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 ectodomain-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 beta2-TM with a TM of other integrin beta-subunits. With the exception of beta4 substitution, others presented beta2-integrins with modified phenotypes, either in their expression or ligand-binding properties. Subsequently, we adopted alphaLbeta2 for follow-on experiments because its conformation and affinity-state transitions have been well defined as compared with other members of the beta2-integrins. Replacement of beta2- with beta3-TM generated a chimaeric alphaLbeta2 of an intermediate affinity that adhered to ICAM-1 (intercellular adhesion molecule 1) but not to ICAM-3 constitutively. Replacing alphaL-TM with alphaIIb-TM, forming a natural alphaIIb/beta3-TM pair, reversed the phenotype of the chimaera to that of wild-type alphaLbeta2. Interestingly, the replacement of alphaLbeta2- with beta3-TM showed neither an extended conformation nor the separation of its cytoplasmic tails, which are well-reported hallmarks of an activated alphaLbeta2, 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.
Vararattanavech
Ardcharaporn
A
School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore.
Tang
Man-Li
ML
Li
Hoi-Yeung
HY
Wong
Chi-Hang
CH
Law
S K Alex
SK
Torres
Jaume
J
Tan
Suet-Mien
SM
D002460:Cell Line; D018076:DNA, Complementary; D019281:Dimerization; D005434:Flow Cytometry; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D047468:Immunoprecipitation; D016023:Integrins / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D010957:Plasmids; D011485:Protein Binding; D011487:Protein Conformation
D018076:DNA, Complementary; D016023:Integrins; D008565:Membrane Proteins