Mechanical control of cAMP signaling through integrins is mediated by the heterotrimeric Galphas 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 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.

Fig. 1. Experimental System

(A) In magnetic twisting cytometry, ferromagnetic microbeads coated with specific ligands, such as RGD peptide (small triangles) or antibodies, are allowed to bind to cell surface receptors. A strong, brief magnetic field pulse (horizontal arrow B) is applied to align the magnetic dipoles of the beads horizontally (left panel). A subsequent prolonged, weaker field directed upward (vertical arrow B) causes the beads to twist, resulting in a shear stress at the bead-cell membrane interface via the ligated receptors (e.g., integrin heterodimers) (right panel). (B) Cytosolic and nuclear catalytic subunit of protein kinase A (PKA-c) quantitated in Western blots of proteins from bovine capillary endothelial (BCE) cells or human aortic endothelial (HAE) cells cultured with no beads (-) or RGD-coated beads (RGD), in the absence (-) or presence (+) of 10 mins applied stress (twist). (C) Quantitation of nuclear intensity of nuclear phospho-CREB in Human Umbilical Vein Endothelial Cells (HUVEC) bound to fibronectin-coated flexible membrane exposed to no strain or 5 minutes of strain at 15%, 20 cycles/min, as applied using a Flex Cell System and fixed and stained for phospho-CREB by immunofluorescence (* p < .001).

Fig. 2. Gαs is recruited to activated integrins

(A) Fluorescent images of vinculin, paxillin, and Gαs recruitment to the cell membrane region bound to RGD-coated bead (4.5 μm diameter) or K20-beads in the presence or absence of soluble RGD peptide (sRGD) that activates the K20-bound integrin receptors. (B) Intensity of Gαs recruitment from studies shown in A was quantitated by measuring the average pixel intensity within a 1.5 μm wide annulus at the bead periphery relative to local background, and normalized against the K20 condition for each G protein subunit [average of 35 beads per condition; * p < .005]. (C) Bead-associated fraction purification with beads coated with HLA, K20, BD15, or RGD probed by Western blot for β1 integrin and Gαs.

Fig. 3. Twisting leads to increased Gαs and AAGTP recruitment to beads

(A) Twisting of RGD-coated beads for 10 minutes (15.6 dynes/cm²) yielded significant increases in local recruitment of Gαs subunits in cells fixed and stained immediately after stress application. Bead staining intensity was quantitated and normalized to no twist levels [average 93 beads per condition; * p < .005]. (B) Cells bound to RGD-beads and briefly incubated with biotin-labeled AAGTP were exposed to control or twisting conditions and subsequent UV light (3 min) to induce AAGTP cross-linking to activated G proteins. After fixation, cells were stained with fluorescent avidin and imaged using fluorescence microscopy; beads appear as dark holes in the fluorescent cytoplasm. Digitally deconvoluted images of representative surface-bound beads, with or without twist, stained for AAGTP. (C) Bead staining intensity of AAGTP measured at the periphery of surface-bound beads and normalized against the `no twist HLA' condition. [Average 143 beads per condition; * p < 0.005 compared to untwisted HLA; ** p < 0.001 compared to corresponding untwisted condition].

Fig. 4. Measurement of bead-associated AAGTP at isolated focal adhesion complexes

(A) Increase in AAGTP incorporation into bead-associated protein fractions with RGD and control HLA beads after biotin-AAGTP incubation and mechanical stress as probed by Western blotting with HRP-avidin. (*p<.005 compared to HLA;**p<.005 compared to RGD no twist) (B) After AAGTP incubation, mechanical stress and UV exposure, isolated proteins associated with cell-bound RGD-beads were subjected to gel electrophoresis and Western blotting with HRP-avidin to identify proteins bound to biotinylated-AAGTP (left panel). In the bead fraction, the primary GTP-binding species migrated at 45 kD, exactly like Gαs, as determined upon reprobing with anti-Gαs (right panel). (C) Integrin-binding beads were bound to bovine capillary endothelial cells and incubated for 0, 5, 10, or 20 minutes in the presence of saponin or 10 minutes in the absence of saponin to exclude AAGTP entrance into cells as a negative control. Lysates were prepared, normalized for protein levels, subjected to gel electrophoresis and probed with HRP-strepavidin to identify AAGTP-biotin incorporation or anti-Gαs antibody to determine Gαs recruitment. The same 45kD molecular weight region is shown in both blots.

Fig. 5. Inhibition of Gαs with melittin inhibits the cAMP response to stress

(A) Percentage of nuclear phospho-CREB positive cells in NIH3T3 cells or (B) KID/KIX activity as measured in B9 cells (* p < .001) subjected to no strain or 5 minutes of mechanical strain (15% strain; 0.3Hz) in the presence or absence of melittin (* p < 0.001).

Fig. 6. Depletion of Gαs using RNAi significantly diminishes phospho-CREB nuclear translocation in response to mechanical strain

(A) Quantitative PCR analysis showing decrease in Gαs transcript in cells treated with Gαs RNAi as compared to the Control (Con RNAi). B2M is shown as a control. (B) Western blot analysis showing downregulation of Gαs protein levels in Gαs RNAi transfected cells compared to Control RNAi (Con RNAi) treated cells. (C) Quantitative analysis of nuclear phospho-CREB intensity showing inhibition of phospho-CREB nuclear translocation in Gαs RNAi knockdown cells compared to Control RNAi (Con RNAi) treated cells (* p < 0.001).