Abstract
The contributions of integrins to cellular adhesive responses depend upon the dynamic regulation of their activation states, which in turn depends on engagement of binding partners by their cytoplasmic tails. It is well-established that not only talin but also kindlin family members are essential for integrin activation, and both must present for optimal integrin activation. Recent studies in humans have specifically emphasized the vital role of kindlin-3 in integrin activation in hematopoietic cells, where kindlin-3 deficiency leads to episodic bleeding, frequent infections and osteopetrosis, consequences of an inability to activate β1, β2 and β3 integrins. Here, we used human erythroleukemia HEL cells to characterize integrin-activating kindlin-3 function and the kindlin-3-β3 integrin interaction in greater detail. Non-stimulated HEL cells are suspension cells, and they do not adhere to fibrinogen readily. Thrombopoetin or PMA stimulation activated β3-containing integrins, the cells adhere and spread on fibrinogen. β3 integrin and kindlin-3 colocalized in focal adhesions and there was enhanced β3 integrin-kindlin-3 association as detected by coimmunoprecipitation. Kindlin-3 knockdown impaired agonist-stimulated adhesion and spreading on fibrinogen. We expressed various β3 CT as chimeric constructs containing the extracellular domain of PSGL-1 and evaluated their effects on β3-mediated cell spreading on fibrinogen. Expression of the wild-type β3 CT chimera impaired β3-mediated cell spreading, and introduction of S752P and Y759A mutations within the kindlin binding core abolished this inhibitory activity. Thus, our data emphasizes a role of direct kindlin-3 and β3 integrin cytoplasmic tail interaction in kindlin-3 induced integrin activation in hematopoietic cells.
Published Version
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call