INTEGRIN SIGNALING Phosphorylation Not Required

The ß subunit of integrins, which mediate adhesion to the extracellular matrix and stimulate intracellular signaling cascades as a result, contain NPxY motifs, which may be recognized by phosphotyrosine binding (PTB) domains. However, Chen et al. show that phosphorylation of the tyrosine residues is not essential for ß1-containing integrin function. Proteins with PTB domains exist in two flavors: those that require phosphorylation of the tyrosine in the NPxY motif to bind and those that can bind through a hydrophobic interaction with the aromatic ring of tyrosine or phenylalanine. Chen et al. created mice in which the tyrosine residues in the two NPxY motifs of ß1 were replaced with either alanine (ß1YA), which cannot bind either type of PTB domain, or phenylalanine (ß1YF), which is only deficient in interactions with PTB domains that require tyrosine phosphorylation. ß1YA mice died in utero before embryonic day 6.5, with the same phenotype as ß1 knockout mice, indicating that the NPxY motif was essential for integrin function. ß1YF mice were born with normal frequency and were fertile. Chimeric mice were created using green fluorescent protein (GFP)-expressing embryonic stem (ES) cells with the ß1YF or ß1YA mutants. The ß1YF cells contributed to all tissues, including hematopoetic cells, whereas the ß1YA cells contributed only to those tissues in which the presence of wild-type cells rescued the mutant cell function (brain, skin, and skeletal muscle). Analysis of ES cells showed that both ß1YF and ß1YA were present on the cell surface but that the ß1YA form was in an inactive conformation that did not bind extracellular matrix ligands. Conditional knockout was used to create platelets with ß1YA and, along with platelets from wild-type mice and the ß1YF mice, these were tested for both inside-out signaling (a signal that converts the integrin from an inactive to an active conformation competent to bind extracellular matrix proteins) and outside-in signaling (signals transmitted by the integrin after binding extracellular matrix proteins). Only the ß1YA platelets were deficient in binding to collagen after stimulation by adenosine diphosphate (ADP), whereas ß1YF platelets were activated by this inside-out signal and bound collagen. ß1YA platelets were also completely deficient in adhering to collagen under conditions of shear stress, whereas the ß1YF platelets showed only a modest defect in this assay for outside-in signaling. Thus, the NPxY motif is essential for ß1 integrin function, but phosphorylation appears to contribute only to the outside-in signaling and even then is not essential.

H. Chen, Z. Zou, K. L. Sarratt, D. Zhou, M. Zhang, E. Sebzda, D. A. Hammer, M. L. Kahn, In vivo ß1 integrin function requires phosphorylation-independent regulation by cytoplasmic tyrosines. Genes Dev. 20, 927-932 (2006). [Abstract] [Full Text]

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