Return to Biochemistry

Download a copy of this study guide

• Membrane and Lipid Structure
• Signal Transduction

Return to top

Distribution of Membranes within Cells (Panel 2): Hepatocyte -vs- Pancreatic Exocrine Cell

• The Plasma Membrane only constitutes 2-5% of membranes in the cell!
• The majority of membrane comes from: The Rough and Smooth Endoplasmic Reticula.
• Hepatocyte: Specialized for metabolism, so it has a higher concentration of inner mitochondrial membrane.
• Pancreatic Exocrine: Specialized for secretion, so it has some (3%) membrane in secretory vesicles. The hepatocyte has virtually none.

Differential Centrifugation: If you homogenize a tissue under conditions where the cell is not bursting (hypertonic), you can spin out the various cellular components to isolate and study them.

Membrane Function: To separate the water-compartments on the outside and inside. Hence the polar parts face both the exoplasmic and cytoplasmic surfaces, and the non-polar part consists of the internal bilayer membrane.

Types and Regions of Membrane Lipids (Panel 3): Lipids constitute generally about half of the weight of biological membranes, the other half being composed of membrane proteins.

• Phosphoglycerides: aka Glycerophospholipids
• Hydrophobic Region: Diacylglycerol
• Hydrophilic Region: A phosphorylated alcohol
• Specific glycerophospholipids found in membranes
• Phosphatidylcholine: Constitutes roughly half of the lipids.
• Phosphatidylserine: Small, but present in most membranes.
• Phosphatidylinositol: Small, but important role in signal transduction
• Phosphatidylethanolamine -- generally second most prevalent.
• Cardiolipin: Found only in inner and outer mitochondrial membranes in eukaryotes, but it is prevalent in bacteria. Hence cardiolipin is further evidence of the endosymbiotic hypothesis.
• Phosphoglycerides spontaneously form membrane bilayers. That is true of any lipid with two fatty-chain tails.
• Sphingomyelin:
• Hydrophobic Region: Ceramide, also with two carbon chains.
• Hydrophilic Region: Phosphorylated alcohol like above.
• Glycosphingolipid: Similar to Sphingomyelin, except that it has sugar residues fot its polar head-group.
• Cholesterol: Fused rings, also found in membranes. Has a very small polar head-group of only an OH, and is responsible for rigidity in membranes.

AMPHIPATHIC: Having both hydrophobic and hydrophilic regions, and thus being able soluble in both polar and non-polar solvents to an extent.

EXOPLASMIC SURFACE: The side of a membrane that doesn't face the cytoplasm. In the case of the mitochondria, nucleus, or ER, that would be defined as the inside or lumen of the organelle.

• Carbohydrates associated with the membrane are found only on the exoplasmic surface of membranes and vesicles, i.e. the outside of the plasma membrane or the inside of vesicles.

Classes of Membrane Proteins (Panel 4): Biological membranes and the proteins associated with them have asymmetry. They are not the same on the outside as on the inside, and they only integrate into the membrane in one orientation.

• Anchored: Proteins with integral lipid anchors in the membrane. The protein is covalently attached to the lipid portion.
• Peripheral: Proteins that are more loosely associated with the membrane, probably not spanning the whole membrane. They could face either the exoplasmic or cytoplasmic surfaces.
• Peripheral proteins require mild conditions to be removed from membrane, e.g. change in pH or salt-concentration.
• Integral: Transmembrane proteins, tightly associated with the membrane.
• Integral proteins require harsh conditions to remove -- detergent or just rip the membrane apart.

DETERGENTS (Panel 5): Detergents have only one fatty tail instead of two, and the polar head group is very polar. Consequently they form micelles spontaneously, rather than a lipid bilayer.

• Two Detergents we should know:

• Sodium Deoxycholate -- a liver bile acid
• Has a ringed structure like cholesterol, but it has a much more polar end.
• Triton X-100 -- synthetic mild detergent

• Purpose of detergents: to solubilize the biological membrane and thus free integral membrane proteins.
• Micelles: Formed by detergents. They are circular with a polar (hydrophilic) perimeter and hydrophobic core.
• Hence they have only one polar surface instead of two, as in the bilayer.
• If you solubilize a membrane with mild detergent, you can form Mixed Micelles, with membrane proteins present in the micelles. Use this to study integral membrane proteins.

