Identification of a Proline-Rich Sequence in the CD2 Cytoplasmic Domain Critical for Regulation of Integrin-Mediated Adhesion and Activation of Phosphoinositide 3-Kinase

Abstract

The CD2 molecule is one of several lymphocyte receptors that rapidly initiates signaling events regulating integrin-mediated cell adhesion. CD2 stimulation of resting human T cells results within minutes in an increase in ␤1-integrin-mediated adhesion to fibronectin. We have utilized the HL60 cell line to map critical residues within the CD2 cytoplasmic domain involved in CD2 regulation of integrin function. A panel of CD2 cytoplasmic domain mutants was constructed and analyzed for their ability to upregulate integrin-mediated adhesion to fibronectin. Mutations in the CD2 cytoplasmic domain implicated in CD2-mediated interleukin-2 production or CD2 avidity do not affect CD2 regulation of integrin activity. A proline-rich sequence, K-G-P-P-L-P (amino acids 299 to 305), is essential for CD2-mediated regulation of ␤1 integrin activity. CD2-induced increases in ␤1 integrin activity could be blocked by two phosphoinositide 3-kinase (PI 3-K) inhibitors or by overexpression of a dominant negative form of the p85 subunit of PI 3-K. In addition, CD2 cytoplasmic domain mutations that abrogate CD2-induced increases in integrin-mediated adhesion also ablate CD2-induced increases in PI 3-K enzymatic activity. Surprisingly, CD2 cytoplasmic domain mutations that inhibit CD2 regulation of adhesion do not affect the constitutive association of the p85 subunit of PI 3-K association with CD2. Mutation of the proline residues in the K-G-P-P-L-P motif to alanines prevented CD2-mediated activation of integrin function and PI 3-K activity but not mitogen-activated protein (MAP) kinase activity. Furthermore, the MEK inhibitor PD 098059 blocked CD2-mediated activation of MAP kinase but had no effect on CD2-induced adhesion. These studies identify a proline-rich sequence in CD2 critical for PI 3-K-dependent regulation of ␤1 integrin adhesion by CD2. In addition, these studies suggest that CD2-mediated activation of MAP kinase is not involved in CD2 regulation of integrin adhesion.T lymphocytes continuously migrate throughout the body, mediating immune responses to foreign antigens. The capacity for normal T cells to function, develop, and migrate depends upon adhesive contacts with other cells as well as with extracellular matrix (ECM) components (76). These adhesive contacts are regulated by and initiate a complex series of elegantly controlled molecular signaling events. Members of the integrin superfamily of adhesion molecules are involved in the cell-cell and cell-ECM interactions that are essential for T-lymphocyte function and migration. The integrins are a family of ␣␤ heterodimeric cell surface receptors with a vast tissue distribution (67). There are at least 20 different integrin heterodimers, with each heterodimer containing 1 of 8 ␤ chains and 1 of 14 ␣ chains. Members of the ␤1 integrin subfamily bind to components of the ECM, such as fibronectin (FN), as well as cell surface counter-receptors, such as VCAM-1 (34). The functional activity of integrins on circulating leukocytes is dynamically regulated by signaling events (21...

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