Abstract
The Ras-dependent activation of Erk kinases by G protein-coupled receptors (GPCRs) is thought to involve tyrosine phosphorylation of docking proteins that serve as scaffolds for the plasma membrane recruitment of Ras guanine nucleotide exchange factors, such as the Grb2-mSos complex. We have investigated the role of two GPCR-regulated tyrosine phosphoproteins, p125(FAK) (FAK) and Shc, in the Ras-dependent activation of Erk kinases by endogenously expressed GPCRs in Rat 1a fibroblasts. Several lines of evidence suggest that tyrosine phosphorylation of FAK and Shc are independently regulated. The GPCRs for lysophosphatidic acid (LPA), thrombin, and bombesin mediate equivalent increases in FAK tyrosine phosphorylation and FAK-Grb2 association. In contrast, only LPA and thrombin receptors significantly stimulate Shc tyrosine phosphorylation and Shc-Grb2 complex formation. Tyrosine phosphorylation of FAK is pertussis toxin-insensitive, can be mimicked by calcium ionophore, and is inhibited by treatment with cytochalasin D, which depolymerizes the actin cytoskeleton. In contrast, tyrosine phosphorylation of Shc is inhibited by pertussis toxin treatment, is not induced by calcium ionophore, and is insensitive to cytochalasin D. In each case, the rapid stimulation of Erk 1/2 correlates with tyrosine phosphorylation of Shc but not of FAK. The dissociation of FAK-Grb2 complex formation from receptor-mediated activation of Erk 1/2 indicates that recruitment of Grb2-mSos to the plasma membrane is not sufficient to mediate rapid Erk activation. Using four mechanistically distinct inhibitors of clathrin-mediated endocytosis, concanavalin A, hypertonic medium, depletion of intracellular potassium, and monodansylcadaverine, we find that GPCR-mediated Erk 1/2 activation is also endocytosis-dependent. Thus, we propose that an additional step involving vesicle-mediated endocytosis is required for the rapid, Ras-dependent activation of Erk kinases in fibroblasts.
Highlights
The Ras-dependent activation of Erk kinases by G protein-coupled receptors (GPCRs) is thought to involve tyrosine phosphorylation of docking proteins that serve as scaffolds for the plasma membrane recruitment of Ras guanine nucleotide exchange factors, such as the Grb2-mSos complex
The adapter protein Grb2 coprecipitated with FAK from lysates of lysophosphatidic acid (LPA), SFLLRN, bombesin- and epidermal growth factor (EGF)-stimulated cells prepared under nondenaturing conditions (Fig. 1, A and D)
The data presented here demonstrate that GPCR-induced tyrosine phosphorylation of FAK and Shc are independently regulated in Rat 1a fibroblasts
Summary
Cell Culture and Transfection—Rat 1a fibroblasts and HEK-293 cells were maintained in minimum essential medium supplemented with 10% fetal bovine serum and 100 g/ml gentamicin at 37 °C in a humidified 5% CO2 atmosphere. Fusion protein complexes were washed twice with ice-cold RIPA buffer and once with PBS, denatured in Laemmli sample buffer, and resolved by SDS-PAGE. Erk 1/2 Phosphorylation—For the determination of Erk 1/2 phosphorylation, monolayers of serum-starved Rat 1a fibroblasts in 6-well culture plates were stimulated as described, washed once with ice-cold PBS, and solubilized by the direct addition of Laemmli sample buffer. Aliqouts of cell lysate (approximately 30 g of protein/lane) were resolved by SDS-PAGE, and Erk 1/2 phosphorylation was detected by protein immunoblotting using 1:20,000 dilution of rabbit polyclonal phospho-MAP kinase-specific IgG (Promega) with alkaline phosphatase-conjugated goat anti-rabbit IgG (Amersham) as secondary antibody. Confluent monolayers of Rat 1 or HEK293 cells in 6-well culture plates were washed twice with potassiumfree buffer (140 mM NaCl, 20 mM HEPES, pH 7.4, 1 mM CaCl2, 1 mM MgCl2, 1 gm/liter dextrose) prior to incubation in hypotonic medium (potassium-free buffer/water (1:1 v/v)) for 10 min. Receptor sequestration was defined as the fraction of total cell surface receptors that are removed from the plasma membrane following agonist treatment
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