Mechanical control of cAMP signaling through integrins is mediated by the heterotrimeric Galphas protein.
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 beta1 integrins is mediated by force-dependent activation of the heterotrimeric G protein subunit Galphas within focal adhesions at the site of stress application. Galphas is recruited to focal adhesions that form within minutes following clustering of beta1 integrins induced by cell binding to magnetic microbeads coated with activating integrin ligands, and beta1 integrin and Galphas co-precipitate when analyzed biochemically. Stress application to activated beta1 integrins using magnetic twisting cytometry increases Galphas recruitment and activates these large G proteins within focal adhesions, as measured by binding of biotinylated azido-anilido-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 Galphas 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 Galphas at these membrane signaling sites.
Alenghat
Francis J
FJ
Departments of Pathology and Surgery, Children's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
Tytell
Jessica D
JD
Thodeti
Charles K
CK
Derrien
Alexandrine
A
Ingber
Donald E
DE
D001011:Aorta / Q000166:cytology; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000502:physiology; D022001:Focal Adhesions / Q000502:physiology; D044385:GTP-Binding Protein alpha Subunits / Q000378:metabolism*; D006801:Humans; D019012:Integrin beta1 / Q000378:metabolism*; D040542:Mechanotransduction, Cellular; D043562:Receptors, G-Protein-Coupled; D015398:Signal Transduction; D013314:Stress, Mechanical
D044385:GTP-Binding Protein alpha Subunits; D019012:Integrin beta1; D043562:Receptors, G-Protein-Coupled; D000242:Cyclic AMP