Cooperative role of the membrane-proximal and -distal residues of the integrin beta3 cytoplasmic domain in regulation of talin-mediated alpha IIb beta3 activation.

Integrin cytoplasmic tails regulate integrin activation that is required for high affinity binding with ligands. The interaction of the integrin beta subunit tail with a cytoplasmic protein, talin, largely contributes to integrin activation. Here we report the cooperative interaction of the beta3 membrane-proximal and -distal residues in regulation of talin-mediated alpha IIb beta3 activation. Because a chimeric integrin, alpha IIb beta3/beta1, in which the beta3 tail was replaced with the beta1 tail was constitutively active, we searched for the residues responsible for integrin activation among the residues that differed between the beta3 and beta1 tails. Single amino acid substitutions of Ile-719 and Glu-749 in the beta3 membrane-proximal and -distal regions, respectively, with the corresponding beta1 residues or alanine rendered alphaIIbbeta3 constitutively active. The I719M/E749S double mutant had the same ligand binding activity as alpha IIb beta3/beta1. These beta3 mutations also induced alphaVbeta3 activation. Conversely, substitution of Met-719 or Ser-749 in the beta1 tail with the corresponding beta3 tail residue (M719I or S749E) inhibited alpha IIb beta3/beta1 activation, and the M719I/S749E double mutant inhibited ligand binding to a level comparable with that of the wild-type alpha IIb beta3. Knock down of talin by short hairpin RNA inhibited the I719M- and E749S-induced alpha IIb beta3 activation. These results suggest that the beta3 membrane-proximal and -distal residues cooperatively regulate talin-mediated alpha IIb beta3 activation.

Novel RUNX1-PRDM16 fusion transcripts in a patient with acute myeloid leukemia showing t(1;21)(p36;q22).

The t(1;21)(p36;q22) is a recurrent chromosome abnormality associated with therapy-related acute myeloid leukemia (AML). Although involvement of RUNX1 has been detected by fluorescence in situ hybridization analysis, the partner gene has not been reported previously. We identified a novel RUNX1 partner gene, MDS1/EVI1-like-gene 1 (PRDM16), in an AML patient with t(1;21). Alternative splicing of the fusion gene generates five different fusion transcripts. In two of them, the PRDM16 reading frame is maintained in the fusion with RUNX1, suggesting that the RUNX1-PRDM16 gene fusion results in the production of a protein that is highly homologous to the RUNX1-MDS1/EVI1 chimeric protein. It is suggested that PRDM16 and MDS1/EVI1 share a common molecular mechanism for the leukemogenesis of RUNX1-associated leukemia. Characterization of the RUNX1-PRDM16 fusion protein and comparison with the RUNX1-MDS1/EVI1 protein will facilitate the understanding of the mechanisms underlying RUNX1-associated leukemia.

Existence of leukemic clones resistant to both imatinib mesylate and rituximab before drug therapies in a patient with Philadelphia chromosome-positive acute lymphocytic leukemia.

Imatinib mesylate and rituximab are molecularly targeted drugs against the BCR-ABL fusion protein and the CD20 antigen, respectively. Although these drugs have excellent anticancer effects, a major concern is drug resistance. We have investigated the case of a patient with Philadelphia chromosome-positive and CD20+ acute lymphocytic leukemia who acquired resistance to imatinib and rituximab. Imatinib therapy resulted in prompt cytogenetic remission, but resistance developed shortly thereafter. Sequencing of the kinase domain of the ABL gene and allele-specific polymerase chain reaction analysis revealed a point mutation resulting in an E255V substitution that was present before the therapy. After the patient received mild chemotherapy followed by rituximab administration, hematologic and cytogenetic remission was sustained for 5.5 months. The recurrent leukemic cells after the rituximab therapy showed not only the E255V mutation in the ABL gene but also loss of the CD20 antigen due to impaired transcription of the CD20 gene. The results of 2-color flow cytometry analysis showed that a small population of CD20(-) leukemic cells existed before the imatinib therapy. These results suggest that leukemic subclones carrying a genetic perturbation of the targeted molecules for both imatinib and rituximab were present before the therapies. The preexistence of primary resistant clones suggests the inability of combination therapy with 2 molecularly targeted drugs to overcome drug resistance in leukemia.

