Limits...
Recreation of the terminal events in physiological integrin activation.

Ye F, Hu G, Taylor D, Ratnikov B, Bobkov AA, McLean MA, Sligar SG, Taylor KA, Ginsberg MH - J. Cell Biol. (2010)

Bottom Line: Here, we reconstructed physiological integrin activation in vitro and used cellular, biochemical, biophysical, and ultrastructural analyses to show that talin binding is sufficient to activate integrin alphaIIbbeta3.Furthermore, we synthesized nanodiscs, each bearing a single lipid-embedded integrin, and used them to show that talin activates unclustered integrins leading to molecular extension in the absence of force or other membrane proteins.Thus, we provide the first proof that talin binding is sufficient to activate and extend membrane-embedded integrin alphaIIbbeta3, thereby resolving numerous controversies and enabling molecular analysis of reconstructed integrin signaling.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, University of California, San Diego, La Jolla, CA 92093, USA.

ABSTRACT
Increased affinity of integrins for the extracellular matrix (activation) regulates cell adhesion and migration, extracellular matrix assembly, and mechanotransduction. Major uncertainties concern the sufficiency of talin for activation, whether conformational change without clustering leads to activation, and whether mechanical force is required for molecular extension. Here, we reconstructed physiological integrin activation in vitro and used cellular, biochemical, biophysical, and ultrastructural analyses to show that talin binding is sufficient to activate integrin alphaIIbbeta3. Furthermore, we synthesized nanodiscs, each bearing a single lipid-embedded integrin, and used them to show that talin activates unclustered integrins leading to molecular extension in the absence of force or other membrane proteins. Thus, we provide the first proof that talin binding is sufficient to activate and extend membrane-embedded integrin alphaIIbbeta3, thereby resolving numerous controversies and enabling molecular analysis of reconstructed integrin signaling.

Show MeSH

Related in: MedlinePlus

THD activates purified αIIbβ3 by binding to the β3 tail. (A) THD induced an increase in PAC1 binding (▴), whereas THD-W359A (■), a mutant deficient in integrin tail binding, failed to do so. The inset on the right depicts SDS-PAGE showing that integrin and THD are comparable. Data are from one of three independent experiments. (B) Activation indices of the subset of liposomes at the mid-point of the forward scatter spectrum (mean FSC = 142) were averaged over three independent experiments. Increases in integrin activation (calculated as AIwith_THD − AIintegrin_alone, where AI stands for activation indices) are depicted. THD increased integrin activation compared with THD-W359A. (C) Requirement of the β3 tail for THD activation. Using the ELISA described in the Materials and methods section, calpain abolished anti–β3-c tail binding but had no effect on anti-β3 or anti–αIIb-c tail binding. SDS-PAGE revealed that calpain did not markedly shift mobility of β3 or αIIb. (D) THD fails to activate αIIbβ3 lacking the β3 C terminus. Increase in integrin activation was calculated as in B. (E) SDS-PAGE shows that integrin and THD incorporation are comparable for calpain-digested and intact integrins. (F) THD mutants do not disrupt protein folding. Differential scanning calorimetry of THD, THD-W359A, and THDF-K322D. All three proteins exhibited similar narrowly defined melting points, indicating that they were well folded.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC2812850&req=5

fig3: THD activates purified αIIbβ3 by binding to the β3 tail. (A) THD induced an increase in PAC1 binding (▴), whereas THD-W359A (■), a mutant deficient in integrin tail binding, failed to do so. The inset on the right depicts SDS-PAGE showing that integrin and THD are comparable. Data are from one of three independent experiments. (B) Activation indices of the subset of liposomes at the mid-point of the forward scatter spectrum (mean FSC = 142) were averaged over three independent experiments. Increases in integrin activation (calculated as AIwith_THD − AIintegrin_alone, where AI stands for activation indices) are depicted. THD increased integrin activation compared with THD-W359A. (C) Requirement of the β3 tail for THD activation. Using the ELISA described in the Materials and methods section, calpain abolished anti–β3-c tail binding but had no effect on anti-β3 or anti–αIIb-c tail binding. SDS-PAGE revealed that calpain did not markedly shift mobility of β3 or αIIb. (D) THD fails to activate αIIbβ3 lacking the β3 C terminus. Increase in integrin activation was calculated as in B. (E) SDS-PAGE shows that integrin and THD incorporation are comparable for calpain-digested and intact integrins. (F) THD mutants do not disrupt protein folding. Differential scanning calorimetry of THD, THD-W359A, and THDF-K322D. All three proteins exhibited similar narrowly defined melting points, indicating that they were well folded.

Mentions: Talin activates αIIbβ3 by binding to the β3 tail (García-Alvarez et al., 2003; Wegener et al., 2007) in cells. We sought to ascertain whether THD activated purified αIIbβ3 by binding to the β3 tail. We first examined the capacity of added THD to activate αIIbβ3 in intact cells and found no effect on PAC1 binding at concentrations up to 25 µM. Second, we found that THD(W359A), a mutant with reduced affinity for the β3 tail (García-Alvarez et al., 2003), failed to activate the purified integrin (Fig. 3, A and B). The sharp melting point of THD(W359A) indicated that the protein was well folded, although the 1.7° lower melting point suggests it was slightly less stable than wild-type THD. (Fig. 3 F). In addition, THD(W359A) was incorporated into the liposomes (Fig. 3 A). Thus, THD required both access to the β3 tail and the capacity to bind to it in order to activate αIIbβ in vitro.


