VIP INDUCES THE TRANSLOCATION AND DEGRADATION OF THE ALPHA-SUBUNIT OFG(S) PROTEIN IN RAT PITUITARY GH(4)C(1) CELLS

It has been shown that G proteins are potential regulatory molecules i n the transmembrane signaling cascade. The aim of this study was to ex amine the possibility of equivalent G-protein redistribution and/or do wn-regulation in a target cell upon agonist stimulation. Short-term (0 -80 min) incubation of rat pituitary GH(4)C(1) cells with vasoactive i ntestinal peptide (VIP, 0.1 mu M) induced a decrease in the levels of G(s) alpha in the membrane fraction, whereas immunoblot analysis and r econstitution assay of adenylyl cyclase clearly showed an increase in the amount of G(s) alpha in the supernatant (cytosolic) fraction, The VIP-induced release of G proteins a subunits from membranes was specif ic for G(s) alpha. The VIP-dependent release of G(s) alpha from membra nes was blocked by a VIP-receptor antagonist, (N-Ac-Tyr, D-Phe)-GRF(1- 29)-NH2 (10 mu M). Pituitary adenylate cyclase-activating polypeptide (PACAP) also stimulated the release of G(s) alpha from membranes of GH (4)C(1) cells. Furthermore, prolonged exposure of cells to VIP (0.1 mu M) for 2-24 h caused a 21-40% decrease in G(s) alpha from membranes a nd a 6% increase in total G(s) alpha in the cytosolic fraction. The ef fect of VIP was dose-dependent with ED50 values of 81.6 +/- 20.0 nM fo r down-regulation and 2.5 +/- 0.3 nM for translocation of G(s) alpha. Concurrent treatment of GH(4)C(1) cells with VIP and cycloheximide ind icated that suppression of protein synthesis de novo did not mimic the effect of VIP. Moreover, the chase experiment of S-35-labeled G(s) al pha clearly demonstrated a more rapid rate of decay in the cells maint ained in the presence of the agonist, These data indicate that VIP-rec eptor activates G(s) alpha protein and induces the release of G(s) alp ha from membranes along with its down-regulation in cellular levels.