Low-affinity binding in cis to P2Y2R mediates force-dependent integrin activation during hantavirus infection

Mol Biol Cell. 2017 Oct 15;28(21):2887-2903. doi: 10.1091/mbc.E17-01-0082. Epub 2017 Aug 23.

Abstract

Pathogenic hantaviruses bind to the plexin-semaphorin-integrin (PSI) domain of inactive, β3 integrins. Previous studies have implicated a cognate cis interaction between the bent conformation β53 integrins and an arginine-glycine-aspartic acid (RGD) sequence in the first extracellular loop of P2Y2R. With single-molecule atomic force microscopy, we show a specific interaction between an atomic force microscopy tip decorated with recombinant αIIbβ3 integrins and (RGD)P2Y2R expressed on cell membranes. Mutation of the RGD sequence to RGE in the P2Y2R removes this interaction. Binding of inactivated and fluorescently labeled Sin Nombre virus (SNV) to the integrin PSI domain stimulates higher affinity for (RGD)P2Y2R on cells, as measured by an increase in the unbinding force. In CHO cells, stably expressing αIIbβ3 integrins, virus engagement at the integrin PSI domain, recapitulates physiologic activation of the integrin as indicated by staining with the activation-specific mAB PAC1. The data also show that blocking of the Gα13 protein from binding to the cytoplasmic domain of the β3 integrin prevents outside-in signaling and infection. We propose that the cis interaction with P2Y2R provides allosteric resistance to the membrane-normal motion associated with the switchblade model of integrin activation, where the development of tensile force yields physiological integrin activation.

MeSH terms

  • Animals
  • CHO Cells
  • Cell Adhesion Molecules / metabolism
  • Cell Line
  • Cricetulus
  • Endothelial Cells
  • GTP-Binding Protein alpha Subunits, G12-G13 / metabolism
  • Hantavirus Infections / metabolism*
  • Humans
  • Integrin beta Chains / metabolism
  • Integrin beta3 / metabolism*
  • Integrin beta3 / physiology
  • Nerve Tissue Proteins / metabolism
  • Orthohantavirus / isolation & purification
  • Platelet Glycoprotein GPIIb-IIIa Complex / metabolism
  • Protein Binding
  • Protein Domains
  • Receptors, Purinergic P2Y2 / genetics
  • Receptors, Purinergic P2Y2 / metabolism*
  • Semaphorins / metabolism
  • Signal Transduction

Substances

  • Cell Adhesion Molecules
  • Integrin beta Chains
  • Integrin beta3
  • Nerve Tissue Proteins
  • Platelet Glycoprotein GPIIb-IIIa Complex
  • Receptors, Purinergic P2Y2
  • Semaphorins
  • integrin beta5
  • plexin
  • GTP-Binding Protein alpha Subunits, G12-G13