Membrane Trafficking and cell signalling by xiaoyounan

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									                              MIMS Lecture Notes
A little tit bit :

Campath-1 is a therapeutic antibody that recognises a receptor (CD52) on mature
lymphocytes but not on stem cells. The antibody depletes the B and T lymphocytes
and is used to treat lymphomas and as an immunosupressant for transplants. It was
formed from a mouse antibody but when it was grafted onto a human backbone it
became inactive and required specific research into its specific structure to correct
this.

To be read in conjunction with the handout from Dr Irvine.

Protein Transport

        Directional trafficking is the general term used for the transport of vesicles and
         movement of proteins.
        Mechanisms underlying trafficking are crucial to protein secretion and
         absorption and the synthesis of organelles.
        Malfunctions are responsible for diabetes, cystic fibrosis and artherosclerosis.
        Protein synthesis, example Insulin:
             o RNA from nucleus moves to cytoplasm and is taken up by a ribosome
             o Translation takes place and the first few amino acids are put together –
                 this is in effect the “address” of the protein.
             o Once the address is created the ribosome and protein complex will
                 bind to a signal recognition particle which halts synthesis.
             o The SRP, ribosome, peptide complex then binds with the SRP receptor
                 in the ER membrane. The SRP is then discarded and the ribosome sits
                 on a translocation channel and resumes translation which now strings
                 into the cisternae of the ER.
             o The protein grows in length and when synthesis is complete a
                 peptidase cleaves the protein from the ribosome and the protein is then
                 freely soluble in the ER.
             o Insulin at high concentration is not soluble and forms crystals – this is
                 how it is stored.
        Many proteins are glycosylated (by enzymes) in the lumen of the rough ER
         even while the protein is being synthesised.
        The transport from the ER to the golgi body occurs by the budding off of
         vesicles from the ER. The vesicle pinches off and the ER reseals.
             o COP proteins coat the vesicles. COPI directs vesicles from the golgi to
                 the ER. COPII from the ER to the golgi.
             o Vesicles can move by diffusion but very slow so they are actively
                 transported on the microtubules (made of tubulin and are part of the
                 cytoskeleton).
             o Motor proteins which use ATP are part of the vesicle coating and are
                 responsible for movement along the microtubules the two main ones
                 being:
                      Kinesins
                      Dyamins
        o There is a potential difference along a microtubule, dynein moves
            vesicles towards the negatively charged end and kinesin toward the
            positively charged end.
        o Vesicles must then fuse with the golgi body releasing their proteins.
        o SNARES are responsible for vesicle recognition. vSNARES on the
            vesicle bind with tSNARES on the target membrane providing
            specificity (there are different types of SNARES depending on the
            contents of the vesicle).
        o COPS must be stripped off vesicles before the SNARES may interact.
        o SNARES consist of several polypeptide chains which are attached to
            each other and when the t and v SNARES bind with one another they
            contribute different numbers of polypeptides to from a complex.
        o This is a highly exergonic process and is favourable.
   Budding from the golgi produces the vesicles that are destined for the cell
    membrane or return to the ER. Again this uses SNARES but of course they
    will be different.
   Synaptobrevin is a vSNARE found in brain synaptic vesicles it interacts with
    two tSNARES:
        o SNAP-25
        o Syntaxin
        o These three peptides form a complex – the process is controlled by
            calcium.
        o Synaptotagmin is a calcium binding protein which regulates synaptic
            vesicle fusion with plasma membranes.
        o Specific proteases e.g. those produced by botulins, hydrolyse syntaxin
            and SNAP-25 hence preventing vesicle release in brain synapses.
   Cystic Fibrosis is a problem with chloride ion transport.
        o The faulty protein is that which consists the chloride channel
        o A missing phenylalanine results in a misfolding, although the channel
            works fine it is unable to get from the golgi to the plasma membrane
        o DEFECT IN CELL TRAFICKING.
   Endocytosis is when vesicles bud off plasma membrane into the cell.
        o The vesicles are coated in clathrin (a COP) and fuse with an endosome
            which then recycles the receptor and returns it to the cell membrane
            and directs the enveloped substances to the appropriate regions.
        o Receptor desensitisation is when receptors are endocytosed from the
            plasma membrane to reduce the effect of a hormone or
            neurotransmitter on a cell.
        o Receptor sensitisation occurs when a hormone or neurotransmitter is
            persistently low and receptors are returned to the cell membrane to
            increase the cell’s sensitivity to it.
        o Low density lipoproteins are taken up by endocytosis in the liver. This
            is done by LDL receptors which when they are bound are endocytosed.
        o Hypercholesteralaemia is when the faulty receptor mechanism prevents
            endocytosis of LDL.
        o Viruses can enter cells by endocytosis by binding with receptors and
            stimulating the process.
        o Marcophages envelope bacteria by endocytosis.
Receptors and Signal Transduction