Seven-Helix Receptor-Protein (Panel 12):

• General Characteristics:
• NH₃⁺-Terminus is always exoplasmic!
• COO^--Terminus is always cytosolic!
• There are glycosylation sites on Asn-Residues, only on the exoplasmic surface.
• This is N-Linked Glycosylation, and it occurs only post-translationally.
• The integral portions of the proteins (seven passes through membrane) are alpha-helices, with non-polar-moieties facing the outside, i.e. facing the non-polar membrane.
• There is usually a lipid anchor somewhere within the protein.
• There are often phosphorylation sites (for protein kinases) somewhere within the cytosolic domain of the protein.
• Interestingly, each amino acid is composed of about 20 amino acids, or the same thickness of lipids we would find in a typical biological membrane.
• 3D-Perspective / Tertiary Structure(Panel 19):
• The seven alpha helices are more or less arranges in a circle through the membrane, providing a pocket on the exoplasmic surface for the hormone to bind to.
• In that orientation, the non-polar residues generally point outside the surface (next to the fats) and the polar residues generally point inside the circle -- toward each other.

PORIN (Panel 20): Exception to the alpha-helix rule. Porin is a transmembrane protein composed of beta-strands.

• It is made of 16 beta-strands, arranged to form a barrel.
• It is found on the outer mitochondrial membrane.
• But it still forms a circle, with the hydrophobic residues pointing outward, interaction with the membrane, and the hydrophilic residues pointing inside, forming a hydrophilic pore.

LIPID ANCHORS (Panel 14):

• Palmitate: 16 Carbons, no double-bonds. It is post-translationally esterified to Cys residues in certain transmembrane proteins, to form the lipid anchor. What proteins:
• Insulin Receptor
• Ras-Protein
• beta-Adrenergic Receptor
• Rhodopsin
• Glycosyl Phosphatidylinositol Anchors: Here, the protein itself (usually a large one) is covalently attached to the membrane, through a glycosyl moiety.
• This tethers protein at some distance away from the membrane, allowing it to interact with soluble substances in the exoplasm.
• Acetylcholinesterase is an example of a protein that is covalently attached to the membrane through a sugar group.

Distribution of Lipids in Cells: There is asymmetry of lipid-distribution between the exoplasmic and cytoplasmic surfaces of different cells.

• HUMAN ERYTHROCYTE MEMBRANE: Relatively, there is a majority of one lipid or the other on each respective side of the lipid-bilayer.
• Cytosolic:
• Phosphatidylethanolamine
• Phosphatidylserine
• Exoplasmic:
• Phosphatidylcholine
• Sphingomyelin
• CHOLESTEROL -- most of the cholesterol in the human RBC is associated with the exoplasmic surface.
• RAT LIVER CELL MEMBRANE:
• Cytosolic:
• Sphingomyelin
• Phosphatidylethanolamine
• Phosphatidylinositol + Serine
• Exoplasmic:
• Phosphatidylcholine
• Rule of Thumb: Glycosylated Sphingolipids are always on the exoplasmic surface. That is, both the fat and sugar group are associated with that surface.

LIPID-MOTIONS IN THE MEMBRANE (Panel 8): You can use C-13 NMR to measure the motion of C-13-tagged lipids in the bilayer.

• Flexing of Polar Head -- 10^-8 sec frequency
• Lateral Diffusion -- 10^-8 sec frequency
• Flexing of Acyl Chains -- 10^-10 sec frequency.
• By far the greatest amount of motion is in the middle, hydrophobic part of bilayer due to flexing of the acyl chains.
• Rotational Diffusion -- 10^-8 sec frequency
• Transverse Diffusion -- flip-flopping in the membrane -- 10⁺⁵ sec. It happens hardly at all.

