Integrin beta 3 cytoplasmic tail is necessary and sufficient for regulation of alpha 5 beta 1 phagocytosis by alpha v beta 3 and integrin-associated protein.

Abstract

Abstract. Using a K562 cell transfection model, we have previously described a novel relationship between the integrins 11v133 and ot5131. %133 ligation was able to inhibit ot5131-mediated phagocytosis without effect on o~5131-mediated adhesion. The 11v133-dependent inhibition apparently required a signal transduction cascade as it was reversed by inhibitors of serine/threonine kinases. Now, we have studied the mechanisms of signal transduction in this system and have found that the 133 cytoplasmic tail is both necessary and sufficient for initiation of the signal leading to inhibition of a5131 phagocytosis. Ligation of integrin-associated protein (IAP), which has been implicated in 11v133 signal transduction, mimics the effects of 11v133 ligation only when the 133 integrin with an intact cytoplasmic tail is present. Although fibronectin-mediated phagocytosis requires the high affinity conformation of 115131, ligation of Otv133/IAP does not prevent acquisition of this high affinity state. We conclude that CXv133/IAP ligation initates a signal transduction cascade, dependent upon the 133 cytoplasmic tail, which inhibits the phagocytic function of eL5131 at a step subsequent to modulation of integrin affinity. INTEGRIN functions are modulated in many cells. This is best understood in the regulation of leukocyte adhesion and in platelet activation (9,16,24,37). In these cell types, integrins have at least two conformational states. One, exhibiting a low affinity for ligand, is expressed by unactivated cells. The second state, which has a much higher affinity for ligand, is expressed by cells after activation. The signals which initiate the transition from low to high affinity states include thrombin and ADP stimulation in platelets (17,19,37), and a variety of serpentine receptor ligands in leukocytes, including the bacterial peptide f-Met-Leu-Phe, the complement activation fragment C5a, the product of arachidonate metabolism LTB4, and chemokines such as 16,20,27,36).In leukocytes, integrin function also can be regulated by ligation of another integrin. Recently, we have developed a model in which to study the mechanism of 133 "crosstalk" with other integrins. In K562 cells transfected with 11v[33, ligation of this integrin inhibits the phagocytic function of the integrin fibronectin receptor, 115131, which is endogenously expressed in K562 (2). This effect requires signal transduction, since it can be reversed by the serine/threonine kinase inhibitor H7 and the more specific protein kinase C inhibitor Calphostin C. Surprisingly, 11v[33 ligation affects phagocytosis without affecting the adhesive functions of 115131, suggesting that the signal transduction discretely inhibits high affinity 115131 function (phagocytosis) without altering the low affinity receptor function (adhesion) (18).We now have used this model to dissect the roles of the integrin 11 and 13 chains and IAP in initiation of the signal transduction cascade. We show that tXlib133 is as effective as 11v133 for initiation of this signal. Deletion of t...

Distinct Ligand-binding Modes for Integrin αvβ3-Mediated Adhesion to Fibronectinversus Vitronectin

“…In the experiments described, we believe that the ␣ 5 ␤ 1 integrin is inactive and does not contribute for several reasons: (a) specific antibodies to ␣ v ␤ 3 reduced the adhesion of the K␣ v ␤ 3 (and ␤ 3 mutant) cells to fibronectin to the same level as BSA; (b) antibodies to ␤ 1 integrin had no effect even on the PMA-stimulated cells; and (c) although ␣ 5 ␤ 1 on these cells can be passively activated by either Mn 2ϩ or activating monoclonal antibodies (28), there are no other known mechanisms of activation, and these elements are not used for this report. Cross-talk between ␣ v ␤ 3 and ␣ 5 ␤ 1 has been described in these cells, but the assays of cell adhesion, cell migration, and ␣ 5 ␤ 1 -mediated phagocytosis described require the addition of Mn 2ϩ (31,32). In those studies the presence of the ␤ 3 cytoplasmic domain suppressed (not activated) ␣ 5 ␤ 1 function.…”

Competition for Talin Results in Trans-dominant Inhibition of Integrin Activation

“…Integrin activation is an important component of cell adhesion, migration, phagocytosis, and integrin-mediated signaling; therefore, the titration of talin may contribute to the transdominant inhibition of these processes by blocking integrin activation. However, because the exogenous activation of integrins does not reverse this inhibition fully (11)(12)(13)56), either sequential activation and inactivation or additional activationindependent processes also are involved in these processes. Talin binds to most integrin ␤ tails and connects integrins to the actin cytoskeleton.…”

Ligand-dependent Activation of Integrin αvβ3

“…Cells and Materials-K562 cells were stably transfected with cDNA encoding wild type (K␣ v ␤ 3 ), Y747F,Y759F (K␣ v ␤ 3 Y747F,Y759F) ␤ 3 or wild type (K␣ V ␤ 5 ) ␤ 5 together with ␣ V and maintained as previously described (7,8). The anti-␤ 3 monoclonal antibodies 7G2 and LIBS-1 were gifts of Eric J.…”