Integrins Regulate the Linkage between Upstream and Downstream Events in G Protein-coupled Receptor Signaling to Mitogen-activated Protein Kinase

Sarah M Short,José L Boyer,R.L Juliano

doi:10.1074/jbc.275.17.12970

Abstract

Receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs) can both activate mitogen-activated protein kinase (MAPK), a critical intermediate in the transduction of proliferative signals. Numerous observations have demonstrated that integrin-mediated cell anchorage can regulate the efficiency of signaling from RTKs to MAPK. Recently, a relationship between integrins and GPCR signaling has also emerged; however, little is understood concerning the mechanisms involved. Here, we investigate integrin regulation of GPCR signaling to MAPK, focusing on the P2Y class of GPCRs that function through activation of phospholipase Cbeta. P2Y receptor signaling to the downstream components mitogen-activated protein kinase kinase and MAPK is highly dependent on integrin-mediated cell anchorage. However, activation of upstream events, including inositol phosphate production and generation of calcium transients, is completely independent of cell anchorage. This indicates that integrins regulate the linkage between upstream and downstream events in this GPCR pathway, just as they do in some aspects of RTK signaling. However, the P2Y pathway does not involve cross-activation of a RTK, nor a role for Shc or c-Raf; thus, it is quite distinct from the classical RTK-Ras-Raf-MAPK cascade. Rather, integrin-modulated P2Y receptor stimulation of MAPK depends on calcium and on the activation of protein kinase C.

Highlights

Normal cells require both soluble mitogenic factors and anchorage to a solid substratum in order to proceed through the cell cycle, while transformed cells have abrogated the anchorage requirement [1]
G Protein-coupled Receptor Activation of mitogen-activated protein kinase (MAPK) Is Dependent on Integrin-mediated Anchorage—As with stimuli that act through receptor tyrosine kinases, certain G protein-coupled receptors (GPCRs) agonists can activate MAPK
When ECV304 cells were stimulated with EGF, with the P2Y receptor ligand ATP, or with LPA, MAPK was strongly activated in cells that were adherent to substrata coated with fibronectin, but not in suspended cells (Fig. 1A)

Summary

EXPERIMENTAL PROCEDURES

Materials—ATP and UTP were from Amersham Pharmacia Biotech. LPA and angiotensin II (ATII) were from Sigma. Cell Culture, Adherence to Fibronectin, and Lysate Preparation— ECV304 cells, a human endothelial-like cell line [45, 46] were maintained as described previously [42]. Mouse 3T3 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. PC12 cells were maintained in Dulbecco’s modified Eagle’s medium-H and 10% heat-inactivated horse serum ϩ 5% fetal bovine serum. Immunocomplexes were incubated for 30 min at room temperature in a kinase buffer (10 mM Tris, pH 7.5, 10 mM MgCl2, 1 mM dithiothreitol, 10 ␮M ATP, 5 ␮Ci of [␥-32P]ATP (3000 Ci/ml)) containing 5–10 ␮g of myelin basic protein (MBP) as a substrate. Raf kinase activity was measured through a two-coupled enzyme assay combining recombinant MEK (0.5 ␮g), and ERK2 (1.25 ␮g) in the kinase reaction buffer [47]. Calcium transients in individual cells were recorded and processed using an InCyt Im2 imaging system (Intracellular Imaging Inc., Cincinnati, OH)

RESULTS

The Locus of Integrin Regulation of GPCR Signaling

DISCUSSION