VIP Induces the Translocation and Degradation of the α Subunit of Gs Protein in Rat Pituitary GH4C1 Cells

It has been shown that G proteins are potential regulatory molecules in the transmembrane signaling cascade. The aim of this study was to examine the possibility of equivalent G-protein redistribution and/or down-regulation in a target cell upon agonist stimulation. Short-term (0–80 min) incubation of rat pituitary GH4C1 cells with vasoactive intestinal peptide (VIP, 0.1 μM) induced a decrease in the levels of Gsα in the membrane fraction, whereas immunoblot analysis and reconstitution assay of adenylyl cyclase clearly showed an increase in the amount of Gsα in the supernatant (cytosolic) fraction. The VIP-induced release of G proteins α subunits from membranes was specific for Gsα. The VIP-dependent release of Gsα from membranes was blocked by a VIP-receptor antagonist, (N-Ac-Tyr, D-Phe)-GRF(1–29)-NH2 (10 μM). Pituitary adenylate cyclase-activating polypeptide (PACAP) also stimulated the release of Gsα from membranes of GH4C1 cells. Furthermore, prolonged exposure of cells to VIP (0.1 μM) for 2–24 h caused a 21–40% decrease in Gsα from membranes and a 6% increase in total Gsα in the cytosolic fraction. The effect of VIP was dose-dependent with ED50 values of 81.6±20.0 nM for down-regulation and 2.5±0.3 nM for translocation of Gsα. Concurrent treatment of GH4C1 cells with VIP and cycloheximide indicated that suppression of protein synthesis de novo did not mimic the effect of VIP. Moreover, the chase experiment of 35S-labeled Gsα clearly demonstrated a more rapid rate of decay in the cells maintained in the presence of the agonist. These data indicate that VIP-receptor activates Gsα protein and induces the release of Gsα from membranes along with its down-regulation in cellular levels.