PHASE TRANSITIONS IN LIPIDS / MEMBRANE FLUIDITY (Panels 9, 11):

• Ca⁺² increases the membrane rigidity. Adding calcium makes the membrane less fluid.
• Ca⁺² associates with the negative charges in the polar heads to make them crystallize.
• Heat denatures the lipids, so to speak, making them more fluid.
• Dipalmitoylphosphatidylcholine (DPPC) bilayer undergoes a formal phase transition change at 42C under limited conditions.
• CHOLESTEROL: As cholesterol is added to the graph, it makes the melting point of the bilayer less concrete. It spreads out the melting point.
• RESULT: Cholesterol modulates membrane fluidity. It makes rigid membranes become fluid more slowly, and fluid membranes become rigid more slowly.
• Cholesterol maintains an intermediate state of fluidity in the membrane.
• Factors which affect membrane fluidity (Panel 10):
• Physical Factors: Temperature, Pressure, membrane potential.
• Increase temp ------> Increase fluidity
• Chemical Factors:
• Polar Head-Groups: Different lipids have different phase transitions.
• Cain-Length: Increase chain-length ------> Decrease fluidity
• Unsaturations: Increase number of double-bonds ------> Increase fluidity, i.e. lower the melting point.
• Anesthetics increase fluidity -- make membranes so fluid that the arrangement of membrane protein-receptors is disrupted.

HUMAN ERYTHROCYTE MEMBRANE (Panel 21): Unlike some other membranes, 90% of Erythrocyte membrane proteins are in fact on the cytosolic surface.

• Most protein in RBC Membrane are peripheral proteins.
• The following proteins, however, are integral proteins:
• Anion Transporter
• Glucose Transporter
• Glycophorin A -- Glycophorin does not show up an the PAGE-Gel because its carbohydrates require a different kind of chemistry to separate it out.
• ERYTHROCYTE CYTOSKELETON (Panel 22):
• Spectrin Dimers form a lattice network that underlies the cytosolic surface of membrane. This is the core of the cytoskeleton.
• Geodesic Dome is formed by the interaction of spectrin dimers with other membrane proteins. Spectrin here plays the role of the "spokes" of the dome, while its contact with other proteins are the "nodes."
• JUNCTIONAL COMPLEX: All of the following interact to give the cytoskeleton mechanical power.
• Actin: Arrayed in long filaments, interacts with the spectrin for mechanical power.
• Myosin:
• Ankyrin:
• Tropomyosin: Dimer of tropomyosin intersperses with actin monomers.
• Tropomodulin: Helps stabilize the actin filament.
• Spectrin: Has alpha and beta subunits.
• CAPILLARIES: The purpose of the cytoskeleton is to squeeze down the RBC from its biconcave shape, so that it can fit through capillaries.
• This occurs without any change in RBC surface area, so that the internal physiology of the cell is not disturbed.
• This occurs by contraction and relaxation of actin/myosin fibers.

DIFFUSION OF PROTEINS THROUGH MEMBRANES EXPT (Panel 24):

• Three species are put into three different types of membranes. The chemical species are:
• Phosphatidylethanolamine
• Rhodopsin
• Anion Transport Protein

• Put them into an artificial membrane, and they all have the same rate of diffusion, no matter what their size.
• Under this condition they are 100% mobile.
• Rhodopsin is much larger than the lipid, yet it moves at same rate.
• Put them into biological membranes, and Rhodopsin and the lipid slow down a little.
• The Anion Transport Protein slows down a lot because f the cytoskeleton -- it keeps the protein immobile
• Rhodopsin slows down a little because of its size.
• Put them into Cytoskeleton-Deficient Membranes, and the Anion Transport Protein no longer slows down more than the other two.
• CONC: The cytoskeleton impedes the movement of the Anion-Transport Protein in the Red Blood Cell.

HEREDITARY SPHEROCYTOSIS (Panel 25): Cytoskeleton deficiency which makes the RBC unable to squeeze through capillaries, resulting in hypersensitivity to osmotic lysis and consequently hemolytic anemia.

• Spheroidal Erythrocytes -- instead of the biconcave shape.
• Multiple Biochemical Causes:
• Deficiency of Spectrin
• Unstable Spectrin beta-Subunit.
• Deficiency of Anion Transport Protein
• Deficiency of Ankyrin
• Deficiency of Band 4.1 protein.
• Most often found in Northern European Populations. The above include both autosomal dominant and recessive variants.

