VIP induces the translocation and degradation of the alpha 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 microM) induced a decrease in the levels of Gsalpha in the membrane fraction, whereas immunoblot analysis and reconstitution assay of adenylyl cyclase clearly showed an increase in the amount of Gsalpha in the supernatant (cytosolic) fraction. The VIP-induced release of G proteins alpha subunits from membranes was specific for Gsalpha. The VIP-dependent release of Gsalpha from membranes was blocked by a VIP-receptor antagonist, (N-Ac-Tyr,D-Phe)-GRF(1-29)-NH2 (10 microM). Pituitary adenylate cyclase-activating polypeptide (PACAP) also stimulated the release of Gsalpha from membranes of GH4C1 cells. Furthermore, prolonged exposure of cells to VIP (0.1 microM) for 2-24 h caused a 21-40% decrease in Gsalpha from membranes and a 6% increase in total Gsalpha 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 Gsalpha. 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 Gsalpha 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 Gsalpha protein and induces the release of Gsalpha from membranes along with its down-regulation in cellular levels.
Yajima
Y
Y
Department of Molecular Biology, The Tokyo Metropolitan Institute of Medical Science, Bunkyo-ku, Tokyo, 113-8613, Japan. yajima@rinshoken.or.jp
Akita
Y
Y
Saito
T
T
Kawashima
S
S
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line; D002478:Cells, Cultured; D015536:Down-Regulation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D010902:Pituitary Gland / Q000378:metabolism*; D051381:Rats; D014660:Vasoactive Intestinal Peptide / Q000494:pharmacology*
D014660:Vasoactive Intestinal Peptide; D019204:GTP-Binding Proteins