Abstract
Integrins function as bi-directional signaling transducers that regulate cell-cell and cell-matrix signals across the membrane. A key modulator of integrin activation is talin, a large cytoskeletal protein that exists in an autoinhibited state in quiescent cells. Talin is a large 235-kDa protein composed of an N-terminal 45-kDa FERM (4.1, ezrin-, radixin-, and moesin-related protein) domain, also known as the talin head domain, and a series of helical bundles known as the rod domain. The talin head domain consists of four distinct lobes designated as F0-F3. Integrin binding and activation are mediated through the F3 region, a critically regulated domain in talin. Regulation of the F3 lobe is accomplished through autoinhibition via anti-parallel dimerization. In the anti-parallel dimerization model, the rod domain region of one talin molecule binds to the F3 lobe on an adjacent talin molecule, thus achieving the state of autoinhibition. Platelet functionality requires integrin activation for adherence and thrombus formation, and thus regulation of talin presents a critical node where pharmacological intervention is possible. A major mechanism of integrin activation in platelets is through heterotrimeric G protein signaling regulating hemostasis and thrombosis. Here, we provide evidence that switch region 2 (SR2) of the ubiquitously expressed G protein (Gα13) directly interacts with talin, relieves its state of autoinhibition, and triggers integrin activation. Biochemical analysis of Gα13 shows SR2 binds directly to the F3 lobe of talin's head domain and competes with the rod domain for binding. Intramolecular FRET analysis shows Gα13 can relieve autoinhibition in a cellular milieu. Finally, a myristoylated SR2 peptide shows demonstrable decrease in thrombosis in vivo Altogether, we present a mechanistic basis for the regulation of talin through Gα13.
Highlights
Integrins function as bi-directional signaling transducers that regulate cell-cell and cell-matrix signals across the membrane
Because of the well established importance of integrin signaling in hemostasis and thrombosis, the mechanisms of integrin regulation remain an area of intense investigation
To identify the precise region where switch region 2 (SR2) binds to talin head domain (THD), chemical cross-linking of biotinylated SR2 (VGGQRSERKRWFECFDS, biotinylated (b)-G␣13SR2Pep) or SR2 scrambled peptide (GCRKEVFSDRQWFGSRE, b-G␣13SR2RandomPep) to recombinant THD was employed (Fig. 1A)
Summary
Integrins function as bi-directional signaling transducers that regulate cell-cell and cell-matrix signals across the membrane. In the anti-parallel dimerization model, the rod domain region of one talin molecule binds to the positively charged face of the F3 lobe on an adjacent talin molecule, achieving the state of autoinhibition [15]. Our group reported that G␣13SR2 (where SR2 is one of three conformationally sensitive switch regions present in G␣ subunits) directly binds to talin and thereby modulates platelet integrin ␣IIb3 activation [27]. This finding provided the first evidence for a G␣13/talin interaction and represented a novel regulatory pathway. This new mechanism for talin activation by G␣13 has important implications regarding platelet integrin activation and in vivo thrombosis, and underscores the critical importance of G␣13 in numerous developmental and pathological states because G␣13 and talin are ubiquitously expressed
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