Abstract
In mouse Balb/c3T3 fibroblasts, insulin-like growth factor (IGF)-II activates a calcium-permeable cation channel through a cell surface IGF-II receptor (Kojima, I., Nishimoto, I., Iiri, T., Ogata, E., and Rosenfeld, R. G. (1988) Biochem. Biophys. Res. Commun. 154, 9-19; Matsunaga, H., Nishimoto, I., Kojima, I., Yamashita, N., Kurokawa, K., and Ogata, E. (1988) Am. J. Physiol. 255, C442-C446). In the action of IGF-II, a pertussis toxin (or islet-activating protein; IAP)-sensitive GTP-binding protein (G protein) is inferred to be involved (Nishimoto, I., Hata, Y., Ogata, E., and Kojima, I. (1987) J. Biol. Chem. 262, 12120-12126). In the present study, we examined the direct coupling of the IGF-II receptor with G proteins. In broken Balb/c3T3 cell membranes, 10 nM IGF-II rapidly attenuated the IAP-catalyzed ADP-ribosylation of a 40-kDa protein in a manner requiring magnesium ion. The IGF-II-mediated attenuation in the IAP substrate activity was 80% recovered after washing off IGF-II and inhibited by coexisting guanosine 5'-O-(2-thiodiphosphate), while either aluminum fluoride solution (10 mM NaF plus 100 microM AlCl3) or 100 microM guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) reproduced the action of IGF-II. When purified IAP substrate G proteins (Gi1, Gi2, G0) were incubated with IGF-II in the presence of membranes from IAP-treated Balb/c3T3 cells, the attenuation in the IAP substrate activity was evident in Gi2, but not in Gi1 or G0. On the other hand, 10 nM insulin had no effect on the modification of the 40-kDa IAP substrate in Balb/c3T3 cell membranes, whereas 10 nM IGF-I elicited a slow onset of the IAP sensitivity attenuation from the 40-kDa protein. However, the specific involvement of the IGF-II receptor in the modification of the IAP substrate induced by low concentrations of IGF-II was suggested by the observations that (i) IGF-I receptor-lacking cell membranes were effective for the Gi2 modification by IGF-II, (ii) the ability of membranes to mediate the action of IGF-II was markedly attenuated in IGF-II receptor-lacking cell membranes, and (iii) agonistic anti-IGF-II receptor antibody mimicked the action of IGF-II on the 40-kDa protein in Balb/c3T3 cell membranes in a dose-dependent manner similar to that observed in the antibody-induced blocking of membrane IGF-II binding.(ABSTRACT TRUNCATED AT 400 WORDS)
Highlights
From the Life Science Laboratory, Fourth Departmentof Internal Medicine, University of Tokyo School of Medicine, 3-286 Mejirodai, Bunkyo-ku, Tokyo 112,Japan, the $Department of Life Science, Faculty of Science, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 227, Japan, and the TDepartment of Pharmaceutical Science, Faculty of Pharmacology, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku Tokyo 113,Japan
Membranes-To clarify which receptor acts as theprincipal receptor mediating the action of IGF-I1in the cell membranes, we examined by the effect of IGF-I and insulin on the IAP substrate activity of the 40-kDa protein (Fig. 2)
When Balb/ c3T3 cell membranes were incubated with insulin, no significant change was observed in the IAP substrate activity of the 40-kDa protein in a range of insulin concentrations from 10 to 100 nM
Summary
54 SF cells by a monoclonal antibody against rat IGF-I1 (the purity of MSAIII-2 was 70%) or 1 mgof synthetic IGF-I was coupled to. ZAP-sensitiveG Proteins from Brain Tissues-G proteins serving For the experiments using ADP-ribosylated Gi, inreconstituted as IAP substrates were purified to homogeneity from porcine brain and dissolved in buffer A (50 mM HEPES/NaOH (pH 7.4), 0.1 mM vesicles, vesicles were incubated the presence or absence of 10 p~. Reconstituted vesicles were incubated with or without IGF-I1 in buffer containing 20 mM. 20 mM HEPES/NaOH (pH 7.4) and 0.1 mM EDTA for 1 h at room temperature in the presence or absence of 10 p~ unlabeled IGF-I1 and mixed with DSS for a final concentration of 500 p ~A.fter a 1-h incubation at 4 "C, the reaction was stopped by the addition of. Lanes 1 and 2 show the cases of 0- and 5-min incubation of membranes with IGF-11, respectively.
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