Mechanical control of cAMP signaling through integrins is mediated by the heterotrimeric Gαs protein

Abstract

Mechanical stresses that are preferentially transmitted across the cell surface via transmembrane integrin receptors activate gene transcription by triggering production of intracellular chemical second messengers, such as cAMP. Here we show that the sensitivity of the cAMP signaling pathway to mechanical stresses transferred across β1 integrins is mediated by force-dependent activation of the heterotrimeric G protein subunit Gαs within focal adhesions at the site of stress application. Gαs is recruited to focal adhesions that form within minutes following clustering of β1 integrins induced by cell binding to magnetic microbeads coated with activating integrin ligands, and β1 integrin and Gαs co-precipitate when analyzed biochemically. Stress application to activated β1 integrins using magnetic twisting cytometry increases Gαs recruitment and activates these large G proteins within focal adhesions, as measured by binding of biotinylated azidoanilido-GTP, whereas application of similar stresses to inactivated, integrins or control histocompatibility antigens has little effect. This response is relevant physiologically as application of mechanical strain to cells bound to flexible extracellular matrix-coated substrates induce translocation of phospho-CREB to the nucleus, which can be attenuated by inhibiting Gαs activity, either using the inhibitor melittin or suppressing its expression using siRNA. Although integrins are not typical G protein-coupled receptors, these results show that integrins focus mechanical stresses locally on heterotrimeric G proteins within focal adhesions at the site of force application, and transduce mechanical stimuli into an intracellular cAMP signaling response by activating Gαs at these membrane signaling sites.

Macrophages require Skap2 and Sirpα for integrin-stimulated cytoskeletal rearrangement

“…Therefore, we employed a bead assay that incorporated integrin ligands and monoclonal antibodies to examine further the role of Skap2 in integrinmediated cytoskeletal rearrangement. Polystyrene beads were coated with polyRGD (pRGD) (Alenghat et al, 2009;Miyamoto et al, 1995), which binds to a broad range of integrins, including b 1 , b 2 , and b 3 (Plow et al, 2000). Skap2 +/2 and Skap2 2/2 BMMs bound these beads similarly, consistent with their equivalent levels of surface integrin expression (Fig.…”

Strategies Targeting cAMP Signaling in the Treatment of Polycystic Kidney Disease

“…107 Although integrins are not conventional GPCRs, they interact with heterotrimeric G proteins through noncanonical pathways to activate or inhibit cAMP signaling in an extracellular matrix-and cell type-specific manner (Figure 1). 108,109 Integrins may also regulate the activity of PDEs in compartmentalized cAMP pools. 110 Polycystin-1 and possibly, polycystin-2 could function in the focal adhesion complexes to regulate these cAMP pools, which is described in other subcellular compartment like the cilia.…”

Mechanosensitive mechanisms in transcriptional regulation

“…These adhesion complexes can trigger transcriptional changes in the nucleus through modulating downstream signaling cascades (Ingber, 2006;Mammoto et al, 2008;Orr et al, 2006). Mechanical signals can, thereby, elicit changes in the concentration of second messengers, such as Ca 2+ flux, inositol trisphosphate (InsP 3 ), diacylglycerol (DAG), cAMP and nitric oxide, or activate protein signaling molecules, such as MAPKs and Rho-family small GTPases, which are involved in controlling virtually all cellular processes, including migration, growth, differentiation and matrix remodeling (Alenghat et al, 2009;Balligand et al, 2009;Huang and Ingber, 2002;Mammoto et al, 2008;Martinac, 2004;Orr et al, 2006;Papachristou et al, 2009;Sadoshima and Izumo, 1993).…”