinase activity.
  • Kinase activation often involves activation loop phosphorylation.

Intracellular Proteins cont.

  • Activities of kinases and phosphatases can be:
    • Stimulated or inhibited indirectly by a receptor.
    • Regulated by their own phosphorylation.
    • Regulated by direct contact with other proteins.
    • Regulated by the concentrations of various second messengers (such as cAMP or Ca^{++}).
  • ~3% of yeast genes are kinases or phosphatases.
  • ~3% (700/25,000) human genes are kinases or phosphatases (~600 k's, ~100 p's).

GTP-Binding Proteins

  • GTP-binding proteins are frequently used in signal transduction as on/off switches.
    • "on" when they bind GTP.
    • "off" when they bind GDP.
  • In the cell, [GTP] is ~10x higher than [GDP].
  • Exchange:
    • Active ("on")
    • Inactive ("off")
  • Ras is an important GTPase switch protein.
  • GEF (Guanine nucleotide exchange factor)
  • GAP (GTPase accelerating protein)

GTP-Binding Proteins (switch proteins)

  • Ras: associated with many human cancers.
  • Ras-like proteins: a superfamily.
    • Ran is a member: nuclear transport, NPC.
  • Trimeric G proteins that interact with cell surface receptors.
  • All have surfaces that interact with effector proteins by way of protein-protein interactions.

Guanine Nucleotide Binding Proteins

  • GTP-bound "on" state.
  • GDP-bound "off" state.
  • Switch I and Switch II regions.
  • Interaction with effector proteins.

Adapter Proteins

  • Adapter Proteins:
    • Do not have intrinsic enzyme activity.
    • Have docking sites for effector proteins.
    • Docking sites include SH2, SH3, and PTB domains.
    • SH stands for src homology.
    • PTB stands for Phospho-Tyrosine Binding.
    • SH2, PTB bind phosphotyrosine.
    • SH3 binds proline-rich sequences.

Signal Transduction Cascades

  • Second messengers often play a role in amplification.
  • Feedback repression is also often used.

Kinetics of Receptor (R) and Ligand (L) Interactions

  • The dissociation constant is a measure of the affinity of a receptor for its ligand
  • Ka = [R][L]/[RL]
  • → when 50% R has bound ligand:
  • [R] = [RL]
  • [R]/[RL] = 1
  • Ka = [L]
  • Lower K_d means higher affinity binding
  • Tight interactions are usually around 10^{-9} to 10^{-10} M
  • Weak interactions are at 10^{-5} to 10^{-7} M
  • Many Receptor-Ligand interactions are weak

Kinetics of Receptor (R) and Ligand (L) Interactions(cont)

  • If K_d = 10^{-7} M and [ligand] = 10^{-9} M in blood
    • Then ~1% of receptors are occupied
  • Increase [ligand] 10x to 10^{-8} M,
    • receptor occupancy increases to ~10%
  • Increase [ligand] 100x to 10^{-7} M,
    • receptor occupancy increases to ~50%
  • This is important for:
    • detecting and characterizing receptors
    • understanding physiologically relevant ligand concentrations
    • characterizing receptor agonists and antagonists
    • developing pharmaceuticals
    • Manipulating the formula and doing an L binding curve allows the number of receptors per cell to be estimated.

Example of one way to study a receptor in cultured cells

  • Agonists: mimic the function of hormones by binding to their receptors to induce a normal response.
  • Antagonists: bind to receptors but do not activate hormone-induced responses acting as inhibitors of the natural hormone.

Cardiac Smooth Muscle

  • Bronchial smooth muscle cells have β2 adrenergic receptors: bind catecholamines to relax.
  • Heart smooth muscle cells have β1 adrenergic receptors: bind catecholamines to increase the heart rate and contraction force.
  • β1 and β2 adrenergic receptors are GPCRs.
  • Agonists (and their effectiveness): Isoproterenol > norepinephrine > epinephrine. Isoproterenol: binds around 10x tighter (Kd ~ 10x lower).
  • Antagonist: Alprenolol: binds around 104x tighter (Kd ~ 10^4x lower), used for cardiac arrhythmia and angina slows heart contractions.

Binding Assays

  • Detect receptors, determine their affinity and specificity for ligands, and determine the number of receptors/cell.
  • Activation of only a fraction of the receptors often induces the maximal physiological response.
  • Ligand concentrations are usually much less than the Kd, so an increase in [ligand] usually leads to a proportional increase in receptor occupancy.