      Hydrophobic molecules work slowly as they must be attached to a protein for
       transport (as are not soluble in the blood) but can move through membranes
       freely e.g. oestrogen which is a lipid.
      Most hormones are hydrophilic so they can move around the body quickly but
       cannot cross membranes unless endocytosed (slow) so they must bind to an
       appropriate receptor to mediate an intracellular response.
      Hormones e.g. Acetyl Choline are tiny compared to their receptors (analogy –
       bee stinging an elephant) and it is the ability of proteins to change their
       properties (allostery) in response to a small molecular interaction which
       permits this.

Ligand Gated Ion Channel

      E.g. Nicotinic acetylcholine receptor (receptors are named by the endogenous
       agonist which may not be specific to that receptor and a non endogenous
       agonist (drug) which is specific to that receptor).
      Muscarine acetylcholine receptor is also an acetylcholine receptor but is not
       affected by nicotine as the nicotinic one is not affected by muscarine. It
       mediates its response to acetylcholine in a different way, by G-protein
       coupling.
      Nicotinic Choinergenic receptors:
           o Pentameric (5 subunits)
           o Are ligand gated so the fastest and easiest method of action
           o Found in skeletal muscle and neurones as the change in conformation
               takes only µs after binding.
      Glutamate is the main neurotransmitter in the brain (an amino acid) which acts
       on a variety of ligand gated ion channels. There are four main types each
       responding to glutamate in different ways three of them being:
           o NMDA receptors – calcium
           o AMPA receptors – sodium (some calcium)
           o KAINATE receptors – sodium
      Agonists mediate a response by activating a receptor.
      Antagonists block responses by blocking the receptor.
      GABA (gamma amino butyric acid) is a neurotransmitter in the brain
           o Interacts with GABA a and b receptors
           o GABA a gates chloride ions which repolarises cells (as negative charge
               is flowing into the cell).
           o GABA is still an agonist of its receptor even though it has an
               ‘antagonistic’ effect to that of Ach.
      Cells can be ‘calmed’ by either:
           o Finding an antagonist to block Ach receptors. This is concentration
               specific and if enough is added the cell will eventually be calmed as all
               the receptors will be blocked preventing Ach from exerting its effect.
           o Finding an agonist of GABA receptors. There will reach a point when
               all the GABA receptors are bound and hence further addition of
               agonist will have no further effect of calming the cell.
      Opiates are agonists of inhibitory receptors so there will be a point when
       administering more opiates will have no more effect.
      Ligand gated channel advantages
          o Speed – therefore used for skeletal muscle
      Ligand gated channel disadvantages
          o Ka = Kon/Koff (Kon is the rate of binding and Koff is the rate of release).
          o Kon is limited by diffusion of the binding molecule so can’t be changed
          o Koff controlled by affinity of receptor for agonist so affinity must be
              low so agonist dissociates from receptor quickly.
          o This limits ligand gated channels to areas of high concentration e.g.
              synaptic cleft where enzymes are also present to rapidly remove the
              neurotransmitter. Unsuitable for application in blood.

