The Transmembrane Domains of β and IX Subunits Mediate the Localization of the Platelet Glycoprotein Ib-IX Complex to the Glycosphingolipid-enriched Membrane Domain

J Biol Chem. 2015 Sep 4;290(36):22155-62. doi: 10.1074/jbc.M115.668145. Epub 2015 Jul 22.

Abstract

We have previously reported that the structural elements of the GP Ib-IX complex required for its localization to glycosphingolipid-enriched membranes (GEMs) reside in the Ibβ and IX subunits. To identify them, we generated a series of cell lines expressing mutant GP Ibβ and GP IX where 1) the cytoplasmic tails (CTs) of either or both GP Ibβ and IX are truncated, and 2) the transmembrane domains (TMDs) of GP Ibβ and GP IX were swapped with the TMD of a non-GEMs associating molecule, human transferrin receptor. Sucrose density fractionation analysis showed that the removal of either or both of the CTs from GP Ibβ and GP IX does not alter GP Ibα-GEMs association when compared with the wild type. In contrast, swapping of the TMDs of either GP Ibβ or GP IX with that of transferrin receptor results in a significant loss (∼ 50%) of GP Ibα from the low density GEMs fractions, with the largest effect seen in the dual TMD-replaced cells (> 80% loss) when compared with the wild type cells (100% of GP Ibα present in the GEMs fractions). Under high shear flow, the TMD-swapped cells adhere poorly to a von Willebrand factor-immobilized surface to a much lesser extent than the previously reported disulfide linkage dysfunctional GP Ibα-expressing cells. Thus, our data demonstrate that the bundle of GP Ibβ and GP IX TMDs instead of their individual CTs is the structural element that mediates the β/IX complex localization to the membrane GEMs, which through the α/β disulfide linkage brings GP Ibα into the GEMs.

Keywords: lipid raft; platelet glycoprotein Ib; protein expression; shear stress; transmembrane domain.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Binding Sites / genetics
  • Blotting, Western
  • CHO Cells
  • Cricetinae
  • Cricetulus
  • Glycosphingolipids / metabolism*
  • Humans
  • Membrane Microdomains / metabolism*
  • Mutation
  • Platelet Glycoprotein GPIb-IX Complex / genetics
  • Platelet Glycoprotein GPIb-IX Complex / metabolism*
  • Protein Binding
  • Protein Subunits / genetics
  • Protein Subunits / metabolism
  • Receptors, Transferrin / genetics
  • Receptors, Transferrin / metabolism
  • Stress, Mechanical
  • von Willebrand Factor / metabolism

Substances

  • Glycosphingolipids
  • Platelet Glycoprotein GPIb-IX Complex
  • Protein Subunits
  • Receptors, Transferrin
  • von Willebrand Factor