Characterization of GPCRs in extracellular vesicle (EV)

Methods Cell Biol. 2017:142:119-132. doi: 10.1016/bs.mcb.2017.07.004. Epub 2017 Sep 11.

Abstract

Extracellular vesicle (EV) are tiny membranous vesicles usually <500nm in size that recently emerged as a new paradigm in human intercellular signaling. EVs have shown a promising role in development of diagnostic markers in many pathophysiological disorders. The presence of chemosensory and therapeutically relevant G protein-coupled receptors (GPCRs) on EV membranes is poorly characterized. Here, we compare different methods including ultracentrifugation and polymer-charge-based separation to isolate EVs from cell culture media and human saliva. The presence of bitter taste GPCRs (T2R4 and T2R38) and a class A GPCR angiotensin II type 1 receptor on these EVs was characterized by qPCR, ELISA, and immunotransmission electron microscopy.

Keywords: Angiotensin II type 1 receptor; Bitter taste receptor; Electron microscopy; Exosomes; Extracellular vesicle; G protein-coupled receptor; Polyethylene glycol; Protamine chloride.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • Enzyme-Linked Immunosorbent Assay
  • Extracellular Vesicles / metabolism*
  • Humans
  • Microscopy, Electron, Transmission / methods
  • Microscopy, Immunoelectron / methods
  • Oligopeptides / chemistry
  • Real-Time Polymerase Chain Reaction
  • Receptor, Angiotensin, Type 1 / chemistry
  • Receptor, Angiotensin, Type 1 / isolation & purification*
  • Receptor, Angiotensin, Type 1 / ultrastructure
  • Receptors, G-Protein-Coupled / chemistry
  • Receptors, G-Protein-Coupled / isolation & purification*
  • Receptors, G-Protein-Coupled / metabolism
  • Receptors, G-Protein-Coupled / ultrastructure
  • Ultracentrifugation / methods

Substances

  • AGTR1 protein, human
  • Oligopeptides
  • Receptor, Angiotensin, Type 1
  • Receptors, G-Protein-Coupled
  • taste receptors, type 2
  • FLAG peptide