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BMS301 - Membrane Receptors > Cytokine Receptors (JAK/STAT) > Flashcards

Cytokine Receptors (JAK/STAT) Flashcards

1
Q
  1. do cytokine receptors have intrinsic tyrosine kinase activity, or are they associated with a cytoplasmic tyrosine kinase?
  2. why is the phosphorylation of substrates beneficial?
  3. name the 4 components of the JAK/STAT pathway.
  4. describe the stages of receptor activation and signal transduction
A
  1. they are associated with cytoplasmic tyrosine kinases (JAKs)
  2. because it is a rapid and reversable change (i.e. doesnt involve the destruction and synthesis of new substrates
  3. JAKs (tyrosine kinases), ligand (cytokine), receptor and STAT
    1. ligand binding induces a conformational change in the receptor
    2. the conformational change brings the 2 JAKs together, so they can become transphosphorylated. This causes an increase in their kinase activity and creates p-Tyr docking sites
    3. STATs, which contain an SH2 domain bind to the p-Tyr sites on JAK and become phosphorylated
    4. phosphorylates STATs dissociate from JAKs. Their free SH2 domain binds to a p-Tyr site on another STAT, promoting hetero- or homo-dimerisation
    5. STAT dimers translocate to the nucleus, where they bind to target gene promotes, initiating the transcription of target genes
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2
Q
  1. what physiological role do EPO and STAT5a mediate?
  2. what physiologocal role do EPO and STAT6 mediate?
  3. what physiological role do IL-6 and STAT3 mediate?
  4. name 6 other physiological role do ILs and STATs mediate
  5. what type of cancers are most commonly caused by overactivation of the pathway and why?
A
  1. haematopoesis of erythrocytes
  2. haematopoesis of T helper cells
  3. involution (organ shrinkage) of post-lactating mammary glands
  4. stem cell maintenance, cell proliferation, immunity and inflammation. Neuronal regeneration and fertility
  5. blood cancers (leukaemia), due to their role in blood cell production
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3
Q
  1. Name 7 examples of IL ligands
  2. what does every dimer pair contain?
  3. what is the specificity of each receptor to certain ligands governed by?
A
  1. IL-6, IL-11, cardiotrophin like cytokine, LIF, cardiotrophin-I, cillary neurotrophic factor and OSM
  2. gp130 and another receptor protein, either a non signalling alpha receptor or a full length, signalling beta receptor
  3. the type of receptor that couples with gp130
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4
Q
  1. Name the 2 domains that make up the extracellular domain
  2. how many of one of these domains bind to 3 sites on the ligand?
  3. Name 2 other speicialist motifs that are found in one of these domains, and what they do.
  4. what do IC domains contain that enables them to interract with JAKs.
  5. why can only one JAK bind to alpha receptors when dimerised with gp130?
A
  1. cytokine binding motif and Ig like repeats
  2. 2
  3. the cytokine binding motifs contain fibronectin type III motifs (which contain spacially conseved cysteines) and the WSXWS sequence, which bind to the ligand
  4. Box domains
  5. they lack an intracellular domain which lacks the Box motifs. Their IC domains however are not required for the functionality of the receptor, as when removed, the resulting receptor is still functional.
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5
Q
  1. what is the HOPtuml mutant?
  2. what is the phenotype of the mutant at 17oC?
  3. what is the phenotype of the mutant at 25oC?
  4. What are myoproliferative neoplasms?
  5. what mutation was identified in sufferers of this condition?
  6. In which domain is this mutation found? What does this domain normally do?

7. what do both of these pieces of evidence show?

