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Identifying Protein-protein Interaction Sites Using Peptide Arrays
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An amyloid-like C-terminal domain of thrombospondin-1 displays CD47 agonist activity requiring both VVM motifs

J F McDonald1, J M Dimitry, W A Frazier

  • 1Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Box 8231, 660 South Euclid Avenue, St. Louis, Missouri 63110, USA.

Biochemistry
|August 20, 2003
PubMed

Insights

Thrombospondin-1

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Background:

  • Thrombospondin-1 (TSP1) is a matricellular protein involved in various cellular processes.
  • The C-terminal domain (CBD) of TSP1 contains VVM motifs implicated in CD47 agonism.
  • CD47 is a receptor that plays a role in immune regulation and cell signaling.

Purpose of the Study:

  • To characterize the structure and function of the recombinant C-terminal domain (rCBD) of TSP1.
  • To investigate the role of VVM motifs in rCBD's interaction with CD47 and melanoma cells.
  • To elucidate the signaling pathways involved in rCBD-mediated melanoma cell responses.

Main Methods:

  • Expression and purification of recombinant TSP1 C-terminal domain (rCBD).
  • Circular dichroism (CD) spectroscopy and thioflavin T binding assays to assess secondary structure.
  • Cell binding assays using C32 melanoma cells.
  • Cell spreading assays on vitronectin.
  • Mutagenesis of VVM motifs within rCBD.
  • Pertussis toxin and heparin inhibition studies.

Main Results:

  • rCBD adopted a beta-sheet rich structure, similar to amyloid proteins, and exhibited CD47-dependent melanoma cell binding at low concentrations.
  • rCBD strongly stimulated melanoma cell spreading on vitronectin, mediated by G(i) signaling, and this effect was blocked by pertussis toxin.
  • Mutations in VVM motifs reduced cell spreading stimulation, indicating their importance in CD47 activation, while cell binding was only modestly affected.

Conclusions:

  • The VVM motifs within the TSP1 rCBD are crucial for CD47-mediated signaling and melanoma cell spreading.
  • rCBD's structural properties, including beta-sheet enrichment, are linked to its biological activity.
  • TSP1's C-terminal domain acts as a potent CD47 agonist, influencing melanoma cell behavior through specific structural motifs.

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