Catalytic Receptors

Question 1

Catalytic receptor subfamilies

Answer 1

Receptor Tyrosine Kinase (RTK)
- have intrinsic tyrosine kinase activity
Receptor Serine/Threonine Kinase
- contain intrinsic serine/threonine kinase activity
Cytokine Receptors
- receptors that associate with proteins that have tyrosine kinase activity
Receptor Gyanylyl Cyclases
- have intrinsic cyclase activity
Receptor Protein Tyrosine Phosphatases
- have intracellular phosphotyrosine phosphatase activity

Question 2

catalytic receptors basic structure

Answer 2

• extracellular binding domain
• 1 transmembrane helix
• intracellular catalytic domain

Question 3

general signalling

Answer 3

• receptor is activated
• protein phosphorylation
• gene transcription
• protein synthesis
• cellular effects
• hours - slow process

Question 4

Receptor Tyrosine Kinases

- general structure, ligands

Answer 4

• most receptor tyrosine kinases consist of single polypeptides monomer in the unbound state
• the insulin receptor exists as dimers consisting of two pairs of polypeptide chains linked . by disulphide bonds
• ligands for the ~60 different receptor tyrosine kinases are growth factors

Question 5

Receptor Tyrosine Kinase structure

Answer 5

• extracellular region is composed of different domains
• transmembrane domain is a hydrophobic segment of 22 to 26 amino acids (single transmembrane helix)
• the intracellular domain primarily consists of the catalytic domain and various phosphorylation sites

Question 6

Dimerisation and autophosphorylation of receptor tyrosine kinases

Answer 6

Growth factor binding induced receptor dimerisation, which results in receptor autophosphorylation as the two polypeptide chains phosphorylate one another

Question 7

Stimulation of tyrosine kinase activity by receptor dimerisation

Answer 7

1. Autoinhibition through activation loop and/or juxtamembrane domain
2. Activation loop movement allows ATP binding
3. Ligand-binding results in dimerisation
4. Cross-phorylation of activation loops
5. . Conformational change, activation
6. Phosphorylation of additional sites

Question 8

Receptor tyrosine kinase signalling

Answer 8

Multiple pathways - add later

• activate RAS –> RAF –> MEK –> ERL –> ELK1 –>enters nucleus and binds to stuff
  Ras pathway most economical
• alternate path
• uses PIP2
• regulates gene transcription
• PI3K pathway
  
  • Shc –> PI3K –> AKT –> survival, growth/differentiation
• MAPK pathway
  
  • Shc –> Ras-GTP –> MAPK –> survival, growth/differentiation, synaptic transmission, long-term potentiation
• PLC pathway
  
  • PLC –> DAG + IP3 –> Ca2+ –> CaM –> CaMK II (growth/differentiation), CaMK IV –> CREB (gene expression)

Question 9

Termination of receptor tyrosine kinase signalling

Answer 9

2 ways to turn off signalling - phosphorylation or internalisation

• phosphorylated proteins do not hydrolyse spontaneously
• Need an enzyme to take phosphate off receptor
  
  • phosphatases remove phosphate groups, e.g. protein tyrosine phosphatase 1B
• removing all the phosphates turns the RTK signalling off
• Receptor protein tyrosine phosphatase are membrane bound
• protein tyrosine phosphatase are soluble proteins
• activated receptors are endocytosed through clathrin-coated pits and then recycled to the cell surface or degraded

Question 10

Receptor Serine/Threonine Kinases

Answer 10

• ligands are cytokines that belong to the transforming growth factor beta (TGFbeta) and bone morphogenetic protein (BMP) families
• the functional complex at the cell surface consists of two ‘type II’ and two ‘type I’ RTSK
• iIn the absence of ligand, type II and type I receptors exist as homodimers at the cell surface
• signal through Smad proteins
• type I and II contain a kinase domain
• type I contains a Gly-Ser (GS) sequence upstream from the kinase domains

