Permissive transmembrane helix heterodimerization is required for the expression of a functional integrin

The current paradigm is that integrin is activated via inside-out signalling when its cytoplasmic tails and TMs (transmembrane helices) are separated by specific cytosolic protein(s). Perturbations of the helical interface between the alpha- and beta-TMs of an integrin, as a result of mutations, affect its function. Previous studies have shown the requirement for specific pairing between integrin subunits by ectodom ai n -exchange analyses. It remains unknown whether permissive alpha/beta-TM pairing of an integrin is also required for pairing specificity and the expression of a functionally regulated receptor. We performed scanning replacement of integrin beta 2-TM with a TM of other integrin beta-subunits. With the exception of beta 4 substitution, others presented beta 2-integrins with modified phenotypes, either in their expression or ligand-binding properties. Subsequently, we adopted alpha L beta 2 for follow-on experiments because its conformation and affinity-state transitions have been well defined as compared with other members of the beta 2-integrins. Replacement of beta 2- with beta 3-TM generated a chimaeric alpha L beta 2 of an intermediate affinity that adhered to ICAM-1 (intercellular adhesion molecule 1) but not to ICAM-3 constitutively. Replacing alpha L-TM with alpha IIb-TM, forming a natural alpha IIb/beta 3-TM pair, reversed the phenotype of the chimaera to that of wild-type alpha L beta 2. Interestingly, the replacement of alpha L beta 2- with beta 3-TM showed neither an extended conformation nor the separation of its cytoplasmic tails, which are well-reported hallmarks of an activated alpha L beta 2, as determined by reporter mAb (monoclonal antibody) KIM127 reactivity and FRET (fluorescence resonance energy transfer) measurements respectively. Collectively, our results suggest that TM pairing specificity is required for the expression of a functionally regulated integrin.