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Membranes pt. 2

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Unit 4 pt. 2

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Membranes pt. 2

  1. 1. Proteins & Signaling Membranes ~ Part II
  2. 2. Maintaining Homeostasis <ul><li>Cells must communicate with their external environment </li></ul><ul><li>Monitoring external conditions determines cellular responses </li></ul><ul><li>Example – E. coli: </li></ul><ul><ul><li>If the bacteria detects a high concentration of lactose, it synthesizes proteins to import and metabolize lactose </li></ul></ul><ul><ul><li>If it detects a higher concentration of glucose, it it synthesizes proteins to import and metabolize gluose </li></ul></ul><ul><li>Membrane proteins help gather information about the environment </li></ul>
  3. 3. Cellular Communication <ul><li>In multi-cellular organisms, communication is more complex </li></ul><ul><li>Each cell communicates with dozens of other cells </li></ul><ul><li>Determines: </li></ul><ul><ul><li>When it should grow </li></ul></ul><ul><ul><li>When it should differentiate or die </li></ul></ul><ul><ul><li>When it should release protein products needed by other cells </li></ul></ul>
  4. 4. Mechanisms of Cell Communication
  5. 5. Communication Through Contact
  6. 6. Two Types of Membrane Proteins <ul><li>The membrane is a barrier </li></ul><ul><li>Prevents interchange of materials </li></ul><ul><ul><li>Special channels are needed to transport some materials into & out of the cell </li></ul></ul><ul><li>It also prevents free exchange of information </li></ul><ul><ul><li>Special receptors are needed to gather information </li></ul></ul><ul><li>Therefore the cell membrane has 2 major types of proteins: </li></ul><ul><ul><li>Transporters </li></ul></ul><ul><ul><li>Receptors </li></ul></ul>
  7. 7. Intrinsic & Extrinsic Proteins <ul><li>Intrinsic membrane proteins </li></ul><ul><ul><li>Embedded in the lipid bilayer </li></ul></ul><ul><ul><li>Some extend through it </li></ul></ul><ul><ul><li>Transmembrane proteins </li></ul></ul><ul><li>Extrinsic membrane proteins </li></ul><ul><ul><li>Absorbed to the surface of the lipid bilayer </li></ul></ul><ul><ul><li>Can be separated from the lipid bilayer without destroying the membrane </li></ul></ul>
  8. 8. Transmembrane Proteins <ul><li>Intrinsic proteins that extend from one side of the membrane to the other are transmembrane proteins. </li></ul><ul><li>Cells pump ions in and out through their plasma membranes. </li></ul><ul><li>More than half the energy that we consume is used by cells to drive the protein pumps in the brain that transport ions across plasma membranes of nerve cells. </li></ul><ul><li>How can ions be transported across membranes that are effectively impermeable to them? </li></ul>
  9. 9. Ligand Gated Ion Channel
  10. 10. Domains <ul><li>Many transmembrane proteins have three different domains : </li></ul><ul><li>A hydrophilic domain at the N-terminus </li></ul><ul><ul><li>consists of hydrophilic amino acids </li></ul></ul><ul><ul><li>pokes out in the extracellular medium </li></ul></ul><ul><li>A hydrophobic domain in the middle of the amino acid chain </li></ul><ul><ul><li>often only 20-30 amino acids long </li></ul></ul><ul><ul><li>threaded through the plasma membrane </li></ul></ul><ul><ul><li>made of amino acids having hydrophobic side chains </li></ul></ul><ul><li>A hydrophilic domain at the C-terminus </li></ul><ul><ul><li>protrudes into the cytoplasm. </li></ul></ul>
  11. 11. Glycoproteins <ul><li>Many transmembrane proteins are glycoproteins </li></ul><ul><li>Sugar side chains are covalently attached to the hydrophilic domains that protrude into the extracellular membrane. </li></ul><ul><li>A typical mammalian cell may have several hundred distinct types of glycoproteins studding its plasma membrane. </li></ul><ul><li>Each glycoprotein has its extracellular domain glycosylated with a complex branching bush of sugar residues covalently linked to the asparagine side chains. </li></ul><ul><ul><li>Some glycoproteins may have 2 or 3 asparagine- linked sugar side chains, others may have dozens. </li></ul></ul>
  12. 12. Multi-membrane Spanning Proteins <ul><li>Some transmembrane proteins have multiple transmembrane domains. </li></ul><ul><li>Hydrophilic domains alternate with hydrophobic domains. </li></ul><ul><li>The protein chain weaves back and forth between opposite sides of the plasma membrane. </li></ul><ul><li>Called serpentine membrane proteins b/c they are snake-like </li></ul><ul><ul><li>A common structure in many serpentine transmembrane proteins involves 7 hydrophobic domains inserted into the plasma membrane, separated by hydrophilic regions that are looped out alternatively into either the cytoplasm or the extracellular space = 7 membrane spanning proteins </li></ul></ul>