G-protein coupled/linked receptors

       GPCRs also known as 7-transmembrane spanning serpantine domain
       receptors (now that’s a mouthful Mr Bond) that cross the membrane 7 times in
       a ring conformation (not a straight line like diagrams will suggest) often
       forming channels.
      Are the largest family of receptors (about 200) and are the biggest group
       targeted by drugs.
      E.g. adrenergic receptors, histamine acts this way.
      Muscarinic acetylcholine are G-protein coupled receptors but although they
       mediate the same response as nictotinic acetylcholine receptors they do so via
       different mechanisms.
       o There are many sub types of receptors and current research is
           concentrating on agonist specificity on receptor subtypes.
      GABA-b and glutamate receptors are also GPCRs.
      When a cell differentiates it makes decisions on which receptors are going to
       be expressed so it can have a particular response to varying physiological
       conditions.
      Responses by a cell are defined by:
                What receptors the cell is expressing
                What Transduction mechanisms are employed
      Cells generally express a small range of the receptors available to them in the
       genome.
      In essence G-proteins are little information transfer switches.
                They are TRIMERIC – have a beta and gamma subunit which are
                   always bound and an alpha subunit which binds to GDP and GTP.
                The receptor is off when GDP is bound to the alpha subunit and all
                   three subunits are bound to one another.

      When adrenalin for example binds with the GPCR the receptor, it
       conformationally changes shape and is now able to bind with the trimeric G
       protein.
      It activates the G-protein by causing the bound GDP to be converted to GTP,
       splitting it into an alpha and beta/gamma subunits. Both subunits can be
       active.
      Effectors are downstream in the signalling chain.
      Effectors are the proteins that activated G-proteins influence.
      G-proteins diffuse slowly and the whole process of agonist binding and GDP
       to GTP conversion is quite slow – 50mS from binding to a measurable effect.
      G-proteins are controlled as the alpha subunits have GTPase activity and
       hydrolyse GTP to GDP hence turning themselves off.


      Advantages of GPCRs:
       o G proteins can interact with more than one effector therefore
           diversification of signals is possible.
       o The beta gamma subunits are very important as they can act synergistically
           with the alpha subunit or have different effects.
       o Proteins expressed in the cell are different depending on what G-protein
           they will interact with.
       o Amplification is possible as one G-protein can stimulate more than one
           enzyme molecule.
       o G-protein coupled receptors have a high affinity for their agonist therefore
           turn off slowly. This is important as the process is catalytic so the binding
           of one agonist molecule can activate many G-proteins.
       o The significance of this is that a full response by the cell can be obtained
           with just 5% (for example) of agonist binding to the GPCRs.
       o GPCRs have a higher affinity than ligand gated channels for their agonist
           but it is still low enough to allow the receptor to be turned on and off very
           quickly. The hormone only needs be present in small quantities to take its
           effect as it needn’t all bind to mediate a full response, in essence a
           compromise.
      Concluding, G-proteins are a large group as they diversify, amplify and
       therefore can compromise between high and low affinity allowing reasonably
       rapid responses.

Effectors of activated G-proteins

      There are two types of GTP switches, both are on when bound to GTP and off
       when bound to GDP:
           o Trimeric G-proteins (discussed above)
           o Monomeric G-proteins (small)
      Almost every process uses small G-proteins – there are about 100 in the
       family.
      An example of a small G-protein is RAS:
           o RAS is a cancer causing gene (oncogene) and is though to be involved
               in 50% cancers.
           o When it mutates the G-protein that controls cell division is
               permanently on as it has lost its GTPase abilities.
      Target proteins are a large family of proteins which are the effectors of G
       proteins.
      They are specific in their effect, an example are the calcium and potassium ion
       channels which are often activated/deactivated by the beta/gamma subunits.
   Adenylate cyclase catalyses ATP conversion to cAMP (full name is
    3’5’cAMP) is G-protein controlled
   There are around nine adenylate cyclases and all are stimulated by the
    stimulatory GTP bound alpha subunit (sGTP). They are also inhibited by
    inhibitory alpha subunits, regulated by the beta/gamma subunit or Ca2+
    controlled.
   All second messenger pathways must have off switches.
   Phosphodiesterase which is calcium controlled regulates the breakdown of
    cAMP to 5’AMP.
   Protein Kinase A is activated by cAMP and regulates proteins by
    phosphorylation.
   PKA phosphorylates serine and threonine residues (have –OH groups) but
    they must be in the correct conformation for the kinase to act.
   Calcium ion channels in the heart are activated by PKA.
   Beta blockers block beta adrenergic receptors therefore preventing cAMP
    production.