ION CONCENTRATIONS ACROSS MEMBRANES (Panel 27):

• Na⁺ -- higher conc on the outside, except during depolarization.
• Mg⁺² -- higher concentration on the inside
• Ca⁺² -- higher concentration on outside.
• Intentionally low concentration is maintained on inside, for effective signal transduction.
• H⁺ -- equal concentrations maintained, with pH around 7.4
• Cl^- -- higher conc on outside
• K⁺ -- higher concentration on inside, except during depolarization.

ERYTHROCYTE MEMBRANE TRANSPORT PROTEINS (Panel 28-30):

• Glucose-Transport Protein:
• Structural Features
• A Band 4.5 protein
• 12 passes through membrane
• One N-Linked oligosaccharide
• Uniport is stereospecific for D-Glucose with lower specificity for other glucoses.
• Single subunit -- one chain
• Anion Transport Protein:
• Structural Features
• Band 3 protein
• 12 passes through membrane
• One N-Linked oligosaccharide
• It is related to Type-A (A-Group antigen) in this case.
• Binds other cytoskeleton proteins, including ankyrin and band 4.1.
• Single subunit -- one chain
• Function: Increase intracellular concentration of Cl^- and lower pH (increase H⁺ via CO₂ equilibrium)
• HCO₃^- goes out
• Cl^- comes in
• Na/K ATPase Transporter:
• Structural Features
• Multiple subunits -- two alpha-subunits and two beta-subunits
• Multiple N-linked Glycosylation sites.
• Functional features
• Inhibited by glycosides such a ouabain and digitoxigenin
• Active antiport: It maintains the proper N⁺ and K⁺ transmembrane balance. Per ATP hydrolyzed:
• 3 Na⁺ go out of the cell.
• 2 K⁺ come back in the cell

INTRACELLULAR TRAFFICKING AND PROTEIN SORTING (Panel 31):

• PRIMARY SORTING EVENTS -- take place cotranslationally, on the ribosome
• Membrane Docking
• Signal Sequence -- usually near or at the N-Terminus. It interacts with the Signal Recognition Particle in the cytosol.
• The Signal-SRP Complex then directs the entire translation machinery to dock on the ER membrane.
• This causes the polypeptide to be translocated across the ER membrane, so that it is now being translated into the ER lumen.
• The N-Terminus is the first end to be exuded into the ER-Lumen, since it is translated first.
• Cytosolic Destination -- either a lack of signal, or no interaction between the signal and the SRP.
• The protein ends up in the cytosol.
• SECONDARY SORTING EVENTS -- the fate of the protein once it has crossed the ER membrane. The Signal Sequence is cut off now, before further trafficking. Two things can happen now:
• Integral Membrane Destiny: It remains in the ER membrane to become an integral membrane protein somewhere in the cell.
• If the protein stays in the ER Membrane, it may go to the plasma membrane, or it may be packed into a secretory vesicle for eventual exocytosis.
• Secretion: It goes to the ER-Lumen or membrane, where it will be packaged into a secretory vesicle for exocytosis.
• Virtually all proteins that are secreted as extracellular hormones are folded in the ER lumen and then directed outside by exocytosis (i.e. secretion).
• SECRETORY VESICLES -- may contain come of both the integral ER-Membrane proteins and portions of the ER-Lumen.
• MAINTAIN ORIENTATION: Proteins that go to the ER-membrane, whether they remain in the membrane or are packaged into secretory vesicles, maintain their orientation from the time they are translated until their final destiny.
• So if they start with NH₃ in the cytosolic domain, e.g., then it will remain on the cytosolic side (the outside of the ER, outside of vesicles, inside of the plasma membrane) throughout its course!
• TRAFFICKING SIGNALS (Panel 32)
• COTRANSLATIONAL SIGNAL: Translated into ER-membrane and then cleaved.
• Sequence homology = Lys-Arg-(hydrophobic segment)-Ala/Gly
• Ala-Gly is at the cleavage site of the signal peptide.
• POST-TRANSLATIONAL SIGNALS
• ER-Lumen Destination: Lys-Asp-Glu-Leu-COO^- (KDEL) sequence at the C-Terminus. In this case it is not cleaved.
• Lysosome Destination -- N-Linked carbohydrate with Mannose-6-Phosphate (M6P). It is cleaved.
• Peroxisome Destination -- Ser-Lys-Leu-COO^- (SKL) sequence. It is rarely cleaved.