A
  1. a temperature sensitive GOF mutation in JAK
  2. stem cells over self renew and progenitors over differentiate into normal blood cells
  3. stem cells over self renew and progenitors over differentiate into lamellocytes. Lamellocytes engulf each other, producing a tumor like growth
  4. a group of bone marrow disorders in which excess blood cells are produced (type of leukaemia)
  5. V617F mutation in JAK2.
  6. pseudokinase domain. This domain normally negatively regulates the kinase domain
  7. GOF mutations of JAK cause over-proliferative phenotypes in both humans and Drosophila.
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6
Q
  1. Name the 3 types of Drosophila blood cells and what they do.
  2. when and where does the first round of haematopoesis occur?
  3. when and where does the second round of haematopoesis occur?
A
  1. PLASMATOCYTES - macrophages that remove cell debris, bacteria and fungi by phagocytosis

CRYSTAL CELLS - involved in melanysation response (wound healing response)

LAMELLOCYTES - large cells that engulf the eggs of parasitic wasps (Leptopilina boulardii)

  1. during embryogenesis in the head mesoderm
  2. in the larvae, in the medullary zone of the lymph gland.
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7
Q
  1. How are cytokines involved in rheumatoid arthritis?
  2. name 3 treatments of rheumatoid arthritis
A
  1. while TNFα plays a major role in the disease, IL-1, IL-6 and IL-5 are also contributing factors to the inflammatory response. Their receptors are also expressed at high levels in RA patients’ blood
  2. steroidal anti-inflammatories and NSAIDsmethorexate (chemotherapy agent) which blocks the JAK/STAT pathwayRuxolin, a 3rd line drug that is a JAK/STAT inhibitor. It is a highly effective treatment but is seen as cost ineffective
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8
Q
  1. Why are cytokine receptors of great biomedical interest?
  2. what did the hyperactivation of the JAK homologue HOP result in?
  3. what did loss of HOP result in?
  4. what are cysteine residues of the fibronectin type III domains important for?
A
  1. because when disrupted they cause severe defects in vivo (e.g. cancer, IBD, RA etc)
  2. extensive haemocyte proliferation, lamellocyte differentiation and the formation of a melanised tumor.
  3. impaired ability to mount an immune response to parasitation
  4. maintaining 3o structure of the EC domain
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9
Q
  1. What was previously thought about IL-6 binding to its receptor?
  2. give an example of how IL-6 ligands are spacially and temporally expressed
  3. what other component also has a restricted expression pattern?
  4. what does the shared usage of gp130 explain?
A
  1. it was thought to promote gp130 homodimerisation, while beta receptors formed heterodimers with gp130. this idea is now unclear
  2. they are expressed under conditions of inflammation
  3. ligand specific receptors (gp130 is ubiquitously expressed)
  4. the redundant functions of the IL-6 cytokines
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10
Q
  1. name 2 conditions, other than cancer and RA that disregulation of signalling is associated with
  2. name 3 proteins which are involved in the termination and modulation of JAK/STAT signalling and how they function
  3. name a function of a certain motif of the IC domain of IL-6R
  4. how did FRAP analysis show that the interraction between the receptor and JAK is very tight?
  5. what domain of JAK enables it to interract with the receptor?
  6. name 2 other post translational modifications which regulate the activity of STAT
A
  1. IBD and osteoporosis
  2. SHIP2 - tyrosine phosphatase; dephosphorylates JAKs and STATsSOCS - classical feedback inhibitor - activated by STATs; inhibit STATsPIAS proteins - inhibit activated STAT from binding to DNA
  3. dileucine motif directs the receptor to the basolateral membrane of polarised cells
  4. JAK doesn’t diffuse like a normal cytosolic protein, indicating it is anchored by something in the membrane
  5. FERM domain
  6. serine phosphorylation increases the transcription of target genesmethylation gives it a higher tendency to bind to DNA by weakening the assocation with PIAS.
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11
Q
  1. in the identification of the V27F mutation, who were the subjects that were screened?
  2. what was screened for?
  3. what proportion of patients carried the mutation? Was the mutation seen in healthy control subjects?
  4. how was homozygoticity of the mutation shown?
  5. what are the future implications of this study? Any problems?
A
  1. 679 individuals, 480 of which were positive for chronic myoproliferative disease
  2. mutations in genes encoding tyrosine kinases and their downstream components
  3. 27%. Not seen in healthy individuals
  4. ARMS assay (stronger mutant bands than WT), and confirmed by pryosequencing
  5. targeting JAK for the treatment of MPD (likely to cause side effects due to its roles in immunity and cardiovascular functionmutant JAK screening test for individuals with suspected MPD. (problem - only 27% of affected individuals carried V27F mutant)
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12
Q
  1. What is tocilizumab?
  2. what was ELISA used to study?
  3. how was the effect of tocilizumab on IL-6 signalling measured?
  4. how was the specificity of tocilizumab studied?
  5. what were the results of this study?
  6. any critical analysis?
  7. what are the future implications of these findings?
A
  1. a humanised monoclonal antibody against IL-6 receptors
  2. the binding activity of tocilizumab to mIL-6R and sIL-6R, and ts supression of IL-6 binding to the receptors
  3. by measuring the proliferation of IL-6R expressing cell lines
  4. by measuring the proliferation of cell lines expressing other cytokine receptors except IL-6
  5. tocilizumab was able to bind to sIL-6R and mIL-6R, and supressed the binding of IL-6 to its receptorit prevented IL-6 induced proliferationit had no effect on the proliferation of cells expressing other IL receptors
  6. the study was only performed in vitro.
  7. does tocilizumab do the same in vivo? are there any side effects of the drug?
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13
Q