Question 11

Receptor Serine/Threonine Kinase Signalling

Answer 11

1. Ligand binds to receptor complex and induced transphospjorylation by the type II receptor kinases of the GS segments int he type I receptor
2. Type I receptors phosphorylate selected Smads at C-terminal serines
3. Receptor-activated Smads (R-Smads) form a complex with a Smad4
4. Activated Smad complexes translocate into the nucleus where they regulate transcription of target genes

Activated RSTKs can also signal through non-Smad pathway such as the MAP kinase pathway

Question 12

Regulation of Receptor Serine/Threonine Kinase Signalling

Answer 12

• two ways to turn off (same as RTK)
• RSTK inactivated by protein phosphatases
• receptors can be degraded or recycled via clathrin-coated puts or caveolin pathways

Question 13

Cytokine receptor family

Answer 13

• cytokine receptors lack intrinsic tyrosine kinase activity and rely on receptor-associated Janus kinases (JAKs) to transmit their signals to the cytoplasm
• the cytokine receptor superfamily includes the receptors for interleukins, erythropoietin, interferon and growth hormone

Question 14

Cytokine receptor family signalling

Answer 14

1. Receptors dimerise and/or reorient
2. The associated JAKs cross-phosphorylate each other, starting the signalling process
3. STAT proteins are recruited
4. The STATs dissociate from the receptors and form active phosphorylated heterodimers which travel to the nucleus where they regulate transcription of target genes
5. Signals are terminated by the action of specific tyrosine phosphatases and receptor internalisation and then by the actions of induced suppressor of cytokine signalling (SOCS) proteins

Question 15

Growth hormone receptor signalling

Answer 15

Multiple sets of signalling pathways

Canonical pathway (idealised/generalised pathway that represent common properties of particular signalling pathways):

• protein-tyrosine kinase
• JAK signalling via STAT

Src/ERK pathway

Question 16

Receptor Guanylate Cyclase family

Answer 16

• Family members have conserved ligand-binding, catalytic (guanylyl cyclase) and regulatory domain with the exception of NPR-C which has an extracellular binding domain homologous to that of other NPRs, but with a truncated intracellular domain which appears to couple via the Gi/o family of G proteins
• ligand binding domain
• membrane
• kinase homology domain
• guanylyl cyclase domain

two enzyme parts - kinase part, cyclase part
NPR-C is missing these parts

Question 17

Receptor guanylate cyclase signalling

Answer 17

1. hormone binds
2. ATP binds to the Kinase Homology Domain (KHD)
3. conformational change occurs within KHD allowing the guanylyl cyclase domains to come together to form two active sites per dimer and convert GTP to cGMP
4. the conformational change in KHD exposes the phosphorylated residues to a protein phosphotase, which de phosphorylates the receptor
5. dephosphorylated receptor is unresponsive to further hormonal stimulation

Question 18

termination of receptor guanylate cyclase signalling

Answer 18

Can be turned off by dephosphorylation or internalisation

Question 19

Receptor protein tyrosine phosphatases

Answer 19

• RPTP are cell-surface proteins with a single TM region and intracellular phosphotyrosine phosphatase activity
• many have constitutive activity when heterologously expressed
• extracellular domains bind components of the extracellular matrix or cell-surface proteins allowing coupling of extracellular adhesion-mediated events to intracellular signalling pathways
• D1 domains displays catalytic activity. D2 domain has not been established

Question 20

models of RPTP activation

Answer 20

Head-to-toe model

• RPTPs are found as homodimers or heterodimers on the cell surface
• the active state of RPTP depends on post-translational modifications such as oxidation or phosphorylation, which cause conformational changes which alter the ligand-binding site and shift the dimers from an active to inactive state

Wedge model of inhibition

• in the inactive dimer the wedge domain of one of the dimer pair occupies the catalytic site of the other RPTP, and vice versa, inhibiting function
• i.e. wedge from dimer 1 blocks dimer 2 active site, wedge from dimer 2 blocks dimer 1 active site

Question 21

RPTPs are regulated by cleavage, oxidation and phosphorylation

Answer 21

• cleavage and extracellular shedding of ectodomains has been reported for several RPTPs
• reversible oxidation of an active state cysteine inactivates RPTPs
• the active state of RPTPs can either be increased or decreased by phosphorylation of serine or tyrosine residues