TYPES OF PARTICLE INTERNALIZATION (Panel 33):

• Phagocytosis -- Envelops a particle and buds it off. Important in immunity. It is non-specific
• Fluid-Phase Pinocytosis -- "Cell-Drinking," also non-specific.
• Receptor-Mediated Endocytosis -- Highly specific -- see below
• Transcellular Vesicular Transport -- the endocytosis of a particle, followed by exocytosis on the other side. It occurs in polarized cells such as intestinal epithelia.
• See Immunoglobulin-G below.

DIFFERENT PROTEIN THAT ARE INTERNALIZED BY RECEPTOR-MEDIATED ENDOCYTOSIS (Panel 34):

• Proteins
• Transferrin -- supplies iron to all cells
• Transferrin is recycled multiple times. It comes in, delivers its Fe⁺², goes to the Golgi, and then is eventually recycled out to the end of the cell.
• Immunoglobulin-G -- an example of Transcellular Vesicular Transport.
• Uptake of this by the fetus allows transfer of immunoglobulins from mother to the growing fetus.
• Peptide Hormones -- May be taken up be endocytosis to take them out of circulation.
• Glycoproteins
• Galactose/Glucose/Mannose-Terminated Proteins -- may be taken up to take them out of circulation.
• Lipoproteins
• Low-Density-Lipoprotein -- endocytosis of this provides cholesterol to the cell.
• The LDL-Receptor is then directed to the Golgi where it is recycled.

RECEPTOR-MEDIATED ENDOCYTOSIS / CLATHRIN COATS: (Panel 35)

• STEPS OF ENDOCYTOSIS
• Ligand binds to membrane receptor
• Receptor internalizes the ligand using Clathrin Pit
• Clathrin Pit then forms a Clathrin-Coated Vesicle.
• Ligand is trafficked to its intracellular destination
• Once internalized, the clathrin coat is sloughed off, which requires ATP.
• After that, the vesicle is now known as an endosome.
• Receptor is processed -- recycled or degraded -- different cellular fates depending on the ligand.
• Again, Transferrin is recycled (Golgi)
• LDL is recycled for a while (Golgi) and eventually disposed of (Lysosome).
• CLATHRIN PITS: Depressions in the plasma membrane generated by Clathrin molecules. These then become sites on the membrane for endocytosis.
• Receptor proteins can laterally diffuse to these endocytosis-specific sites in the membrane.
• This coated pit will bud off into a coated vesicle, in order to internalize the ligand.
• CLATHRIN STRUCTURE -- Triskelion Structure -- very regular cage-like structure, almost like a golf-ball surface.
• Three heavy chains; three light chains; three-fold symmetry.

Return to top

Major Classes of Hormones (Panel 2):

• Polypeptides (proteins) -- mostly growth factors, like insulin, glucagon, nerve growth factor
• Amines -- derived from amino acids
• Thyroxine is modified tyrosine
• Epinephrine
• Serotonin
• Steroids -- derived from cholesterol
• Aldosterone
• Sex hormones
• Retinoic acid
• Eicosanoids -- derived from 20-Carbon polyunsaturated acids
• Prostaglandin-E, Thromboxane-A

AGONIST (Panel 2): Isoproterenol is an agonist for Epinephrine. It binds the beta-Adrenergic receptor with a higher specificity then epinephrine, and it elicits the same adrenergic response.

ANTAGONIST (Panel 2): Propanolol -- binds to the beta-adrenergic receptor with higher specificity than epinephrine, but it does not elicit the response.

General Properties of Hormones (Panel 3):

• They are effective at low concentrations
• They bind to specific receptor proteins
• They generate a cellular response via intracellular second messengers, usually.

Classes of Hormone-Receptors (Panel 5):

• 7-Pass Proteins linked to Heterotrimeric GTP-binding proteins
• Tyrosine Kinase Receptors
• Nitric Oxide Receptors
• Steroid receptors -- cytoplasmic or nuclear

Criteria for the Participation of an Intracellular Messenger (Panel 7):

• The enzyme that catalyzes the synthesis of the messenger (e.g. adenylate cyclase) should respond to the hormone that acts on the cell.
• A change in concentration of the messenger (e.g. cAMP) should precede or occur simultaneously with the intended action of the hormone.
• Inhibitors of the removal or degradation of the messenger (e.g. Phosphodiesterase) should function synergistically with the hormone that promotes its synthesis.
• The biological effect of the hormone should be mimicked directly, by adding messenger and bypassing the initial hormone binding.