name the drosophila homologues of the ligand, the receptor and JAK

A

Ligand = UPD

Receptor = DOME

JAK = HOP

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14
Q

describe the βlue-βlau complimentation technique

A

β-galactosidase normally hydrolyses lactose into galactose and glucose. It also hydrolyses X-gal, which produces a blue ppt

β-galactosidase is a homotetramer. 4 subunits are required for enzymatic activity

Δα and Δω are transcripts of β-galactosidase that individually are unable to form a homotetramer, however together can form a homotetramer required for enzymatic activity.

When Δα and Δω are fused onto protiens that interract, they can perform hydrolysis of X-gal, producing a blue precipitate.

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15
Q

STUDYING WHETHER DOME DIMERISATION IS LIGAND DEPENDENT AND NECESSARY FOR SIGNALLING

  1. name 2 techniques used to determine subcellular localisation of DOME
  2. what was the Df(1)os1A mutant used to show? Why?
  3. what is DomeDN and what was it used to show?
  4. why was ectopic UPD expressed in the amnioserosa, the ectoderm surrounding the tracheal pits and the hindgut?
A
  1. X gal staining and anti-βgalactosidase antibodies
  2. a mutant ligand which causes a UPD deficiency. It was used to show whether UPD induces dimerisation of receptors. (can receptors dimerise in the absense of UPD?)
  3. a mutant Dome which lacks TM and IC domains. It was used to see whether any unknown ligands not deleted by Df(1)os1A can still cause receptor dimerisation
  4. to see if dimerisation is essential for the competency of cells to respond to JAK/STAT
    Dome is dimerised in the aminoserosa, but it doesnt have a mutant phenotype associated with JAK/STAT signalling

JAK/STAT signalling induces the expression of trachealess and Knirps in the tracheal pits. Dome is not dimerised in the surrounding ectoderm

JAK/STAT signalling induces the expression if Vvl in the hindgut where dome is dimerised

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16
Q
  1. where do hybrid receptors localise?
  2. what is the expression pattern of hybrid receptors? In which tissues are they dimerised?
  3. what was the effect of UPD deficiency on X gal staining?
  4. what did the expression of UPD in the amnioserosa result in?
  5. what did the expression of UPD in the surrounding ectoderm result in?
  6. what did the expression of UPD in the hindgut result in?
  7. Name 2 general conclusions made from this study.
A
  1. at the plasma membrane and cytoplam. X gal staining only seen at the PM, suggesting that dimerised receptors are not found in the cytoplasm
  2. ubiquitously expressed. Dimerised in tissues where JAK/STAT signalling is active
  3. no effect on X gal signalling
  4. produces mutant phenotype
  5. unable to induce the expression of Knirps and trachealess
  6. induction of Vvl expression
  7. receptor homodimerisation occurs independently of ligand activityreceptor dimerisation is required for competency of cells to respond to JAK/STAT ligands

BMS301 - Membrane Receptors (6 decks)

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