o Most cells are highly polarised and organised. The methods of targeting,
  restricting and localising of pathways are only just being discovered.
o Scaffold proteins assist in increasing efficiency by keeping proteins involved
  in a pathway in close proximity and hence reducing the inefficiency in
  diffusion.
o Long term responses are mediated in cells by changes in the cell structure and
  proteins (by gene Transduction). PKA can phosphorylate transcription factors
  in the nucleus.
o Transcription factors can be found upstream from a gene in a chromosome and
  control whether the gene is transcribed or not.
o CREs (cAMP response elements) allow cAMP to control whether the gene
  downstream is transcribed.
o cAMP activates PKA, PKA phosphorylates CREB (CRE binding protein),
  activated CREB binds to CRE on the DNA and the gene is activated.

o cGMP is not usually involved in G-protein coupled receptors except in the
  eye. It is formed from GTP by guanylyl cyclase.
o In the absence of light cGMP is bound to sodium channels keeping them open.
  Light acts on Rhodopsin which is a GPCR, the G-protein activates cGMP
  phosphodiesterase and decreases [cGMP] hence closing the sodium channels.
o Guanylyl cylase is not regulated by trimeric G-proteins:
      o cGMP levels often increase when the IP3 pathway is activated.
      o There are two forms of guanylyl cyclase, the membrane bound form
           and the freely soluble form.
      o Endothelial derived relaxation factor (EDRF) now discovered to be
           NO (nitric oxide) and activates soluble guanylyl cyclase in the
           cytoplasm which stimulates cGMP production and therefore relaxes
           blood vessels.
o cGMP acts on protein kinase G (PKG) in a similar way to PKA.
o cGMP is deactivated by a type V phosphodiesterase converting it to GMP.
  Diseases such as cholera and whooping cough are associated with interference
  with G-protein mechanisms.
Calcium as a second messenger
   o Blood serum concentration of calcium is about 2mM.
   o Calcium concentration in the cell is kept low (µm) to stop it binding to
      phosphate groups – such as those in DNA forming insoluble salts.
   o As the concentration of free calcium is kept low it is a great second
      messenger.
   o Calcium isn’t made or broken down, it is released ain by calcium voltage
      gated ion channels or IP3 gated ion channels (in the ER) and is pumped out of
      the cell or into the ER by ATP dependent calcium pumps.
   o Calcium diffuses very poorly and slowly in cells due to its charge nature in
      comparison to cAMP that diffuses rapidly.
   o Calcium therefore stimulates its own release by calcium induced calcium
      release acting of ryanodine (called so because plant alkaloids act on them)
      receptors in the ER.
   o Phosphoinositide specific phospholipase C which hydrolyses membrane
      bound phosphatidyl inositol 4,5-bisphosphate, producing IP3 (inositol
      triphosphate) and membrane bound diacyl glycerol is activated by the GTP
      bound alpha subunit of a G protein Gq.
   o IP3 is a second messenger that releases calcium from the ER – it acts on IP3
      activated calcium channels in the ER.
   o Where calcium release occurs within a cell its site of release can be controlled
      by expression of IP3 receptors in the ER so in polarised cells such as
      pancreatic acinar cells the ER at the excretory end of the cell contains IP3
      receptors whereas the rest of the ER does not.

Effectors of calcium
       o The adaptor protein calmodulin is a calcium binding protein which
           controlls muscle contraction and neurotransmitter release
       o CaM kinases are activated by calmodulin activated PKS.
       o As CREB (see earlier) is phosphorylated by CaM kinases calcium can
           control some gene expression too.
       o Calcium and DAG cooperate to activate protein kinase C.

								
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