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Blood, 15 December 2008, Vol. 112, No. 13, pp. 5007-5015.
Prepublished online as a Blood First Edition Paper on September 22, 2008; DOI 10.1182/blood-2008-03-144543.
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IMMUNOBIOLOGY
Genetic perturbation of the putative cytoplasmic membrane-proximal salt bridge aberrantly activates  4 integrins
Yoichi Imai1,2,*,
Eun Jeong Park1,2,*,
Dan Peer1,2,
António Peixoto2,3,
Guiying Cheng2,3,
Ulrich H. von Andrian2,3,
Christopher V. Carman4, and
Motomu Shimaoka1,2
1 Department of Anesthesia, Harvard Medical School, Boston, MA;
2 Immune Disease Institute, Boston, MA;
3 Department of Pathology, Harvard Medical School, Boston, MA; and
4 Beth Israel Deaconess Medical Center and Department of Medicine, Harvard Medical School, Boston, MA
 4 integrins play a pivotal role in leukocyte migration and tissue-specific homing. The ability of integrins to bind ligand is dynamically regulated by activation-dependent conformational changes triggered in the cytoplasmic domain. An NMR solution structure defined a putative membrane-proximal salt bridge between the IIbβ3 integrin cytoplasmic tails, which restrains integrins in their low-affinity state. However, the physiological importance of this salt bridge in 4 integrin regulation remains to be elucidated. To address this question, we disrupted the salt bridge in murine germ line by mutating the conserved cytoplasmic arginine RGFFKR in 4 integrins. In lymphocytes from knock-in mice (4-R/AGFFKR), 4β1 and 4β7 integrins exhibited constitutively up-regulated ligand binding. However, transmigration of these cells across VCAM-1 and MAdCAM-1 substrates, or across endothelial monolayers, was reduced. Perturbed detachment of the tail appeared to cause the reduced cell migration of 4-R/AGFFKR lymphocytes. In vivo, 4-R/AGFFKR cells exhibited increased firm adhesion to Peyer patch venules but reduced homing to the gut. Our results demonstrate that the membrane-proximal salt bridge plays a critical role in supporting proper 4 integrin adhesive dynamics. Loss of this interaction destabilizes the nonadhesive conformation, and thereby perturbs the properly balanced cycles of adhesion and deadhesion required for efficient cell migration.
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