(page 2 of 2)

This work started a few years ago after Ginsberg's group discovered a salt bridge between the alpha and beta tails of the integrin. Salt bridges are favorable interactions formed by two oppositely-charged ionized groups within a protein, and Ginsberg knew that this salt bridge probably had a stabilizing effect on the interface between the two subunits—locking them in place, so to speak.

Ginsberg demonstrated that if he mutated the amino acid residues that formed this salt bridge disrupting these contacts, the integrins became activated.

This led him to speculate that under normal conditions, some sort of association between the inner tails of the alpha and beta subunits of the integrin held the protein in an inactive position.

"Whatever was activating [the integrins]," says Ginsberg, "was doing it by pulling the tails apart."

Around the same time, Calderwood arrived at TSRI as a postdoctoral fellow in the Ginsberg lab. Calderwood and Ginsberg began asking what cellular proteins might be disrupting the two tails of the integrin subunits, and they soon focused on talin.

Talin is a large intracellular protein more than 2,000 amino acids long and a major cytoskeleton protein on the inside of cells. Most of the talin protein binds to actin—the filamentous cellular protein that makes up the cytoskeleton and gives a cell its shape.

But Calderwood and Ginsberg discovered a small domain on the amino-terminus end of talin that binds to the beta-subunit tail of the integrin.

A Fruitful Collaboration

This week's report in Science shows that talin, indeed, is essential to integrin activation. The report is the result of a fruitful collaboration between Calderwood, Ginsberg and several other scientists at TSRI and The Burnham Institute.

TSRI Professor Sanford Shattil and his former TSRI postdoctoral fellow Seiji Tadakoro contributed their expertise with a technique called RNA interference.

RNA interference involves delivering small, 20- to 30-base pieces of double-stranded RNA into a cell. Once inside the cell, these short sequences anneal to complementary regions of cellular RNA and trigger an intracellular response that specifically destroys the target RNA. The technique allows scientists to selectively shut off normal cellular genes and permits them to study the impact of the absence of the corresponding gene products on cellular function.

The team used RNA interference to remove the talin from a type of cell called a megakaryocyte, a precursor of platelets, which TSRI postdoctoral fellow Koji Eto derived from embryonic stem cells. These megakaryocytes have the same machinery as platelets and respond to certain stimuli the same way that platelets do.

One of these stimuli is the chemical adenosine 5' diphosphate (ADP). When platelets are exposed to ADP, they become activated and the integrins on their surface switch from low to high affinity. The same is true of the megakaryocytes.

However, Calderwood and his colleagues showed that when the talin was removed from the megakaryocytes by RNA interference, the ADP no longer worked.

"It could not activate the integrins," says Calderwood, adding that they were able to rescue the activation by adding talin back into the cells from which it had been removed.

"This is a great example of a [scientific] collaboration," says Shattil. "It provided the critical evidence that talin was required for integrin activation."

The TSRI scientists also collaborated with Robert C. Liddington and Jose M. de Pereda of The Burnham Institute, with whom they had previously solved the crystal structure of talin bound to the cytoplasmic domain of integrin. This structure enabled Liddington and de Pereda to suggest places to mutate the talin and the beta subunit of the integrin to selectively disrupt the interaction between the two proteins.

"When [TSRI Research Assistant] Vera Tai introduced those mutations into full-length integrins, those integrins are inactive," says Calderwood. In the paper, the team also points out that overexpressing talin normally activates integrins. Overexpressing the mutant form of talin has no effect.

Further Questions

The importance of this discovery is enhanced by the fact that talin binds to almost all of the various tails of the beta subunits of integrins (eight of which are known).

The next step for Calderwood, Ginsberg, Shattil and their colleagues is to ask how the cell controls talin binding.

Figuring out these mechanisms is particularly interesting from a therapeutic point of view, since integrins are involved in such major killers as heart disease and cancer. Because talin binding is the final step in integrin activation, it might be a good target for keeping the integrins from becoming active.

"It's theoretically possible to perturb this interaction pharmaceutically," says Calderwood.


1 | 2 |



Surface representation of the integrin-activating domain of talin, colored by electrostatic potential, with the integrin beta3 tail (shown as sticks) bound. Provided by R.C. Liddington and J.M. de Pereda. Click to play movie