SIGNAL TRANSDUCTION VIA SECOND MESSENGERS:

• THE SUBUNITS OF THE HETEROTRIMERIC G-PROTEIN
• alpha-Subunit: It binds the guanine nucleotide
• beta-Subunit:
• Always stays together with gamma-subunit, remaining on the cytosolic surface of the membrane.
• It may have secondary action once the alpha-subunit has been displaced.
• gamma-Subunit: Has a lipid-anchor that goes into the membrane, so that it always remains on the cytosolic surface of the membrane.
• HORMONE ACTIVATION OF beta-ADRENERGIC RECEPTOR: There are only two types of adrenergic hormones -- epinephrine and norepinephrine, yet there are over 30 different adrenergic receptors.
• The hormone binds to the 7-pass beta-adrenergic receptor protein, initiating a conformational change in the receptor.
• This conformational change allows the alpha-Subunit of the attached GTP-Binding protein to let go of GDP and accept a new GTP.
• The alpha-Subunit, with GTP attached, detaches from the Heterotrimer, and then can have some sort of secondary action on another enzyme.
• The betagamma-Subunit, still attached to the membrane, may have some secondary action on another enzyme.
• GTPase spontaneously cleaves GTP ------> GDP on the alpha-Subunit. This is carried out by the alpha-subunit.
• There is a "clock" that ticks down to hydrolyze the GTP. Each G-Protein has its own intrinsic clock.
• This causes the alpha-subunit to reattach to the betagamma-Subunit.
• In the meantime, the hormone has left the beta-Adrenergic receptor.
• MAJOR CLASSES OF HETEROTRIMERIC G-PROTEINS: The class is based on the gene-product -- i.e. different genes encode each respective alpha-subunit. The main difference is usually in the alpha-subunit.
• G_s: (G-Stimulatory)
• Linked to beta-Adrenergic Receptors, sensitive to Catecholamines
• Effector proteins are Adenylate Cyclase, Ca⁺²-channels, and Na⁺-Channels
• G_i (G-Inhibitory)
• Linked to alpha₂-Adrenergic Receptor, sensitive to Catecholamines
• Effector proteins are Adenylate Cyclase, Ca⁺²-channels, and Na⁺-Channels
• G_q
• Linked to alpha₂-Adrenergic Receptor, sensitive to Catecholamines
• Effector Protein is Phospholipase-C
• G_o -- opiate receptor proteins
• Effector protein is Phospholipase-C
• G_t -- Rhodopsin effector protein
• SECONDARY ACTION OF beta-ADRENERGIC RECEPTOR (Panel 13): ADENYLYL CYCLASE
• The activated alpha-Subunit forms a complex with Adenylyl Cyclase, to catalyze the conversion of ATP ------> 3'-5'-cAMP
• Adenylyl Cyclase is a transmembrane protein and remains in the membrane during this conversion.
• cAMP then activates Protein Kinase A (PKA), which in turn phosphorylates other target enzymes in the pathway to somehow change the metabolism or enzymatic activity of the cell.
• Phosphodiesterase then quickly breaks down cAMP, stopping the activation of Protein Kinase A.
• Cholera Toxin -- Ribosylates the G_s subunit, thereby blocking it open. RESULT = Hyperproduction of cAMP.
• Pertussis Toxin -- Blocks open G_i subunit. Result = over-inhibition of cAMP.
• Methylxanthine -- Caffeine -- Inhibits phosphodiesterase. RESULT = Higher level of cAMP in cells.
• SECONDARY ACTION OF alpha₁-ADRENERGIC RECEPTORS (G_Q) (Panel 14): PHOSPHOLIPASE-C
• The activated betagamma-Subunit (NOT alpha-Subunit!) forms a complex with Phospholipase-C, to cut apart a LIPID -- Phosphatidylinositol-4,5-biphosphate into two different parts.
• Diacylglycerol (DAG) -- one of the lipid-products
• DAG stays with the membrane and stimulates Protein Kinase C (PKC) to phosphorylate Ca⁺²-dependent target-enzymes.
• Phorbol Esters mimic the actions of DAG and thereby activate Protein Kinase C.
• Inositol Triphosphate (IP₃) -- the other lipid-product
• IP₃ goes to the ER, where it stimulates the release of Ca⁺².
• Lithium inhibits the degradation of IP₃ by inhibiting phosphatases. This keeps Ca⁺² channels in the ER open longer. RESULT = higher overall intracellular concentration of calcium.
• Calcium
• Activates Protein Kinase C (PKC) so that it can phosphorylate target enzymes.
• Activates Ca⁺²-Calmodulin Kinase to phosphorylate other target enzymes.
• Ionophores can independently increase the Ca⁺² concentration in a cell by adding Ca⁺² channels to the plasma.
• AMPLIFICATION (Panel 21): A single molecule of epinephrine ------> 3 molecules of adenylyl cyclase ------> 5 molecules of cAMP each ------> 1 protein kinase ------> multiple target enzymes
• Only one cAMP will activate one PKA -- this is a 1:1 ratio.
• Amplification means that a very low concentration of hormone can have a large intracellular effect.
• MULTIPLE GENES ENCODE THE SUBUNITS
• About 20 different genes encode the alpha-subunit.
• 5 genes encode beta-subunit
• 6 genes encode gamma-subunit
• CROSSTALK: The same receptor may activate or inhibit different G-Proteins by laterally going across membrane. This is known as crosstalk -- one receptor interacting with multiple cytosolic GTP-Binding proteins, yielding multiple effects.
• MCCUNE-ALBRIGHT SYNDROME: A mutation in the G_s protein at Arg201, going to His or Cys.
• RESULT = Intrinsic GTPase activity of the alpha-subunit is inhibited, thus making adenylyl cyclase over activated.
• Strangely, mutant allele is present in all effected endocrine tissues, but is absent in other tissues.
• Cholera Toxin modifies (ribosylates) the same Arg201 residue!
• SYMPTOMS
• Skeleton abnormalities, bone cysts
• Cafe Au Lait spots
• Multiple endocrinopathies (general overdevelopment, hyperthyroidism)

