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J. Biochem, 1998, Vol. 123, No. 6 1024-1030
© 1998 Japanese Biochemical Society

research-article

VIP Induces the Translocation and Degradation of the {alpha} Subunit of Gs Protein in Rat Pituitary GH4C1 Cells

Yukiko Yajima*,1, Yoshiko Akita*, Toshikazu Saito{dagger} and Seiichi Kawashima*

*Department of Molecular Biology, The Tokyo Metropolitan Institute of Medical Science 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613
{dagger}Division of Endocrinology and Metabolism, Jichi Medical School 3311-1 Yakushiji, Minamikawachi-machi, Kawachi-gun, Tochigi 329-0431

1To whom correspondence should be addressed. Fax: +81-3-5685-6609, E-mail: yajima{at}rinshoken.or.jp

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 Gs{alpha} in the membrane fraction, whereas immunoblot analysis and reconstitution assay of adenylyl cyclase clearly showed an increase in the amount of Gs{alpha} in the supernatant (cytosolic) fraction. The VIP-induced release of G proteins {alpha} subunits from membranes was specific for Gs{alpha}. The VIP-dependent release of Gs{alpha} 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 Gs{alpha} 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 Gs{alpha} from membranes and a 6% increase in total Gs{alpha} 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 Gs{alpha}. 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 Gs{alpha} 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 Gs{alpha} protein and induces the release of Gs{alpha} from membranes along with its down-regulation in cellular levels.


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