Identification of G-Proteins Coupling to the Vasoactive Intestinal Peptide Receptor VPAC1 Using Immunoaffinity Chromatography: Evidence for Precoupling
DOI: 10.1006/bbrc.2002.6342
Title: Identification of G-Proteins Coupling to the Vasoactive Intestinal Peptide Receptor VPAC1 Using Immunoaffinity Chromatography: Evidence for Precoupling
Journal Title: Biochemical and Biophysical Research Communications
Volume: 290
Issue: 4
Publication Date: 1 February 2002
Start Page: 1300
End Page: 1307
Published online: online 28 February 2002
ISSN: 0006-291X
Affiliations:

  • a Department of Pharmacology, College of Medicine, University of Vermont, Burlington, Vermont, 05405
  • Abstract: ceptor subtype-specific G-protein interactions were identified using a strategy that exploits an essential initial signaling event, namely the functional and physical association of the receptor with G-protein. An immunoaffinity purification column was constructed using a previously characterized antibody that had been raised against the first extracellular loop of the VPAC1 receptor. VPAC1/G-protein complexes were solubilized from membranes and copurified. Receptor and Gα-proteins were detected in eluates using 125I-VIP labeling and immunoblotting, respectively. Human VPAC1 transfected in HEK293 cells couples to Gs but not Gi3, Gi1/2, or Gq. Rat VPAC1 in brain membranes is coupled to Gs and Gi3. Rat VPAC1 in lung membranes couples to Gs, Gi3, and Gq. Pretreatment of membranes with VIP increased the level of all G-Proteins copurifying with VPAC1. Immunoaffinity chromatography also revealed VPAC1 receptor precoupling to G-protein in the absence of VIP pretreatment. This was confirmed using a cross-linking procedure to capture VIP receptor/G-protein complexes in the native membrane milieu prior to solubilization. Precoupling suggests that there is a significant basal level of VPAC1 receptor activity especially in cells, such as some human malignant tumor cells, that express high levels of receptor.
    Received: 17 December 2001
    Keywords: vasoactive intestinal peptide; vasoactive intestinal peptide receptor; human VPAC1; G-protein; receptor signaling; receptor/G-protein coupling; precoupling; immunoaffinity chromatography; cross-linking

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