Analysis of stimulation-G protein subunit coupling by using active insulin-like growth factor II receptor peptide.
The peptide Arg2410-Lys2423 (peptide 14) of the human insulin-like growth factor II/mannose 6-phosphate receptor directly activates Gi-2, a GTP-binding protein (G protein), and is responsible for Gi-2 activating function of the receptor. To characterize the basic mechanism of couplings between receptor stimulation and subunits of G proteins, we constructed a system consisting of peptide 14 and alpha and beta gamma subunits of Gi-2 in aqueous solution. Peptide 14 significantly increased the rate of guanosine 5'-[gamma-thio]triphosphate binding to isolated Gi-2 alpha from 0.50 +/- 0.03 (mean +/- SE; n = 3) to 0.75 +/- 0.02 mol per mol of Gi-2 alpha per 3 min (n = 3) at 100 microM. In this system, G beta gamma does dependently potentiated the peptide 14 action on Gi-2 alpha; and G beta gamma-induced potentiation reached saturation at a concentration comparable to that of Gi-2 alpha. An antibody specific for the C-terminal decapeptide of Gi-2 alpha reduce peptide 14-stimulated GDP release from Gi-2 to the basal level. This simplified system indicates that (i) the receptor sequence directly interacts with isolated Gi-2 alpha at its C-terminal region and (ii) G beta gamma potentiates the stimulation-G alpha coupling in a stoichiometrical manner for G alpha.
Okamoto
T
T
Fourth Department of Internal Medicine, University of Tokyo School of Medicine, Japan.
Nishimoto
I
I
D000818:Animals; D002417:Cattle; D019204:GTP-Binding Proteins / Q000378:metabolism*; D066298:In Vitro Techniques; D007335:Insulin-Like Growth Factor II / Q000378:metabolism*; D046911:Macromolecular Substances; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*; D017451:Receptors, Somatomedin; D015398:Signal Transduction
D046911:Macromolecular Substances; D010455:Peptides; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface; D017451:Receptors, Somatomedin; D007335:Insulin-Like Growth Factor II; D019204:GTP-Binding Proteins