Insulin activates GTP binding to a 40 kDa protein in fat cells

Abstract

The first steps in insulin action are binding of insulin to its receptor and activation of the insulin receptor kinase. As there is indirect evidence that further signal transduction might involve a guanine-nucleotide-binding protein (G-protein), we studied whether insulin modulates GTP binding to plasma membrane proteins of fat cells and skeletal muscle. We found that insulin rapidly increased (30 s) binding of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) in a dose dependent manner (0.03-2.0 nM). This effect was not altered by pertussis toxin, but it was abolished by cholera toxin treatment of fat cells. Scatchard analysis of the binding data showed that the increased GTP[S] binding is due to a decrease in the Kd for GTP from 100 nM to 50 nM. Furthermore, binding of GTP to these plasma membranes inhibited both the binding of 125I-insulin to the insulin receptor and the stimulation of the insulin receptor kinase, suggesting a feedback interaction between the insulin-stimulated GTP-binding site and the insulin receptor. In order to identify this insulin-stimulated GTP-binding site, plasma membranes were labelled with the photoreactive GTP analogue [alpha-32P]GTP gamma-azidoanilide. We found that insulin selectively stimulated GTP binding to a 40 kDa protein. In conclusion, in plasma membranes of fat cells and skeletal muscle, the insulin receptor interacts with a 40 kDa GTP-binding site. We speculate that this 40 kDa GTP-binding site might be a G-protein which is involved in insulin signal transmission.

GTP‐binding protein‐activator sequences in the insulin receptor

“…Lutterel et al [18] found that insulin promotes guanine nucleotide-binding to the membranes of BC3-H-1 myocytes, where they had earlier reported that pertussis toxin inhibits DNA synthesis and glucose uptake promoted by insulin [10]. Similar findings have been reported using adipocytes [11][12][13]19]. It is also reported that in hepatocyte membranes, insulin attenuates the pertussis-toxin sensitivity of a 40-kDa Gi-like protein [20,21], suggesting that the insulin-bound insR activates the pertussis-toxin-sensitive G protein.…”

“…In hepatocyte membranes, insulin causes a change in a 40-kDa Gi-like protein [20,21], which can be recognized by reduction in pertussis toxin-catalyzed ADP-ribosylation. Insulin promotes the binding of GTP analogs to myocyte and adipocyte membranes [18,19]. InsR is directly associated with a Gi protein of 40 kDa [22].…”

Mitogenic Signaling via G Protein-Coupled Receptors

“…Insulin also inhibits PTX-catalyzed ADPribosylation of Gi by about 50% in isolated rat liver plasma membranes (268) and promotes guanine-nucleotide binding to BC3H-1 myocyte plasma membranes (269). In adipocyte plasma membranes (270), insulin stimulates the binding of GTP to a 40-kDa protein, and binding of GTP leads to a decrease in [ 125 Ilinsulin binding to the receptor, suggesting a feedback interaction between the insulin-stimulated GTPbinding site and the insulin receptor. Further, peptides derived from autophosphorylation sites of the insulin receptor have been shown to directly activate Gi in phospholipid vesicles (271), and this activation is modulated by tyrosine phosphorylation.…”

Tumor necrosis factor-alpha- and hyperglycemia-induced insulin resistance. Evidence for different mechanisms and different effects on insulin signaling.

“…It has been shown that factors such as hyperinsulinemia and hypoinsulinemia as well as agonists, including catecholamines, adenosine, and phorbolesters, are able to regulate insulin receptor function (for review see reference 11). There is also evidence for a regulatory role of G-proteins (12) and other membrane proteins (13). In addition, insulin receptor inhibition by high glucose levels was demonstrated in different cell models (14)(15)(16)(17), and, more recently, a number of studies have shown that the cytokine TNF-␣ might be another important regulator of insulin receptor function (18-21).…”