intracellular signalling - transmitting signals within cells

what is a transmembrane receptor and why are they needed

• Transmembrane receptor: a cell surface receptor that is either anchored in the membrane or passes through the membrane that binds to extracellular ligands

• Proteins, peptides and charged molecules cannot cross the lipid bilayer

• Integral membrane proteins transmit the signal into the cell - these receptors span the membrane

• Ligands may interact directly with receptors or by binding to co-receptors or accessory molecules on the cell surface

• Receptor activation causes a conformational change in the tertiary or quaternary structure that allows the initiation of signalling

what are the 4 ways to transduce a signal

• Hydrophobic proteins - membrane associated

• Hydrophilic proteins - in the cytosol

• Second messengers - cAMP

• Ions - Ca2+

what is the purpose of signal amplification

• Some stages amplify the signal

• Sometimes many signals are needed to activate a pathway

• Some signalling molecules activate any pathways causing distribution

• Some signals might prevent pathways being activated

what are the 3 ways that signalling molecules are controlled

• Post translational modification - phosphorylation

• By regulating whether a G protein has bound to GDP or GTP

• By provision of activators - Ca2+ and cAMP

what are the different ways a protein can be phosphorylated and dephosphorylated, also include a description of the cycle

• Kinase phosphorylates proteins

  • Serine/ threonine kinases

  • Tyrosine kinases

    • non receptory tyrosine kinases

    • receptor tyrosine kinases

• Phosphatases dephosphorylates proteins

• There are some proteins which when phosphorylase’s become inactive

• Only 3 amino acids can be phosphorylated: serine, threonine and tyrosine can be activated by these enzymes as they have free hydroxyl groups

what activates the different kinases and what for

• Serine/ threonine kinases

  • Activated by Ca2+/ calmodulin-dependent protein kinases (CaM kinases) phosphorylate transcription factors and myosin during muscle contraction

• Tyrosine kinases

  • Non receptor tyrosine kinases such as Src family kinases for cell proliferation

  • Receptor tyrosine kinases (RTKs) such as epidermal growth factor receptor

what is the function of GTP binding proteins, when are they used and what affects these proteins

• Molecular switch mechanism which are on when binding GTP and off when binding GDP

• Used by individual proteins (small GTPases), also heterotrimeric G proteins in GPCR signalling

• G protein are always inactive when bound to GDP

• Guanine exchange factors (GEFs) promote exchange of GDP for GTP

• Hydrolyse GTP-GDP by their intrisin GTPase activity. GTPase-activating proteins (GAPs) speed this up

GTP binding proteins switch mechanism cycle describe

what causes uncontrolled signalling and what is the effect

• Ras is a small GTPase

• Mutations can cause loss of GTPase activity so there is an abnormally active intracellular signalling protein

• Ras mutations found in a large proportion of adenocarcinomas

• Yellow signalling protein in diagram is ras

what are the 2 provisional activators of proteins and how do they work

• Calcium

  • Calcium is needed to activate the calmodulin

  • Calmodulin has to bind to Ca2+ to cause it to change shape and then it folds up and wraps around the kinase which activates it by interacting with it

• cAMP

• Binding changes the conformation of the target proteins which changes their activity

3 main categories of membrane receptors

• Linked to ion channels

• Linked to G proteins

• Linked to enzymes

function, structure andproperties of ion channels

• Transport ions along electrochemical gradient

• Specificity of channel is defined by the amino acids lining the channel

• Channels are formed of protein subunits

• Fast regulated opening/closing mechanism

how are ion channels activated

• Change in membrane voltage - voltage gated ion channels: Na+ ion channel

  • Alpha subunit contains 4 homologous domains forming the pore which opens in response to a change in voltage

  • 4 beta subunits traffic the channel and regulate its kinetic properties

  • Na+ channel is too small for K+ and it contains negatively charged amino acids to stop Cl-

    • Alpha subunit has 4 homologous domains which each have 6 transmembrane regions

    • Region 4 has amino acids with positive R groups which sense the voltage across the membrane causing movement of region 4 opening the channel

• Ligand - ligand gated ion channels

  • Transmembrane proteins consisting of a receptor part and a channel which traverses the membrane

