Abstract
The activation of several G protein-coupled receptors is known to regulate the adhesive properties of cells in different contexts. Here, we reveal that Gbetagamma subunits of heterotrimeric G proteins regulate cell-matrix adhesiveness by activating Rap1a-dependent inside-out signals and integrin activation. We show that Gbetagamma subunits enter in a protein complex with activated Rap1a and its effector Radil and establish that this complex is required downstream of receptor stimulation for the activation of integrins and the positive modulation of cell-matrix adhesiveness. Moreover, we demonstrate that Gbetagamma and activated Rap1a promote the translocation of Radil to the plasma membrane at sites of cell-matrix contacts. These results add to the molecular understanding of how G protein-coupled receptors impinge on cell adhesion and suggest that the Gbetagamma x Rap1 x Radil complex plays important roles in this process.
Highlights
G protein-coupled receptors (GPCRs)3 form the largest family of cell surface signal transducing molecules in vertebrates
Rap1induced inside-out signaling has been deemed important in the context of GPCR signaling during platelet and leukocyte activation (13, 29 –33), how this signaling and integrin activation are regulated by GPCRs and whether it extends to other cellular contexts is not well understood
Several GPCRs have been implicated in the control of cell motility and adhesion
Summary
Plasmid Constructs and Reagents—Human G2, Radil, and Rap1GAP cDNAs were cloned from a human brain cDNA library in the pGlue or pIRES-puro-FLAG backbone vector [34]. Samples were immunostained with ␣-HA (HA., Covance) or ␣-FLAG (Sigma)-specific mouse monoclonal antibodies in PBS supplemented with 1% normal donkey serum. 48 h after transfections, cells were fixed in 4% paraformaldehyde and immunostained with ␣-HA monoclonal antibody to label the overexpressed proteins followed by secondary detection with goat-anti-mouse conjugated Alexa 594. 48 h after transfection with cDNA or siRNAs, HT1080 cells were dissociated from culture plates with trypsin, washed twice with PBS, and resuspended in serum free DMEM supplemented with 0.1% bovine serum albumin and 20 mM HEPES, pH 7.4. Beads were washed 3 times in TAP lysis buffer, bound proteins were eluted in 15 l of 2ϫ Laemmli buffer containing -mercaptoethanol (Sigma), and samples were analyzed by Western blot analysis using ␣-Rap1a antibodies. Statistical significance was measured at p Ͻ 0.05 for all analysis
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