Factors that influence the sensitivity of a cell to external signals:

  • Kd of a receptor for a ligand.
  • Ligand concentration (usually much lower than receptor Ka).
  • Number of receptors on the cell surface.
    • More receptors → higher number occupied (even if the % is low).
    • More receptors occupied → stronger physiological response.
  • Epo Receptor example:
    • Kd of Epo Receptor for Epo: 10^{-9} M
    • 100 EpoR occupied 50% max cell response
    • If cell has 1000 EpoR, 100 will be occupied when Epo≈ 10^{-10} M
    • If cell has 200 EpoR, 100 will be occupied when Epo≈ 10^{-9} M
    • Receptors and signal transduction proteins can be purified and studied by various techniques.

Factors that influence the sensitivity of a cell to external signals

  • Kd of a receptor for a ligand.
  • Ligand concentration.
  • Number of receptors on the cell surface.
    • More receptors → higher number occupied (even if the % is low).
    • More receptors occupied → stronger physiological response.
  • Takehome:
    • The right balance between these three factors is important.
    • If the balance is off:
      • A cell will either respond when it should not OR A cell will not respond when it should

G Protein Coupled Receptors (GPCRs)

  • Called "seven-pass" receptors.
  • Amino terminus lies outside the cell.
  • Carboxy terminus lies inside the cell.
  • ~35% of pharmaceuticals target GPCRs!
  • At least 134 GPCRs are targets!
  • 4 Nobel prizes for GPCR research: 1988, 1994, 2004, 2012.
  • 5 Nobel prizes for research related to GPCRs: 1947, 1970, 1971, 1992, 2000.
  • GPCRs are important!!!!!

GPCR target

  • ~35% of pharmaceuticals target GPCRs!
  • At least 134 GPCRs are drug targets!

GPCRs

  • All GPCRs share the same basic structure.
  • Below are 3 (of 5 or more) principal classes of GPCRs that bind their ligands in different ways:

GPCR Structure

  • A typical GPCR (family A).
  • Real orientation of the membrane-spanning helices (H1-H7).
  • Ligand binding region (Family A).
  • Ligand binding causes H5 and H6 to move relative to each other.
  • Loop C3 connecting H5 and H6 changes its conformation binding the G protein.

Glucagon receptor (family B)

  • Glucagon receptor (family B): Same basic structure but with an N-terminal extracellular domain that helps with ligand binding (Glucagon is a peptide hormone).
  • GPCR: Receptor is coupled to trimeric G proteins (Ga/GBy).

Transduction Mechanism

  • G protein = alpha subunit bound to beta and gamma subunits.
  • Agonist binding.
  • GEF for Ga.
  • Effector enzyme protein.
  • Target ion channel protein.
  • G_α has intrinsic GAP activity, but Effector protein can enhance GAP activity.
  • Notice that alpha and gamma are prenylated.

GPCRs

  • Large family of receptors: ~800 encoded in the human genome.
  • Light-activated rhodopsin.
  • Several hundred odorant receptors.
  • Receptors for hormones and neurotransmitters.
  • 3% to 4% of genes!
  • All bind G proteins (alpha subunit of a trimeric complex).
  • G proteins are "switch proteins" that bind GTP/GDP.
  • Classic example of GPCR is the epinephrine receptor:
    • Epinephrine is produced by the adrenal glands (aka adrenaline).
    • Released in times of fright or heavy exercise.
    • Glucose is needed quickly; glucose provided by hydrolysis of glycogen (glycogenolysis).
    • Fatty acids are also mobilized from triacyl-glycerol in adipose cells (lipolysis).

GPCR Activation

  • GPCRs act as the GEF for trimeric G proteins, activating Go by causing the exchange of GDP for GTP.
  • This leads to separation of GBy from Ga
  • This separation can be detected by FRET.

Different G proteins.

  • Different G proteins are activated by different GPCRs and in turn regulate different effector proteins.

GPCRs

  • Gas/Gai Exterior.
  • Effect on adenylyl cyclase.
  • Chimeric receptors (mixing and matching domains from the different receptors) were made to define functions of the individual domains.
  • Region determining specificity of G protein binding (compare chimeras 1 and 2).

Epinephrine (aka adrenaline) binds:

  • β-adrenergic receptors on hepatocytes and adipose cells to release glucose and fatty acids for quick energy (ATP) production (striated muscle cells, too).
  • β-adrenergic receptors on heart muscle cells to increase contraction rates and force; to better transport oxygen and nutrients to muscle cells.
  • β-adrenergic receptors on smooth muscle cells of the intestines causing them to relax to temporarily shut down digestive functions.
  • α₂ adrenergic receptors in smooth muscles lining blood vessels of skin, intestine and kidney to constrict vessels to cut off blood flow to shunt blood to skeletal muscles, heart, and brain.
  • "looks like you've seen a ghost"