Antibodies to a Human α2-C10 Adrenergic Receptor Fusion Protein Confirm the Cytoplasmic Orientation of the V-VI Loop

Patrick Vanscheeuwijck, Yi Huang, Daniel Schullery, John W. Regan

Research output: Contribution to journalArticle

12 Scopus citations

Abstract

DNA encoding the hydrophillic region between transmembrane domains V and VI of the human platelet α2-adrenergic receptor (α2-C10) was amplified using the polymerase chain reaction and was cloned in-frame with a portion of the gene encoding glutathione-S-transferase (GST). Expression of the recombinant plasmid in E. coli resulted in the production of a GST/α2-C10 fusion protein which was purified by preparative SDS-PAGE. Chickens innoculated with the fusion protein produced antibodies that were present in their eggs. In cells expressing the α2-C10, these antibodies recognized the receptor in both Western Blots and indirect immunofluorescence. For the immunofluorescence studies, antibody recognition required permeabilization of the cells with detergent. This evidence establishes the cytoplasmic orientation of the V-VI loop and supports the general model for G-protein coupled receptors.

Original languageEnglish (US)
Pages (from-to)340-346
Number of pages7
JournalBiochemical and Biophysical Research Communications
Volume190
Issue number2
DOIs
StatePublished - Jan 1 1993

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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