Suppression of integrin activation by the membrane-distal sequence of the integrin alphaIIb cytoplasmic tail.

Integrin cytoplasmic tails regulate integrin activation including an increase in integrin affinity for ligands. Although there is ample evidence that the membrane-proximal regions of the alpha and beta tails interact with each other to maintain integrins in a low-affinity state, little is known about the role of the membrane-distal region of the alpha tail in regulation of integrin activation. We report a critical sequence for regulation of integrin activation in the membrane-distal region of the alphaIIb tail. Alanine substitution of the RPP residues in the alphaIIb tail rendered alphaIIbbeta3 constitutively active in a metabolic energy-dependent manner. Although an alphaIIb/alpha6Abeta3 chimaeric integrin, in which the alphaIIb tail was replaced by the alpha6A tail, was in an energy-dependent active state to bind soluble ligands, introduction of the RPP sequence into the alpha6A tail inhibited binding of an activation-dependent antibody PAC1. In alphaIIb/alpha6Abeta3, deleting the TSDA sequence from the alpha6A tail or single amino acid substitutions of the TSDA residues inhibited alphaIIb/alpha6Abeta3 activation and replacing the membrane-distal region of the alphaIIb tail with TSDA rendered alphaIIbbeta3 active, suggesting a stimulatory role of TSDA in energy-dependent integrin activation. However, adding TSDA to the alphaIIb tail containing the RPP sequence of the membrane-distal region failed to activate alphaIIbbeta3. These results suggest that the RPP sequence after the GFFKR motif of the alphaIIb tail suppresses energy-dependent alphaIIbbeta3 activation. These findings provide a molecular basis for the regulation of energy-dependent integrin activation by alpha subunit tails.

Critical residues for ligand binding in blade 2 of the propeller domain of the integrin alphaIIb subunit.

Ligand binding to integrin alphaIIbbeta3 is a key event of thrombus formation. The propeller domain of the alphaIIb subunit has been implicated in ligand binding. Recently, the ligand binding site of the alphaV propeller was determined by crystal structure analysis. However, the structural basis of ligand recognition by the alphaIIb propeller remains to be determined. In this study, we conducted site-directed mutagenesis of all residues located in the loops extending above blades 2 and 4 of the alphaIIb propeller, which are spatially close to, but distinct from, the loops that contain the binding site for an RGD ligand in the crystal structure of the alphaV propeller. Replacement by alanine of Q111, H112 or N114 in the loop within the blade 2 (the W2:2-3 loop in the propeller model) abolished binding of a ligand-mimetic antibody and fibrinogen to alphaIIbbeta3 induced by different types of integrin activation including activation of alphaIIbbeta3 by beta3 cytoplasmic mutation. CHO cells stably expressing recombinant alphaIibbeta3 bearing Q111A, H112A or N114A mutation did not exhibit alphaIibbeta3-mediated adhesion to fibrinogen. According to the crystal structure of alphaVbeta3, the alphaV residue corresponding to alphaIIbN114 is exposed on the integrin surface and close to the RGD binding site. These results suggest that the Q111, H112 and N114 residues in the loop within blade 2 of the alphaIIb propeller are critical for ligand binding, possibly because of direct interaction with ligands or modulation of the RGD binding pocket.

Identification of critical residues for ligand binding in the integrin beta3 I-domain by site-directed mutagenesis.

To define the structural basis of ligand recognition by alphaIIb beta3, we conducted site-directed mutagenesis of residues located on the top surface of the beta3 I-domain that is homologous to the I-domain of several alpha subunits and contains a putative ligand binding site. Here we identify D158 and N215 in beta3 as novel residues critical for ligand binding. Alanine substitution of D158 or N215 abolished binding of a ligand-mimetic antibody and fibrinogen to alphaIIb beta3 induced by different types of integrin activation. CHO cells expressing recombinant alphaIIb beta3 bearing D158A or N215A mutation did not adhere to fibrinogen. These mutations had the same effect on ligand binding to another beta3 integrin, alphaVbeta3. Compared to the alphaI-domain structure, the betaB-betaC loop containing D158 in the beta3 I-domain is quite different in length and sequence. These results suggest that the structure for ligand recognition is different in the betaI- and alphaI-domains.