Identification of G-proteins coupling to the vasoactive intestinal peptide receptor VPAC(1) using immunoaffinity chromatography: evidence for precoupling.
VPAC(1) receptor 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 VPAC(1) receptor. VPAC(1)/G-protein complexes were solubilized from membranes and copurified. Receptor and Galpha-proteins were detected in eluates using (125)I-VIP labeling and immunoblotting, respectively. Human VPAC(1) transfected in HEK293 cells couples to Gs but not Gi3, Gi1/2, or Gq. Rat VPAC(1) in brain membranes is coupled to Gs and Gi3. Rat VPAC(1) in lung membranes couples to Gs, Gi3, and Gq. Pretreatment of membranes with VIP increased the level of all G-proteins copurifying with VPAC(1). Immunoaffinity chromatography also revealed VPAC(1) 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 VPAC(1) receptor activity especially in cells, such as some human malignant tumor cells, that express high levels of receptor.
Martin Shreeve
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Department of Pharmacology, University of Vermont, Burlington 05405, USA. Stephen.Shreeve@uvm.edu
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D008168:Lung / Q000378:metabolism; D046911:Macromolecular Substances; D051381:Rats; D018005:Receptors, Vasoactive Intestinal Peptide / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; D014162:Transfection
D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D018005:Receptors, Vasoactive Intestinal Peptide; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; D019204:GTP-Binding Proteins; C498819:GNAI3 protein, human; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D019205:GTP-Binding Protein alpha Subunits, Gs; C498820:Gnai3 protein, rat; D020962:Heterotrimeric GTP-Binding Proteins