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Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy
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The class I scavenger receptor CD163 promotes internalization of ADAMTS13 by macrophages

Fabian C Verbij1, Nicoletta Sorvillo1, Paul H P Kaijen1

  • 1Department of Plasma Proteins, Sanquin Research and Landsteiner Laboratory, Academic Medical Center (AMC), University of Amsterdam, Amsterdam, The Netherlands.

Blood Advances
|January 4, 2018
PubMed

Insights

Macrophages internalize ADAMTS13 via endocytosis, a process crucial for its clearance. This study identifies scavenger receptor CD163 as a primary receptor mediating ADAMTS13 uptake by macrophages.

Area of Science:

  • Immunology
  • Cell Biology
  • Biochemistry

Background:

  • Macrophage uptake of ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13) is implicated in its circulating clearance.
  • Understanding the specific endocytic pathways and receptors involved is essential for elucidating ADAMTS13 homeostasis.

Purpose of the Study:

  • To investigate the endocytic mechanisms and identify the specific receptors responsible for ADAMTS13 internalization by macrophages.
  • To characterize the interaction between ADAMTS13 and its potential receptors.

Main Methods:

  • Utilized human monocyte-derived macrophages and fluorescently labeled recombinant ADAMTS13 to study uptake via flow cytometry.
  • Employed pharmacological inhibitors (dynasore, mannan) and blocking antibodies (anti-CD206, anti-CD163) to probe endocytic pathways.
  • Identified candidate receptors using pull-down assays followed by mass spectrometry.
  • Validated receptor binding and internalization using surface plasmon resonance and Chinese hamster ovary (CHO) cells expressing CD163.

Main Results:

  • ADAMTS13 internalization was inhibited by dynasore, indicating dynamin-dependent endocytosis.
  • Mannan partially inhibited uptake, but anti-CD206 antibody had no effect, suggesting roles for receptors other than CD206.
  • Mass spectrometry identified scavenger receptor CD163 as a candidate receptor.
  • Blocking CD163 with antibody EDHu-1 significantly reduced ADAMTS13 uptake, particularly in high-CD163 expressing cells and CD163-expressing CHO cells.
  • Surface plasmon resonance confirmed direct binding of ADAMTS13 to specific domains of CD163.

Conclusions:

  • Class I scavenger receptor CD163 is a major endocytic receptor for ADAMTS13 on macrophages.
  • The interaction between ADAMTS13 and CD163 mediates the internalization and likely clearance of ADAMTS13 from circulation.

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