INTRACELLULAR CALCIUM:

• It is normally at very low levels -- 0.01 - 0.1 micron
• Ways in which Ca⁺² is released into cytosol
• Cardiac Muscle, Neurons: Voltage-gated Ca⁺² channels.
• Many cells: IP₃ gated Ca⁺²-channels in ER.
• Skeletal muscle: Ryanodine receptors in SR.
• Intracellular effects of Ca⁺²
• Calmodulin binds up to 4 Ca⁺² / mol of protein.
• Ca⁺² activates multiple Calcium-dependent calmodulin kinases.
• Activates glycogen degradation (glycogenolysis)
• Activates 3'-5'-AMP-Phosphodiesterase
• Activate nitric oxide synthase

PROTEIN KINASE / PHOSPHATASE ACTIVITY: What do the target enzymes do?

• Ser, Thr, Tyr are the amino acids which can become phosphorylated by protein kinases... they all have available alcohol moieties.
• Protein Phosphatase: The opposite of a protein kinase, it is the enzyme that dephosphorylates the target protein.
• TARGET PROTEINS: Usually metabolic proteins, but may be a biosynthetic protein or gene-regulatory protein.
• Regulation of Hormone Binding: 7-helix receptors can be phosphorylated to inhibit ("desensitize") their binding to hormone.
• This phosphorylation occurs on a Ser-residue in the cytosolic domain of the protein.
• Sensitivity can be restored by a protein phosphatase.
• GLYCOGEN SYNTHASE: A complicated, multi-regulated protein kinase cycle.
• Glycogen Synthase has at least 10 amino acids that are subject to phosphorylation.
• Six different enzymes phosphorylate this protein, to convert it from a "more active" to a "less active" form. The enzyme is never really completely turned off. It is a gradient.
• Note that in this case phosphorylation reduces the protein's activity.
• EVERYTHING INHIBITS IT! 3'-5'-cAMP (via PKA), DAG (via PKC), and Ca⁺² (via CAM-Kinases) all have inhibitory effects on this protein.
• Phosphatase-Inhibitor: It is stimulated by phosphorylation, and it acts to inhibit the glycogen synthase activator.
• Thus 3'-5'-cAMP has a doubly-inhibitory effect -- it phosphorylates both species, inhibiting glycogen synthase itself, and activating the phosphatase inhibitor.