Recreation of the terminal events in physiological integrin activation.

Ye F, Hu G, Taylor D, Ratnikov B, Bobkov AA, McLean MA, Sligar SG, Taylor KA, Ginsberg MH - J. Cell Biol. (2010)

THD activates purified αIIbβ3 by binding to the β3 tail. (A) THD induced an increase in PAC1 binding (▴), whereas THD-W359A (■), a mutant deficient in integrin tail binding, failed to do so. The inset on the right depicts SDS-PAGE showing that integrin and THD are comparable. Data are from one of three independent experiments. (B) Activation indices of the subset of liposomes at the mid-point of the forward scatter spectrum (mean FSC = 142) were averaged over three independent experiments. Increases in integrin activation (calculated as AIwith_THD − AIintegrin_alone, where AI stands for activation indices) are depicted. THD increased integrin activation compared with THD-W359A. (C) Requirement of the β3 tail for THD activation. Using the ELISA described in the Materials and methods section, calpain abolished anti–β3-c tail binding but had no effect on anti-β3 or anti–αIIb-c tail binding. SDS-PAGE revealed that calpain did not markedly shift mobility of β3 or αIIb. (D) THD fails to activate αIIbβ3 lacking the β3 C terminus. Increase in integrin activation was calculated as in B. (E) SDS-PAGE shows that integrin and THD incorporation are comparable for calpain-digested and intact integrins. (F) THD mutants do not disrupt protein folding. Differential scanning calorimetry of THD, THD-W359A, and THDF-K322D. All three proteins exhibited similar narrowly defined melting points, indicating that they were well folded.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2812850&req=5

fig3: THD activates purified αIIbβ3 by binding to the β3 tail. (A) THD induced an increase in PAC1 binding (▴), whereas THD-W359A (■), a mutant deficient in integrin tail binding, failed to do so. The inset on the right depicts SDS-PAGE showing that integrin and THD are comparable. Data are from one of three independent experiments. (B) Activation indices of the subset of liposomes at the mid-point of the forward scatter spectrum (mean FSC = 142) were averaged over three independent experiments. Increases in integrin activation (calculated as AIwith_THD − AIintegrin_alone, where AI stands for activation indices) are depicted. THD increased integrin activation compared with THD-W359A. (C) Requirement of the β3 tail for THD activation. Using the ELISA described in the Materials and methods section, calpain abolished anti–β3-c tail binding but had no effect on anti-β3 or anti–αIIb-c tail binding. SDS-PAGE revealed that calpain did not markedly shift mobility of β3 or αIIb. (D) THD fails to activate αIIbβ3 lacking the β3 C terminus. Increase in integrin activation was calculated as in B. (E) SDS-PAGE shows that integrin and THD incorporation are comparable for calpain-digested and intact integrins. (F) THD mutants do not disrupt protein folding. Differential scanning calorimetry of THD, THD-W359A, and THDF-K322D. All three proteins exhibited similar narrowly defined melting points, indicating that they were well folded.
Mentions: Talin activates αIIbβ3 by binding to the β3 tail (García-Alvarez et al., 2003; Wegener et al., 2007) in cells. We sought to ascertain whether THD activated purified αIIbβ3 by binding to the β3 tail. We first examined the capacity of added THD to activate αIIbβ3 in intact cells and found no effect on PAC1 binding at concentrations up to 25 µM. Second, we found that THD(W359A), a mutant with reduced affinity for the β3 tail (García-Alvarez et al., 2003), failed to activate the purified integrin (Fig. 3, A and B). The sharp melting point of THD(W359A) indicated that the protein was well folded, although the 1.7° lower melting point suggests it was slightly less stable than wild-type THD. (Fig. 3 F). In addition, THD(W359A) was incorporated into the liposomes (Fig. 3 A). Thus, THD required both access to the β3 tail and the capacity to bind to it in order to activate αIIbβ in vitro.

Bottom Line: Here, we reconstructed physiological integrin activation in vitro and used cellular, biochemical, biophysical, and ultrastructural analyses to show that talin binding is sufficient to activate integrin alphaIIbbeta3.Furthermore, we synthesized nanodiscs, each bearing a single lipid-embedded integrin, and used them to show that talin activates unclustered integrins leading to molecular extension in the absence of force or other membrane proteins.Thus, we provide the first proof that talin binding is sufficient to activate and extend membrane-embedded integrin alphaIIbbeta3, thereby resolving numerous controversies and enabling molecular analysis of reconstructed integrin signaling.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, University of California, San Diego, La Jolla, CA 92093, USA.

ABSTRACT
Increased affinity of integrins for the extracellular matrix (activation) regulates cell adhesion and migration, extracellular matrix assembly, and mechanotransduction. Major uncertainties concern the sufficiency of talin for activation, whether conformational change without clustering leads to activation, and whether mechanical force is required for molecular extension. Here, we reconstructed physiological integrin activation in vitro and used cellular, biochemical, biophysical, and ultrastructural analyses to show that talin binding is sufficient to activate integrin alphaIIbbeta3. Furthermore, we synthesized nanodiscs, each bearing a single lipid-embedded integrin, and used them to show that talin activates unclustered integrins leading to molecular extension in the absence of force or other membrane proteins. Thus, we provide the first proof that talin binding is sufficient to activate and extend membrane-embedded integrin alphaIIbbeta3, thereby resolving numerous controversies and enabling molecular analysis of reconstructed integrin signaling.

Show MeSH
Related in: MedlinePlus