  13. 13. Receptors <ul><li>Specialized transmembrane proteins that acquire information from the extracellular space </li></ul><ul><li>Relay this information into the cell through the plasma membrane </li></ul><ul><li>Cell surface receptors act as the antennae of the cell. </li></ul><ul><li>Mammalian cells have wide variety of transmembrane receptors </li></ul><ul><li>Two important types: </li></ul><ul><ul><li>Growth Factor Receptors </li></ul></ul><ul><ul><li>G Protein Receptors </li></ul></ul>
  14. 14. Growth Factor Receptors <ul><li>Help the cell determine whether or not it should grow by binding growth factors </li></ul><ul><li>Growth factors may be present in the medium around the cell </li></ul><ul><ul><li>Sometimes called mitogens because they induce the cell to grow and pass through mitosis </li></ul></ul><ul><ul><li>They are polypeptides, often 50-100 amino acids long. </li></ul></ul><ul><li>When present in sufficient quantity, a growth factor (GF) will stimulate a cell to enter into a round of growth and division. </li></ul>
  15. 15. Specificity of Binding <ul><li>GFs bind to cell surface GF receptors. </li></ul><ul><li>Each type of GF binds to the extracellular domain of its own specific receptor </li></ul><ul><ul><li>will not bind to receptors for other growth factors. </li></ul></ul><ul><li>Each type of receptor binds specifically to its own ligand </li></ul><ul><ul><li>accommodates the appropriate growth factor in a lock-and-key fashion </li></ul></ul>
  16. 16. Variety of Ligand: Receptor Pairs <ul><li>Other ligands besides growth factors convey signals from cell to cell through intercellular space. </li></ul><ul><li>There are at least several hundred distinct receptor: ligand pairs in our body </li></ul><ul><li>Each devoted to the binding of a distinct extracellular ligand such as a growth factor to its cognate receptor. </li></ul><ul><li>Each ligand originates elsewhere and is secreted by a cell or cells specialized for its release. </li></ul>
  17. 17. Transmembrane Signal Transduction <ul><li>The binding of a ligand to its receptor is the beginning of the signalling process. </li></ul><ul><li>How does the interior of the cell learn that the ligand has bound? </li></ul><ul><li>How is this translated into information the cell can use? </li></ul><ul><li>Transmission of information by a protein is a form of signal transduction. </li></ul>
  18. 18. An Overview of Cell Signaling
  19. 19. Structure GF Receptor Proteins <ul><li>Outside the cell, they have a ligand-binding N- terminal ectodomain </li></ul><ul><li>Inside is a single membrane-spanning transmembrane domain. </li></ul><ul><li>At their C-termini in the cytoplasm, they have a specialized enzyme domain </li></ul><ul><ul><li>This becomes activated whenever the extracellular domain of the receptor binds a GF ligand. </li></ul></ul><ul><ul><li>In the case of many GF receptors, the cytoplasmic enzyme domain contains protein kinase activity. </li></ul></ul>
  20. 20. Kinases & Signal Transduction <ul><li>Kinases are enzymes that attach phosphate groups to their substrates. </li></ul><ul><li>Protein kinases take the gamma-phosphates from ATP and transfer them to protein substrates, resulting in the phosphorylation of the substrate proteins. </li></ul><ul><li>The phosphate groups are attached to the tyrosine side chains of substrate proteins that communicate with or lie near the cytoplasmic domains of the GF receptors. </li></ul><ul><li>These receptors are considered to have protein tyrosine kinase activity to distinguish them from many other protein kinases that are devoted to other signalling functions. </li></ul>
  21. 21. Sequence of GF Signal Transduction <ul><li>The GF ligand binds to the extracellular domain of its receptor. </li></ul><ul><li>This activates the tyrosine kinase domain at the other end of the receptor in the cytoplasm. </li></ul><ul><li>The tyrosine kinase becomes active and phosphorylates a series of cytoplasmic substrate proteins. </li></ul><ul><li>These are activated or altered functionally as a consequence of being phosphorylated. </li></ul><ul><li>They then send signals further into the cell that result in the cell growing and dividing. </li></ul>
  22. 22. An External Event <ul><li>GF ligand does not need to enter the cell in order for transmembrane signalling to occur. </li></ul><ul><li>All active transmembrane signal transduction occurs while the ligand is still in the extracellular space. </li></ul>
