The β 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 β1-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 β1 were replaced with either alanine (β1YA), which cannot bind either type of PTB domain, or phenylalanine (β1YF), which is only deficient in interactions with PTB domains that require tyrosine phosphorylation. β1YA mice died in utero before embryonic day 6.5, with the same phenotype as β1 knockout mice, indicating that the NPxY motif was essential for integrin function. β1YF 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 β1YF or β1YA mutants. The β1YF cells contributed to all tissues, including hematopoetic cells, whereas the β1YA 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 β1YF and β1YA were present on the cell surface but that the β1YA form was in an inactive conformation that did not bind extracellular matrix ligands. Conditional knockout was used to create platelets with β1YA and, along with platelets from wild-type mice and the β1YF 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 β1YA platelets were deficient in binding to collagen after stimulation by adenosine diphosphate (ADP), whereas β1YF platelets were activated by this inside-out signal and bound collagen. β1YA platelets were also completely deficient in adhering to collagen under conditions of shear stress, whereas the β1YF platelets showed only a modest defect in this assay for outside-in signaling. Thus, the NPxY motif is essential for β1 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 β1 integrin function requires phosphorylation-independent regulation by cytoplasmic tyrosines. Genes Dev. 20 , 927-932 (2006). [Abstract] [Full Text]