Structural features that affect β2 integrin expression and regulation of adhesion
Date of Issue2013
School of Biological Sciences
A hallmark for the Leukocyte Adhesion Deficiency 1 (LAD-1) syndrome is the defect in the β2 integrins due to mutations in the ITGB2 gene. To establish the correlation between LAD-1 genotype and phenotype, as well as better understand integrin function and structure, 19 LAD-1 missense mutants that recently identified were introduced into the pcDNA3 plasmid containing the ITGB2 cDNA. These plasmids were transfected into HEK293T cells together with that of either the αL, αM, αX subunits. These transfectants were subsequently examined for their ability in supporting the expression of the integrins and adhesion functions. Fourteen of these mutants do not support heterodimer expression of integrin LFA-1, Mac-1 and p150,95. Of the remaining 5, the integrins with the β2-D77N, β2-S453N and β2-P648L mutations have normal ligand binding activities. These “mutations” are therefore rare polymorphisms of the β2 subunit. In contrast, integrins with the β2-G150D mutation have no binding activities. LFA-1 with the β2-G716A mutation is constitutively active to bind ICAM-1 and requires only one activating agents to bind to ICAM-3. (It should be noted that wild-type LFA-1 requires one activating agent to bind ICAM-1 and two to bind ICAM-3.) Immunoprecipitation experiment with the reporter mAb KIM127 suggested that the LFA-1 bearing the β2-G716A is in the bent configuration. The epitope of the mAb H52 was mapped to the C-terminal half of the β2 hybrid domain. However, the epitope was abolished by the β2-L105P mutation, which is located in the N-terminal half. Based on this new information, it is concluded that the epitope is conformational. It is established that integrin activation is regulated by the divalent cations. However, Mg2+/EGTA (5 mM Mg2+ and 1.5 mM EGTA) had been shown to promote αLβ2 adhesion to ICAM-1 but not αMβ2 adhesion to denatured BSA. In order to determine the difference between αL and αM which contributes to Mg2+/EGTA sensitivity, a series of αL/αM chimeric integrins were constructed. It was shown that LFA-1 with αM calf-1 domain is not responsive to the Mg2+/EGTA activation. In the reverse experiment with Mac-1 bearing the αL calf-1 domain, the results were not clear cut due to the modified Mac-1 was constitutively active.
DRNTU::Science::Biological sciences::Molecular biology