Abstract
Leukocytes and platelets require stimulation for optimal beta3 integrin receptor function, whereas beta3 function is constitutive in many other cells. The molecular mechanisms that enhance integrin function in stimulated hematopoietic cells are poorly understood. Phosphorylation of the beta3 cytoplasmic tail is a recently described but prevalent phenomenon, with unknown effects on alphavbeta3 function. Here, we show that mutation of the beta3 cytoplasmic tail tyrosine 747 to phenylalanine (Y747F) prevents beta3 tyrosine phosphorylation in two cell lines. Whereas this mutation has no effect on alphavbeta3-mediated adhesion in a cell with constitutive beta3 function, it completely abolishes adhesion and clot retraction by a cell that requires stimulation for beta3 function. Ligand-induced conformational change as detected by LIBS-1 antibody occurs normally in Y747F mutant alphavbeta3. Thus, tyrosine 747 of beta3 is required for stimulation of alphavbeta3-mediated adhesion, probably due to its phosphorylation. Because the motif in beta3 required for tyrosine phosphorylation is shared by several integrin beta-chains, this may be a conserved mechanism for regulation of integrin-dependent adhesion.
Highlights
Integrin receptors are plasma membrane heterodimers expressed on all nucleated cells, critical for development and normal homeostasis because of their essential role in mediating cell adhesion
One of the better studied models for integrin activation involves the 3 integrins [3, 11,12,13,14]. 3 integrins ␣v3 and ␣IIb3 are expressed on many bone marrow-derived cells including platelets, neutrophils, macrophages, lymphocytes, and eosinophils. ␣IIb3 is the major integrin on the platelet surface, and, following platelet stimulation, ␣IIb3 is activated to bind fibrinogen, leading to platelet aggregation and eventually to clot retraction [15]
After recloning into pBSIIKS via PstI, the process was repeated using 5Ј-TCCTGCAATCCTGGCGCTAGCTGATAATGATCTGAG-3Ј and 5Ј-CTCAGATCATTATCAGCTAGCGCCAGGATTGCAGGA-3Ј to introduce a silent NheI site 3Ј of the 3 termination codon. This product was cloned into pIAP93 via PstI digests yielding pBLY100, containing the complete coding sequence of 3 with the silent introduction of NdeI and NheI restriction sites at amino acid position 714 of 3 and just 3Ј of the termination codon, respectively. pBLY100 was used as the template for the introduction of Tyr 3 Phe mutations by nested PCR using oligonucleotides bridging the NdeI and NheI sites, 5Ј-CTTGCCGCCCTGCTCATATGGAAACTCCTC-3Ј and 5Ј-AATCCTGGCGCTAGCTGATAATGATCTGAG-3Ј, respectively, with 5Ј-ACGTGGCCTCTTTAAACAGTGGGTTGTTGG-3Ј and 5Ј-CCAACAACCCACTGTTTAAAGAGGCCACGT-3Ј for Y747F and with 5Ј-TATCATTAAGTGCCCCGGAACGTGATATTG-3Ј and 5Ј-CAATATCACGTTCCGGGGCACTTAATGATA-3Ј for Y759F
Summary
Integrin receptors are plasma membrane heterodimers expressed on all nucleated cells, critical for development and normal homeostasis because of their essential role in mediating cell adhesion. Stimulation of leukocytes and platelets results in activation of integrin-mediated ligand binding, cell adhesion, and cell spreading [11, 12]. 3 phosphorylation was detected under baseline conditions in human endothelial cells, which have constitutively active 3 integrins, and was induced by Mn2ϩ in monocyte-derived macrophages and by Mn2ϩ or thrombin in human platelets (Fig. 1B).
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