Signal Transduction Mechanisms:

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Chapter 14

• Signal Transduction Mechanisms
• II. Messengers and Receptors

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Signal Transduction
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Cellular Communication

• Cells must communicate with one another at either
  a distance or nearby
• Communication is determined by chemical signals
  and cellular receptors
• Nervous system will regulate tissue function by
  its innervation of the tissue
• Regulation is by the release of chemical
  messengers

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Types of Chemical Signals

• Classified by distance between made and use
• Eendocrine signals produced at a great distance
  from the target tissue hormones
• Paracrine signals released locally, acting on
  nearby tissues growth factors
• Autocrine signals act on cells that secreted it

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Signal Transduction

• Messenger (ligand) binds to the receptor on cell
  surface, steroid hormones bind on internal
  receptors
• 1 messenger is the ligand that binds the
  receptor and results in the production of other
  molecules within the cell receiving signal
• 2 messenger is the small molecules or ions that
  relay signals from one location in the cell to
  another
• The 2 messenger then can affect expression of
  genes or changes in the identity or function of
  the cell

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Messengers

• Amino acids or derivatives of them
• Peptides
• Proteins
• Fatty acids
• Lipids
• Nucleosides
• Nucleotides

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Hydrophobic Messengers

• Act on receptors in the nucleus or cytosol
• Receptors function to regulate transcription of
  genes
• Messengers
• Steroid hormones derivative of cholesterol
• Retinoids derivative of Vitamin A

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Hydrophilic Messengers

• Not important in what the message is but that
  they provide the correct molecular shape and
  bonding characteristics to fit in the binding
  site
• Ligand binds to the binding site or pocket by
  several non-covalent bonds
• The fit between the receptor and the ligand must
  be perfect to achieve the specificity

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Receptor Binding

• Ligand and its cognate receptor interaction is
  similar to enzyme/substrate interaction
• When a receptor has a ligand bound, it is said to
  be occupied
• Amount of receptor occupied is proportional to
  the ligand free in the cytosol
• Can increase the ligand to increase the number
  of receptors bound until all are bound then no
  matter what the ligand no increase in binding
  will occur saturation

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Receptor Affinity

• ligand in solution and the number of receptors
  occupied
• When almost all receptors are occupied when
  ligand is low the receptors affinity is HIGH
• When it takes high ligand to occupy most
  receptors, then the affinity is LOW
• Dissociation constant (Kd) is the free ligand
  required to produce ½ the receptors occupied
• High affinity receptors have low Kd
• Low affinity receptors have high Kd
• Ligand must be in the range of Kd to effect the
  target tissue

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Receptor Down-Regulation

• Cells can sense the changes in ligand
• Ligand bound to the receptor can cause
  desensitization or tolerance of the target cell
  to the ligand and will then require higher
  ligand to elicit a response
• Desensitization is due to changes in properties
  or location of the receptor known as receptor
  down-regulation
• Desensitization leads to tolerance loss of
  effectiveness

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3 Methods of Down-Regulation

• 1) Remove the receptor from the cell surface
• Receptor-mediated endocytosis
• Reduced receptor will yield diminished cellular
  activity
• 2) Alterations in the receptor that lowers the
  affinity for the ligand or 3) alterations so
  cannot initiate changes in cell function
• Altered by phosphorylation
• Remain on the surface but cannot respond to ligand

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Drug Design

• Drug companies take advantage of ligand binding
  to create therapeutic drugs that either activate
  or inhibit receptors on the target cell
• Antagonists prevents binding of ligand and
  receptor activation
• Agonists activates the receptor

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Signal Transduction

• Cognate receptor binds ligand
• Causes changes in cellular activities
• Initiates a pre-programmed sequence of events and
  uses cellular processes that are rarely used
• Signal amplification caused by small amount of
  signal with a big response from the cell

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2 Types of Receptors

• G proteins
• Protein kinases

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G-Protein Linked Receptor Family

• Change in the receptor causes a change in the G
  protein
• G protein is a guanine-nucleotide binding protein
• Signal transduction occurs when an active G
  protein binds the target (enzyme or channel
  protein) that then alters the targets activity

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G-Protein Linked Receptor

• Most have similar structure but with significant
  AA differences
• 7 trans-membrane spanning ? helices with
  alternating intra- and extra-cellular loops
• N terminus is extracellular and C terminus is
  intracellular
• Unique message binding site on extracellular
  surface
• Loop between 5th and 6th ? helix is specific for
  G proteins
• Different ligands mean different signal
  transduction pathways

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Activation of G Proteins

• On or off is determined by whether the G
  protein has GTP or GDP bound
• 2 classes of G proteins
• Large heteromeric 3 subunits - G? G? G?
• Small monomeric Ras (not called G protein)
• G? is the largest subunit and that which binds
  GTP or GDP
• G? binds GTP and detaches from G?? (permanently
  bound together)
• Gs stimulates signal transduction
• Gi inhibits signal transduction

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G Protein Activation

• Messenger binds at binding site, causes
  conformation change in the receptor and then the
  G protein binds
• G? releases GDP and acquires a GTP and is now
  active detaches from the complex
• Either G? or G?? can initiate transduction
  depending on the cell and the G protein
• Each part can effect by binding an enzyme or
  other protein
• Persists only as long as G? is bound to GTP and
  subunit is separated
• G? cleaves its own GTP, only active for a short
  time, re-associates with G??