SIGNAL TRANSDUCTION VIA TYROSINE KINASES (Panel 24-25):

• STRUCTURE
• They are one-pass proteins.
• There is a cystine-rich domain on the exoplasmic side.
• There is an immunoglobulin-like domain on the exoplasmic side.
• HORMONES that interact with them: Generally growth factors
• Platelet-Derived Growth Factor (PDGF)
• Epidermal Growth Factor
• Insulin
• AUTOPHOSPHORYLATION: Process of Tyrosine Kinase Activation
• Platelet-Derived Growth Factor (PDGF) interacts with the tyrosine-kinase receptor as a dimer.
• This causes local domains on the cytosolic side of the receptor to autophosphorylate neighboring Tyrosine residues. The receptor is now activated.
• Phospholipase-C is then attracted to the phosphates on the activated receptors. The phosphate-groups are transferred to the SH2-Domains of Phospholipase-C.
• Phospholipase-C then has its standard transduction pathway of splitting PI ------> IP₃ + DAG ------> higher Ca⁺² levels.
• Thus there are two ways to activate this pathway in cells, via alpha₁-Adrenergic Receptors and Tyrosine Kinase Receptors.
• INSULIN RECEPTOR (Panel 26): Another autophosphorylating tyrosine-kinase receptor
• Insulin has a lot of different downstream target proteins. Generally it promotes anabolic activities (glycogenesis, lipogenesis, and glucose uptake) by phosphorylating effector proteins.
• Ultimate targets of insulin action:
• Alter the gene expression of glucose metabolizing enzymes.
• Cause membrane translocation of glucose transporters (to effect glucose transport), of cell-surface receptors, or of Na/K-ATPases.

SIGNAL TRANSDUCTION VIA STEROID HORMONES (Panel 27):

• Any hormone that crosses the biological membrane and interacts with cytosolic (or nuclear) components directly is classified as a steroid-like hormone. These include:
• Thyroxine (T₃ or T₄) -- a modified tyrosine.
• Vitamin-D
• Retinoic Acid.
• STEROID HORMONE RECEPTOR:
• DNA-Binding Domain: It contains a region of protein that interacts directly with the Hormone-Response Element (HRE) of the DNA.
• Zinc Finger: The domain is often of the zinc-finger motif. In this case it contains 2 independent sites of 4 Cysteines each, which bind around a central zinc atom.
• Leucine Zipper -- another protein structure
• Homeobox
• In the glucocorticoid receptor, Val, Ser, and Gly are specific residues which specify it to bind to only certain regions of DNA.
• alpha-Helix region actually binds to the DNA. This region is separate from the zinc-finger.
• Hormone-Receptor Domain binds the hormone.
• RECEPTOR ACTIVATION: The hormone binds to the hormone-receptor, which causes the Zinc-Finger to open, freeing the alpha-helix region allowing it to bind to the DNA.

NITRIC OXIDE: (Panel 33-35)

• Nitric Oxide Synthase: Makes nitric oxide from arginine.
• Citrulline is the byproduct.
• Guanylyl Cyclase: NO causes vasodilation!!!
• Acetylcholine (vasodilator) or Bradykinin stimulate the release of Ca⁺² in the vascular endothelium.
• Ca⁺² in turn stimulates NO-Synthase in the vascular epithelium.
• NO diffuses out of the endothelium and into the neighboring smooth muscle cell.
• In the smooth muscle cell, NO stimulates Guanylyl Cyclase to make cGMP from GTP.
• Via secondary signal transduction pathway, cGMP ultimately causes vasodilation.
• Nitrosyl Cation can interact with heme-groups and affect immunotoxicity.
• NO is used to treat pulmonary problems in newborns and adults, but only in limited quantities.
• It must be carefully balanced with oxygen tension. Too much oxygen can cause oxidation of nitrogen and problems as a result.
• At higher concentrations, NO results in bleeding and platelet dysfunction.
• Methemoglobin can form by NO binding to hemoglobin, with higher specificity than O₂

Return to top