Abstract
The activation of α/β heterodimeric integrins is the result of highly coordinated rearrangements within both subunits. The molecular interactions between the two subunits, however, remain to be characterized. In this study, we use the integrin α(L)β(2) to investigate the functional role of the C-linker polypeptide that connects the C-terminal end of the inserted (I) domain with the β-propeller domain on the α subunit and is located at the interface with the βI domain of the β chain. We demonstrate that shortening of the C-linker by eight or more amino acids results in constitutively active α(L)β(2) in which the αI domain is no longer responsive to the regulation by the βI domain. Despite this intersubunit uncoupling, both I domains remain individually sensitive to intrasubunit conformational changes induced by allosteric modulators. Interestingly, the length and not the sequence of the C-linker appears to be critical for its functionality in α/β intersubunit communication. Using two monoclonal antibodies (R7.1 and CBR LFA-1/1) we further demonstrate that shortening of the C-linker results in the gradual loss of combinational epitopes that require both the αI and β-propeller domains for full reactivity. Taken together, our findings highlight the role of the C-linker as a spring-like element that allows relaxation of the αI domain in the resting state and controlled tension of the αI domain during activation, exerted by the β chain.
Highlights
Integrins are a large family of ␣/ heterodimeric cell surface receptors that mediate interactions with other cells or the extracellular matrix
In integrins that lack ␣I domains, the activated I domain directly interacts with the ligand through a metal ion-dependent adhesion site (MIDAS)
The ␣I domain remains in an inactive state, whereas the I domain together with the “leg” region of the integrin is stabilized in a pseudoliganded, active state, as shown by the induction of activationdependent epitopes and induction of the extended conformation with the open headpiece [6] (Fig. 1D)
Summary
Antibodies, Small Molecules, and Recombinant ICAM-1— The sources of the mouse anti-human ␣L mAbs TS2/4, TS1/22, CBR LFA-1/1, and the mouse anti-human 2 mAb CBR LFA1/2 have been described previously [9, 10]. The transfected 293T cells were detached, resuspended in assay buffer B, and labeled with 1–2 g/ml 2Ј,7Ј-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF AM) (Invitrogen) at 37 °C for 30 min in the dark. After this labeling step, the cells were washed once and resuspended in assay buffer B containing 1 mM CaCl2 and 1 mM MgCl2 (resting condition) or 1 mM CaCl2, 1 mM MgCl2, and activating mAbs (10 g/ml KIM127 and 10 g/ml CBR LFA-1/2) or 1 mM MnCl2 alone (activating conditions). The cells were incubated at room temperature for 30 min, washed in assay buffer C containing 1 mM CaCl2/1 mM MgCl2 and subjected to immunofluorescence flow cytometry. Soluble multimeric human myeloma IgG1 complexes with anti-human IgG were prepared as described above and exposed to the transfected cells
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