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G Protein Activation
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Second Messengers

• Most important G proteins release or form 2nd
  messengers
• Usually cAMP or Ca2 ions
• cAMP is made from cytosolic ATP by adenylyl
  cyclase

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Activation of Adenylyl Cyclase

• Adenylyl cyclase is in the membrane as an
  inactive enzyme without G? of the Gs bound
  (stimulatory G protein)
• Ligand binding stimulates the release of GDP from
  Gs? and binds GTP
• Gs? leaves the complex and binds adenylyl cyclase
  now active and can convert ATP to cAMP
• Response is quick but Gs? hydrolyzes GTP to GDP
  to shu down G protein
• High levels of cAMP is broken down by
  phosphodiesterase
• Helps to shut off the transduction pathway

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cAMP Dependent Kinase

• Also called protein kinase A (PKA)
• cAMP binds to PKA and causes the detachment of 2
  regulatory units from the 2 catalytic subunits
• PKA can then phosphorylate wide range of enzymes
  on Ser or Thr
• Specific for each cell

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Response to cAMP

• Increases in cAMP cause many different responses
• In liver cells breakdown glycogen
• In heart cells strengthens contratraction
• In smooth muscles contraction inhibited
• In blood platelets inhibits mobilization during
  clotting
• In intestinal cells secretes salts and water
  into lumen
• Can see the above when you increase the cAMP
  production directly or by inhibiting
  phosphodiesterase
• Phosphodiesterase inhibitors methylxanthines
  found in caffeine, tea and soft drinks

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Diseases Caused by cAMP

• Vibrio cholera - Gs? cant cleave GTP to GDP and
  not turned off
• large amount of salt and water leave in the
  intestine, leads to dehydration and possible
  death
• Bordetella pertussis toxin acts on Gi
  (inhibitory G protein) so cant inhibit adenylyl
  cyclase
• not sure how it causes whooping cough

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Other 2nd Messengers

• By-products of inositol phospholipids (PIP2) in
  membrane caused by cleavage with phospholipase C
  when activated
• Inositol triphosphate (IP3) and diacylglycerol
  (DAG)

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Pathways Triggered by IP3 and DAG
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Inositol-Phospholipids Ca Pathway

• Ligand binds membrane receptor and activates
  specific G protein called GP (P is for PLC) which
  in turn activates a form of phospholipase C (PLC)
  known as C? making IP3 and DAG
• IP3 diffuses quickly to IP3-receptor
  (ligand-gated Ca channel) and binds
• Ca channel opens and Ca rushes in binds
  calmodulin and complex activates desired
  physiological processes

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Inositol-Phospholipids Ca Pathway (cont)

• DAG stays in membrane and activates protein
  kinase C (PKC) can phosphorylate on Ser or Thr
  on target proteins
• Activates
• stimulates cell growth
• regulation of ion channels
• changes in the cytoplasm
• increases cellular pH
• effects on secretion of proteins and other
  substances

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Ca Regulation

• Ca levels are low in cytosol (10,000 x lower than
  outside cell) because of Ca pumps in the membrane
  that pumps it out and the ER that sequesters Ca
  in the lumen
• Some cells have Na/Ca exchangers
• Mitochondria also pump Ca into their matrix
• IP3 receptor channels function to increase Ca
  levels from intracellular stores
• Ryanodine receptor channel releases Ca in the
  sarcoplasmic reticulum to stimulate muscle
  contraction in skeletal and cardiac muscle
• Ca induced Ca release

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Ca-CalmodulinComplex

• Calmodulin is a protein that regulates a variety
  of cellular processes, has 4 binding sites for Ca
  at 2 locations in the protein
• When Ca is bound, the complexes conformation
  changes and it can wrap around the target protein
• Calmodulin will bind Ca only when the Ca
  increases due to a certain stimulus levels go
  down, release Ca
• Calmodulin binding proteins are enzymes like
  kinases and phosphatases cell response depends
  on binding proteins in cell

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Ca and Egg Fertilization

• When the sperm penetrates the egg, a wave of Ca
  release spreads across the surface
• 2 functions
• stimulates fusion of cortical granules so surface
  changes so that no additional sperm enter the egg
• Egg activation resumption of metabolic
  processes to initiate embryo development

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Wave of Ca Moving Across Egg
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Nitric Oxide (NO)

• NO synthase breaks down arginine into NO and
  citrulline
• NO is a signal molecule in the endothelium of the
  smooth muscle of the cardio-vascular system
  involves G proteins
• Allows for the relaxation of the muscle fibers

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Mechanism of NO

• Acetylcholine binds to receptor (G-protein
  linked) and get IP3 produced in the endothelial
  cells
• IP3 causes release of Ca from ER
• Ca binds calmodulin that stimulates NO synthase
  to make NO
• NO diffuses out to adjacent smooth muscle
• Activates guanylyl cyclase to make cGMP from GTP
  (similar to cAMP generation)
• cGMP activates protein kinase G and
  phosphorylates appropriate protein for muscle
  relaxation

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Drugs Affecting NO

• Nitroglycerine is given to patients with angina
  constriction of blood vessel causing decrease
  blood flow and chest pain
• NO is released in response to the drug acting as
  acetylcholine
• Viagra inhibits the cGMP phosphodiesterase so
  cGMP levels stay high and smooth muscle is
  relaxed and blood vessels remain dilated and you
  can sustain an erection longer