  23. 23. Mechanism of Kinase Activation <ul><li>How does the association of GF ligand outside the cell cause tyrosine kinase activation inside the cell? </li></ul><ul><li>Some considerations: </li></ul><ul><ul><li>There are many copies of each type of GF receptor molecule that are displayed on the surface of a given cell. </li></ul></ul><ul><ul><li>These receptor molecules, while tethered in the plasma membrane via their hydrophobic transmembrane domains, diffuse laterally through the plane of the plasma membrane. </li></ul></ul>
  24. 24. Dimerization <ul><li>When a GF ligand binds to a single receptor molecule, it encourages the dimerization of the receptor with another receptor molecule floating in the plasma membrane. </li></ul><ul><li>Often the GF ligand itself has two receptor-binding ends </li></ul><ul><ul><li>enables it to serve as a bridge between the two receptors </li></ul></ul><ul><ul><li>attracts two receptors </li></ul></ul><ul><ul><li>encourages their dimerization </li></ul></ul><ul><ul><li>stabilizes the resulting receptor/ dimer pair. </li></ul></ul>
  25. 25. Passing the Message <ul><li>Dimerization pulls the cytoplasmic domains of the two receptor molecules closer. </li></ul><ul><li>The tyrosine kinase (TK) of one receptor molecule then phosphorylates the kinase domain of the second receptor molecule </li></ul><ul><li>This phosphorylation results in a steric shift in the 3-dimensional structure of the phosphorylated kinase domain </li></ul><ul><li>This causes its functional activation. </li></ul>
  26. 26. Tyrosine Kinase Receptor Dimers
  27. 27. The Final Steps <ul><li>The two kinase domains phosphorylate and thereby activate each other. </li></ul><ul><li>Once they are activated, they phosphorylate nearby cytoplasmic substrate proteins that then pass signals further into the cell. </li></ul>
  28. 28. Phosphorylation Cascade
  29. 29. 7 Membrane-spanning Serpentine Receptors Varied Functions <ul><li>Receptors on cells of the tongue convey taste. </li></ul><ul><li>Hundreds of receptor types in our nose convey information about odors. </li></ul><ul><li>A carotenoid molecule related to vitamin A binds rhodopsin in the rods and cones of our eyes. </li></ul><ul><ul><li>It picks up photons which alters its conformation, and causes the receptor to which it is bound to release signals into the rod/cone cytoplasm that result in our perception of light. </li></ul></ul><ul><li>Baker's yeast cells communicate their sexual identity to each other by release of polypeptide mating factors that bind this type of receptor </li></ul><ul><li>Epinephrine controls the “flight or fight response” </li></ul>
  30. 30. Exchange of Yeast Mating Factors
  31. 31. A G-Protein Receptor
  32. 32. The Role of Epinephrine <ul><li>Also known as adrenaline </li></ul><ul><li>Released by the adrenal glands above the kidneys in response to stressful stimuli. </li></ul><ul><li>Epinephrine travels through the blood stream and binds to specific receptors on cells in various tissues throughout the body. </li></ul><ul><li>This results in the mammalian fight / flight reaction. </li></ul><ul><li>This includes: </li></ul><ul><ul><li>increased heart rate, </li></ul></ul><ul><ul><li>decreased blood flow to gut </li></ul></ul><ul><ul><li>increased blood flow to skeletal muscles </li></ul></ul><ul><ul><li>increased blood glucose </li></ul></ul>
  33. 33. Tracing One Action <ul><li>Epinephrine acts at many sites to produce a wide array of physiologic changes </li></ul><ul><li>One of these is increased blood glucose </li></ul><ul><li>Epinephrine causes liver and muscle cells to break down glycogen and release the resulting glucose into the circulation </li></ul><ul><li>We will trace this one action of epinephrine </li></ul>
  34. 34. How Epinephrine Acts <ul><li>Epinephrine binds to its receptor on the surface of a variety of cell types throughout the body. </li></ul><ul><li>This beta adrenergic receptor is a 7 membrane-spanning, serpentine receptor embedded in the plasma membranes of these cells. </li></ul><ul><li>As is the case with the growth factor receptors, the epinephrine ligand is not internalized into the cell. </li></ul><ul><li>While bound for a short period of time to its receptor, epinephrine causes the latter to release biochemical signals into the cell cytoplasm. </li></ul>
  35. 35. The Epinephrine Receptor <ul><li>These receptors do not depend upon receptor dimerization to transduce signals across the plasma membrane. </li></ul><ul><li>Instead, single receptor molecules will change their 3 dimensional steric configuration in response to ligand binding. </li></ul><ul><li>This steric shift affects the configuration of the cytoplasmic domains of the receptor (the loops of receptor protein that protrude into the cytoplasm). </li></ul>
