Functional and immunological evidence for stable association of solubilized vasoactive-intestinal-peptide receptor and stimulatory guanine-nucleotide-binding protein from rat liver.
We have reported the solubilization of complexes between vasoactive intestinal peptide (VIP) and its receptor from rat liver in a GTP-sensitive form of Mr 150,000 [Couvineau, A., Amiranoff, B. & Laburthe, M. (1986) J. Biol. Chem. 261, 14482-14489]. In the present study, we demonstrate a stable association of solubilized VIP receptor and stimulatory guanine nucleotide-binding protein (Gs protein), taking advantage of the ability of the glycoproteic VIP receptor (Mr 48,000), and the inability of the Gs protein, to adsorb to wheat germ agglutinin (WGA). 125I-VIP-receptor complexes solubilized in Triton X-100 were adsorbed on WGA-Sepharose, extensively washed and the radioactivity retained was eluted with 1 mM GTP showing that: (a) radioactivity corresponds to free 125I-VIP and (b) alpha s (Mr 42,000) and beta (Mr 35,000) subunits of Gs protein are detectable in the GTP eluate by immunoblotting using antisera against these subunits. Such an effect of GTP implied that a stable ternary complex consisting of VIP, receptor and Gs protein had been adsorbed to WGA-Sepharose. When Triton-solubilized 125I-VIP-receptor complexes were adsorbed on WGA-Sepharose, then retained material was specifically eluted with 0.3 M N-acetylglucosamine, analysis of the sugar eluate showed the following results. (a) GTP induces the dissociation of 125I-VIP-receptor complexes of Mr 150,000 contained in the eluate indicating that 125I-VIP-receptor-G protein complexes had been adsorbed to the WGA column. (b) The Mr-42,000 alpha s subunit can be specifically ADP-ribosylated by cholera toxin. (c) Immunoblotting using antisera against the alpha s and beta subunits of Gs protein, reveals Mr-42,000 and Mr-35,000 components corresponding to alpha s and beta subunits, respectively. (d) Affinity cross-linking using dithiobis(succinimidyl-propionate) of 125-I-VIP-receptor complexes eluted from the WGA column reveals a major band corresponding to Mr 150,000. Immunoblotting using antisera against the beta-subunit shows the presence of the beta subunit (Mr 35,000) in this Mr-150,000 component. In conclusion, these data provide functional and immunochemical evidence for the physical association of solubilized VIP-receptor complexes with alpha s and beta subunits of Gs protein.
Couvineau
A
A
Unité de recherche sur la différenciation et la neuroendocrinologie de cellules digestives, Institut National de la Santé et de la Recherche Médicale, Villejui, France.
Rouyer-Fessard
C
C
Voisin
T
T
Laburthe
M
M
D000246:Adenosine Diphosphate Ribose / Q000302:isolation & purification; D000818:Animals; D001345:Autoradiography; D001665:Binding Sites; D002846:Chromatography, Affinity; D019204:GTP-Binding Proteins / Q000302:isolation & purification* / Q000502:physiology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000031:analogs & derivatives*; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000302:isolation & purification; D015151:Immunoblotting; D008099:Liver / Q000032:analysis*; D051381:Rats; D011964:Receptors, Gastrointestinal Hormone / Q000302:isolation & purification* / Q000502:physiology; D018005:Receptors, Vasoactive Intestinal Peptide; D012685:Sepharose / Q000031:analogs & derivatives; D012995:Solubility; D013873:Thionucleotides / Q000302:isolation & purification; D014660:Vasoactive Intestinal Peptide / Q000302:isolation & purification*
D011964:Receptors, Gastrointestinal Hormone; D018005:Receptors, Vasoactive Intestinal Peptide; D013873:Thionucleotides; C034882:guanosine 5'-O-(1-thiotriphosphate); C042758:wheat germ agglutinin-sepharose 6MB; D000246:Adenosine Diphosphate Ribose; D014660:Vasoactive Intestinal Peptide; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D012685:Sepharose; D019204:GTP-Binding Proteins