  • Opens in response to binding of a ligand

  • Receptors are often classified based on which agonists they bind

  • Nicotinic acetylcholine receptor

    • 5 subunits: 2 alpha, beta, betagamma and gamma

    • Transmembrane region M2 of each subunit forms the channel

    • 2 acetylcholine molecules bind the alpha subunits causing movement of the M2 helices opening the channel

    • When the channel is closed, the leu side chains close the channel, when the M2 helices are made smaller due to twisting of these helices so channel opens

what is the role of ion channels in ion signalling - include how Ca2+ is mediated

• Ca2+ regulates secretion, transcription factor activities, skeletal muscle contraction

• Effects of Ca2+ are mediated through:

  • CAM kinases

  • Calcineurin

• Ca2+ is normally kept at low levels in the cytoplasm by ATP dependent pumps

• Ca2+ channels are ligand or voltage gated. Activation causes a transient increase in Ca2+

structure of GPCR

• GPCRs typically have 7 transmembrane domains

• They do not form an ion channel pore

• There is a G protein free floating along the inner leaflet of the membrane which consists of 3 subunits

how are GCPRs activated

• When a ligand binds to the receptor the affinity for the G protein increases

• The receptor and the G protein may sometimes already be in a complex at the membrane

• Receptor activation changes the conformation of the internal portion of the receptor releasing GDP

how to GPCRs initiate signalling

• GDP attached to the alpha subunit is replaced by GTP

• The alpha subunit and the bg Complex dissociate and then each can initiate further signalling

• GTP is then hydrolysed to GDP and bg Recombines with an alpha ready to associate with another GCPR

example of GPCR pathway

adenylate cyclase and cAMP, beware of which alpha subunit is acitvated for each of the different pathways

phospholipase C activation

• Activates G alphaQ

• Activated PLC which cleaves inositol phospholipids in the membrane that releases IP3 which opens calcium channels

• Release of calcium leads to secretion which activates PKC

  • Diacylglycerol also activates PKC

how do GPCRs activate ion channels - use example of slowing down heart rate

• Acetylcholine binds to the GPCR (muscarinic M2 receptor in the heart), which activates Gi subunit which inactivates adenylyl cyclase from functioning

• the activated bg Unit binds to the K+ channel causing it to open. K+ flows out and the cell becomes hyperpolarised and the heart slows down

  • Subunit was activated because it was bound to GTP, when this is hydrolysed back to GDP it becomes inactive. When this happens it will come across the bg Subunit on the membrane and they will now recombine and the whole system is turned off again

how does cholera affect signalling pathways

• Acute bacterial infection of the intestine

• Bacterium Vibrio Cholerae produces cholera toxin which stops this signalling pathway from being able to turn off

• Inhibits GTPase activity of the subunit G alphaS (stimulates adenylyl cyclase)

• Prolonged signalling so lots of cAMP which keeps activating the CL- transporter so causes water and Cl- to move out of the cells lining the intestine

• Results in diarrhoea, severe dehydration and death

how does whooping cough affect signalling pathways

• Bacterial infection of the lungs

• Bordetella pertussis bacterium releases an active adenylyl cyclase domain so activates the signalling pathways by producing cAMP

• Pertussis toxin renders G alphaI inactive (G alphaI inhibits adenylyl cyclase)

  • Prevents natural way of turning off adenylyl cyclase so it cant bind to receptor so lots of stimulation

• Modifies G alphaI preventing association with GPCRs

• Prolonged signal stimulates coughing

how do receptors linked to enzymes work

• Either have enzymatic ability built into receptor (so are tyrosine kinase receptor) or first step in signalling will be a kinase (serine, threonine, tyrosine)

• Usually have a single membrane spanning domain and come together

• Response usually requires receptor dimerisation (2 parts of receptor coming together)

  • Homo dimers - 2 of same protein coming together to form receptor

  • Heterodimers - 2 slightly different proteins

• Cytoplasmic enzymes that induce signalling are normally protein tyrosine kinases

  • Got kinase activity built into the intracellular domain

  • Close proximity allows the kinase activity to get activated and they will phosphorylate each other and add lots of phosphate groups at different sites which will then allow first steps in signal transduction cascade to bind as it is now right shape and charge for signalling molecules to dock

  • Ligand binding activated enzyme activity within the cytoplasmic domain

  • Tyrosine residues in the intracellular domains are auto-phosphorylated in response to the signal