  36. 36. Cytoplasmic Signal Transduction <ul><li>The receptor communicates with the cytoplasm by stimulating a second protein </li></ul><ul><li>This is known as a G protein (G = guanine) </li></ul><ul><li>The G protein normally lies near the receptor in an inactive, quiet state. </li></ul><ul><li>When the receptor is activated by ligand binding, it pokes the G protein. </li></ul><ul><li>The G protein responds by switching itself on, into an active state. </li></ul><ul><li>Once in the active state, the G protein sends signals further into the cell. </li></ul>
  37. 37. The G Protein is Binary <ul><li>The G protein remains in the active state for only a brief period, after which it shuts itself off. </li></ul><ul><li>The G protein's two states (ON or OFF) are determined by guanine nucleotide which it binds </li></ul><ul><ul><li>thus the term G protein </li></ul></ul><ul><li>When it is inactive, it binds GDP </li></ul><ul><li>When active, it binds GTP. </li></ul>
  38. 38. GTP Binding Activates the Protein <ul><li>The resting, OFF form of the G protein sits around with its bound GDP. </li></ul><ul><li>When a ligand-activated receptor pokes it, the G protein releases its bound GDP </li></ul><ul><li>It then allows a GTP molecule to jump aboard. </li></ul><ul><li>The GTP-bound form of the G protein is the active ON state. </li></ul><ul><li>While in the ON state, it releases downstream signals. </li></ul>
  39. 39. Feedback Regulation <ul><li>After a short period of time (seconds or less), the G protein hydrolyzes its own GTP back to GDP . . . </li></ul><ul><li>Thus shutting itself off. </li></ul><ul><li>This hydrolysis represents a negative feedback mechanism </li></ul><ul><li>Ensures that the G protein is only in the active, signal- emitting ON mode for a short period of time. </li></ul>
  40. 40. Structure of the G Protein <ul><li>Composed of 3 subunits: alpha, beta & gamma </li></ul><ul><li>In its inactive OFF state, the 3 subunits are bound together </li></ul><ul><li>The alpha subunit binds the guanine nucleotide, in this case GDP. </li></ul><ul><li>When the beta adrenergic receptor activates the G protein, the alpha subunit releases GDP, </li></ul><ul><li>then binds GTP, </li></ul><ul><li>and falls away from the beta and gamma subunits. </li></ul>
  41. 41. The Signaling Cascade <ul><li>Once GTP is bound, the GTP-bound alpha subunit also loses affinity for the receptor. </li></ul><ul><li>It dissociates from receptor, </li></ul><ul><li>moves over and pokes another nearby protein </li></ul><ul><li>the enzyme adenylate cyclase, </li></ul><ul><li>which is activated by being poked, </li></ul><ul><li>and cyclizes ATP into 3'5' cyclic AMP. </li></ul>
  42. 42. The Second Messenger <ul><li>cAMP is a second messenger </li></ul><ul><li>After G protein encounters adenyl cyclase enzyme, the alpha subunit of the G protein hydrolyzes its bound GTP and releases the adenyl cyclase </li></ul><ul><ul><li>Thus, the G protein reverts to an inactive OFF signalling state. </li></ul></ul><ul><ul><li>The alpha subunit rejoins the beta and gamma subunits </li></ul></ul><ul><li>Adenyl cyclase, no longer poked by the activated a subunit of the G protein, shuts down </li></ul><ul><ul><li>stops making cAMP from ATP </li></ul></ul><ul><li>The whole cycle results in only a brief signaling pulse </li></ul><ul><ul><li>the production of several hundred cAMP molecules </li></ul></ul>
  43. 43. Cyclic AMP
  44. 44. Second Messenger Action <ul><li>Once made, cAMP molecules act as intracellular glycogen </li></ul><ul><li>The high cAMP concentrations enable A kinase to </li></ul><ul><li>phosphorylate and thereby activate an enzyme, that </li></ul><ul><ul><li>activates glycogen phosphorylase, which in turn </li></ul></ul><ul><ul><li>breaks down glycogen into glucose-l-phosphate molecules; and </li></ul></ul><ul><li>phosphorylate glycogen synthase, which </li></ul><ul><ul><li>turns it off, </li></ul></ul><ul><ul><li>preventing the reconversion of the released glucose to glycogen. </li></ul></ul>
  45. 45. cAMP Second Messenger System
  46. 46. Effect of cAMP on Blood Glucose <ul><li>These two changes together ensure the mobilization of glucose through the breakdown of glycogen stored in the liver. </li></ul><ul><li>A number of other reactions are triggered as well that together contribute to the fight/flight response. </li></ul>
  47. 47. Signal Amplification <ul><li>There is enormous signal amplification in this cascade. </li></ul><ul><li>A single epinephrine molecule (present at 1O -10 M) may cause the activation of dozens of alpha subunits of proteins. </li></ul><ul><li>Each of these in turn will activate the synthesis of a single adenylate cyclase, and </li></ul><ul><li>each of these in turn will synthesize hundreds of cAMP molecules. </li></ul><ul><li>Each of these in turn can activate a cAMP-dependent kinase that will </li></ul><ul><li>modify hundreds of target molecules in the cell. </li></ul>

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