Identification of G-Proteins Coupling to the Vasoactive Intestinal Peptide Receptor VPAC1 Using Immunoaffinity Chromatography: Evidence for Precoupling

Abstract 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 Gα-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.