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

P. Vanscheeuwijck, Y. Huang, D. Schullery, John W Regan

Research output: Contribution to journalArticle

12 Citations (Scopus)

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 29 1993

Fingerprint

Adrenergic Receptors
Fusion reactions
Glutathione Transferase
Antibodies
Proteins
Gene encoding
Polymerase chain reaction
Indirect Fluorescent Antibody Technique
G-Protein-Coupled Receptors
Platelets
Detergents
Escherichia coli
Eggs
Fluorescent Antibody Technique
Polyacrylamide Gel Electrophoresis
Chickens
Plasmids
Blood Platelets
Western Blotting
Cells

ASJC Scopus subject areas

  • Molecular Biology
  • Biophysics
  • Biochemistry

Cite this

Antibodies to a Human α2-C10 Adrenergic Receptor Fusion Protein Confirm the Cytoplasmic Orientation of the V-VI Loop. / Vanscheeuwijck, P.; Huang, Y.; Schullery, D.; Regan, John W.

In: Biochemical and Biophysical Research Communications, Vol. 190, No. 2, 29.01.1993, p. 340-346.

Research output: Contribution to journalArticle

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