Identification and characterization of triosephosphate isomerase that specifically interacts with the integrin alphaIIb cytoplasmic domain.
Integrin alphaIIb/beta3 (IIb/IIIa), a platelet fibrinogen receptor, has been shown to play a critical role in thrombosis and hemostasis. However, the mechanisms by which ligands interact with the alphaIIb/beta3 receptor is not very clear at this time. The interaction between the ligand, the receptor and the transmission of extracellular signals may involve the cytoplasmic domains of these integrins. The objective of this investigation was to identify novel proteins that interact with the cytoplasmic tail of alphaIIb. Using alphaIIb cytoplasmic tail as the bait and a yeast two-hybrid system, we have identified three separate clones containing inserts that encoded the same protein with different truncated N-terminals. Sequence analysis showed that the inserts of the three clones encoded a previously identified enzyme: triose phosphate isomerase (TPI). In addition, we demonstrated that TPI failed to interact with the integrin alpha2 tail, beta3 tail and lamin, but showed a weak binding to the alphaV tail which shares the highest homology with alphaIIb tail among the integrin alpha family. Site-directed mutagenesis studies around the homology region indicated that the critical peptide sequence necessary for the interaction between TPI and alphaIIb tail is GFFKRNRPPLEE. Using RT-PCR, we have demonstrated the presence of TPI mRNA in platelets. In addition, experiments were also performed to demonstrate specific binding of TPI to alphaIIb using an ELISA and fusion protein. Taken together, these data suggest that TPI specifically interacts with alphaIIb and may play a critical role in alphaIIb/beta3-mediated platelet function.
Liu
Qiang-Yuan
QY
Du Pont Pharmaceuticals, Route 141 & Henry Road, Wilmington, DE 19880, USA.
Corjay
Martha
M
Feuerstein
Giora Z
GZ
Nambi
Ponnal
P
D000595:Amino Acid Sequence; D001483:Base Sequence; D001792:Blood Platelets / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism*; D017931:DNA Primers; D006801:Humans; D016297:Mutagenesis, Site-Directed; D040201:Platelet Membrane Glycoprotein IIb / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013379:Substrate Specificity; D014305:Triose-Phosphate Isomerase / Q000737:chemistry / Q000378:metabolism*
D017931:DNA Primers; D040201:Platelet Membrane Glycoprotein IIb; D014305:Triose-Phosphate Isomerase