Cell Signalling Research

Question 1

Discoidin domain

Answer 1

• unique set of receptor tyrosine kinases that signal in response to collagen
• Structurally, the DDRs are type I transmembrane multidomain glycoproteins. The extracellular portion of both DDRs contain the Discoidin Domain, a unique structural domain that comprises the collagen binding sites. Like all RTKs, the DDRs contain a kinase domain in the intracellular portion of the receptor that is activated upon collagen binding resulting in receptor autophosphorylation at multiple tyrosine residues present within the intracellular region
• activation is slow and sustained

Question 2

Discoidin domain discovery

Answer 2

• searching for proteins related to the insulin receptor and screened a human placental cDNA library with a P-labelled antisense oligonucleotide against a region conserved in many tyrosine kinases
• discovered new RTK with discoidin like domain in the extracellular region

Question 3

Roles of DDRs

Answer 3

• tissue and organ development

- pathologies associated with overexpression and disregulation

Question 4

DDR Structure Function Studies

Answer 4

• ligand binding assay to monitor binding of DDR to collagen
• solid-phase assay with a color reaction telling you how much DDR antibody has bound to the collagen-DDR complex
• found that the triple helical structure of collagen is needed for binding

Question 5

Identification of Binding site

Answer 5

• use triple helical peptide library to make synthetic peptides in a way that you use 24 residues in each structure running through the whole sequence
• use binding assay to define which peptide sequences bind to the receptor
• find biologically relevant binding site
• alanine scanning to determine key residues in sequence
• key residues are M,F,O with hydroxylated proline
• binding site formed by two chains in triple helical chain

Question 6

SMED

Answer 6

• pathology caused by DDR mutation
• short limbs and abnormal calcifications
• kinase domain mutants not trafficked to cell surface
• another cause is the E113K mutation abolishing collagen binding

Question 7

Disulphide cross-linking study**

Answer 7

• mapped ligand induced RTK dimerisation
• active form is assumed to be dimer
• put cysteines in places you believe dimerization occurs
• use presence of disulphide bridges to determine position/presence of dimerization
• crosslinking pattern reflectes cysteine positions
• in DDR, it existed as a dimer regardless of activation (non monomeric)
• likely constitutively active
• only with collagen do you get pTyr activation but dimer forms regardless: dimer doesn’t affect signal

Question 8

DDR1 Structure

Answer 8

• large cytosolic juxtamembrane region connecting extracellular domain to the kinase
• unique to this RTK
• only the last 1/4 of the JM region was shown to be essential for activation

Question 9

DDR1 Kinase

Answer 9

• JM4 (last part) region reinforces auto-inhibition by the activation loop

Question 10

Kinase Phosphorylation

Answer 10

• PAGE shows auto-phosphorylation of soluble kinase constructs occurs in steps
• Western blot with antibodies showed JM4 phosphorylation preceded activation loop phosphorylation

Question 11

DDR1 Kinase activation

Answer 11

• both JM4 and activation loop must be phosphorylated for full activation
• JM4 has 2 tyrosines needed for activation (phosphoylated)
• mutation of second tyrosine severly affects activity and no activity is recorded when both are inactivated

Question 12

Summary

Answer 12

• A triple-helical peptide library was used to identify the DDR binding site in collagen (unique case usually recombinant dissection of ligand is done)
• X-ray crystallography has revealed the details of the DDR-collagen interaction.
• Disulphide cross-linking experiments ruled out ligand-induced dimerisation or conformational changes as mechanisms of DDR activation.
• Super-resolution microscopy has revealed large-scale DDR redistribution in response to collagen binding.
• Structural and biochemical experiments have shown that the cytosolic juxtamembrane region plays an important role in DDR autoinhibition and activation.