Insulin-like Growth Factors Mediate Heterotrimeric G Protein-dependent ERK1/2 Activation by Transactivating Sphingosine 1-Phosphate Receptors

Abstract

Although several studies have shown that a subset of insulin-like growth factor (IGF) signals require the activation of heterotrimeric G proteins, the molecular mechanisms underlying IGF-stimulated G protein signaling remain poorly understood. Here, we have studied the mechanism by which endogenous IGF receptors activate the ERK1/2 mitogen-activated protein kinase cascade in HEK293 cells. In these cells, treatment with pertussis toxin and expression of a G␣ q/11 -(305-359) peptide that inhibits G q/11 signaling additively inhibited IGF-stimulated ERK1/2 activation, indicating that the signal was almost completely G protein-dependent. Treatment with IGF-1 or IGF-2 promoted translocation of green fluorescent protein (GFP)-tagged sphingosine kinase (SK) 1 from the cytosol to the plasma membrane, increased endogenous SK activity within 30 s of stimulation, and caused a statistically significant increase in intracellular and extracellular sphingosine 1-phosphate (S1P) concentration. Using a GFP-tagged S1P 1 receptor as a biological sensor for the generation of physiologically relevant S1P levels, we found that IGF-1 and IGF-2 induced GFP-S1P receptor internalization and that the effect was blocked by pretreatment with the SK inhibitor, dimethylsphingosine. Treating cells with dimethylsphingosine, silencing SK1 expression by RNA interference, and blocking endogenous S1P receptors with the competitive antagonist VPC23019 all significantly inhibited IGF-stimulated ERK1/2 activation, suggesting that IGFs elicit G protein-dependent ERK1/2 activation by stimulating SK1-dependent transactivation of S1P receptors. Given the ubiquity of SK and S1P receptor expression, S1P receptor transactivation may represent a general mechanism for G protein-dependent signaling by non-G protein-coupled receptors.The insulin-like growth factors type 1 and 2 (IGF-1 and -2) 2 are single chain polypeptides that share structural homology with proinsulin. The actions of IGF-1 and IGF-2 are mediated by binding to two structurally distinct plasma membrane receptors, referred to as the IGF-1 and IGF-2/mannose 6-phosphate (M6P) receptors. Most of the metabolic and mitogenic effects of IGF-1 and IGF-2 are thought to result from binding to the IGF-1 receptor, a receptor tyrosine kinase with structural homology to the insulin receptor. It is composed of two extracellular ␣ subunits and two transmembrane ␤ subunits linked by disulfide bonds (1, 2). Ligand binding to the ␣ subunits activates the intrinsic ␤ subunit receptor tyrosine kinase activity, leading to tyrosine phosphorylation of adapter proteins, such as insulin receptor substrate 1 (IRS-1) and Shc and Gab1 (3-6). Subsequent src homology 2 or protein tyrosine-binding domaindependent recruitment of enzymes with phospholipase, phosphatase, and protein and lipid kinase activity transmits signals intracellularly, including activation of the mitogenic Ras cascade and initiation of phosphatidylinositol 3-kinase (PI3K)/Aktdependent cell survival. Adding to the diversity, in some cell types IGF-1 stimula...

The Insulin-like Growth Factor Type 1 and Insulin-like Growth Factor Type 2/Mannose-6-phosphate Receptors Independently Regulate ERK1/2 Activity in HEK293 Cells

“…7B shows the effects of PTX on IGF-stimulated ERK1/2 activation. Consistent with our previous observations (31,38), pretreatment with PTX inhibited IGF-1-, IGF-2-, and S1P-stimulated ERK1/2 phosphorylation by 47, 66, and 73%, respectively. PMA-stimulated ERK1/2 activation was not affected.…”

“…We have previously shown that both IGF-1 and IGF-2 promote membrane translocation and activation of SK1, increase intracellular and extracellular S1P levels, and activate G protein-coupled S1P receptors in HEK293 cells (31). What remains unclear is how these two structurally distinct receptors activate SK.…”

“…PMA-stimulated ERK1/2 activation was not affected. The incomplete inhibition produced by PTX probably results from coupling of S1P receptors to PTX-insensitive G q / 11 family heterotrimeric G proteins in addition to G i / o family G proteins since we have observed that PTX and a G q / 11 inhibitor peptide additively inhibit S1P-stimulated ERK1/2 activation in HEK293 cells (31,39). Collectively, these data suggest that two distinct receptor types, the IGF-1 receptor tyrosine kinase and the catalytically inactive IGF-2/M6P receptor, share a common mechanism of intracellular signaling involving SK-dependent transactivation of S1P receptors.…”

“…Activity-We recently demonstrated that IGFs can activate ERK1/2 through a novel mechanism involving SK-dependent transactivation of heterotrimeric G protein-coupled S1P receptors (31). To determine whether signals transmitted via the IGF-1 and IGF-2/M6P receptors employed the same or different mechanisms, we exposed HEK293 cells to EC 50 concentrations of IGF-1 or IGF-2 in the presence or absence of either DMS, an inhibitor of SK activity, or pertussis toxin (PTX), which inactivates G i / o family heterotrimeric G proteins.…”

“…We recently reported that in HEK293 cells, exposure to IGF-1 or IGF-2 leads to activation of the ERK1/2 mitogen-activated protein (MAP) kinase cascade through a mechanism involving sphingosine kinase (SK)-dependent "transactivation" of G protein-coupled sphingosine-1-phosphate (S1P) receptors (31). Here, we sought to definitively identify the endogenous receptor(s) that mediate G protein-dependent ERK1/2 activation in response to IGF-1 and IGF-2 in HEK293 cells.…”