Dimerization of the Transmembrane Domain of Integrin αIIb Subunit in Cell Membranes

Renhao Li,Roman Gorelik,Vikas Nanda,Peter B Law,James D Lear,William F Degrado,Joel S Bennett

doi:10.1074/jbc.m314168200

Renhao Li, Roman Gorelik + Show 5 more

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https://doi.org/10.1074/jbc.m314168200

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Abstract

Homo- and hetero-oligomeric interactions between the transmembrane (TM) helices of integrin alpha and beta subunits may play an important role in integrin activation and clustering. As a first step to understanding these interactions, we used the TOXCAT assay to measure oligomerization of the wild-type alpha(IIb) TM helix and single-site TM domain mutants. TOXCAT measures the oligomerization of a chimeric protein containing a TM helix in the Escherichia coli inner membrane via the transcriptional activation of the gene for chloramphenicol acetyltransferase. We found the amount of chloramphenicol acetyltransferase induced by the wild-type alpha(IIb) TM helix was approximately half that induced by the strongly dimerizing TM helix of glycophorin A, confirming that the alpha(IIb) TM domain oligomerizes in biological membranes. Mutating each of the alpha(IIb) TM domain residues to either Ala, Leu, Ile, or Val revealed that a GXXXG motif mediates oligomerization. Further, we found that the residue preceding each glycine contributed to the oligomerization interface, as did the residue at position i + 4 after the second Gly of GXXXG. Thus, the sequence XXVGXXGGXXXLXX is critical for oligomerization of alpha(IIb) TM helix. These data were used to generate an atomic model of the alpha(IIb) homodimer, revealing a family of structures with right-handed crossing angles of 40 degrees to 60 degrees, consistent with a 4.0-residue periodicity, and with an interface rotated by 50 degrees relative to glycophorin A. Thus, although the alpha(IIb) TM helix makes use of the GXXXG framework, neighboring residues have evolved to engineer its dimerization interface, enabling it to subserve specific and specialized functions.

Highlights

By interacting with macromolecular extracellular ligands, integrins mediate essential cell-cell and cell-matrix interactions (1)
In TOXCAT, a chimeric protein consisting of an amino-terminal ToxRЈ DNA binding domain, an maltose-binding protein (MBP) domain, and an intervening TM domain of interest is expressed in the inner membrane of E. coli
The TOXCAT system is an optimized method for overcoming the technical difficulty of studying the homomeric associations of TM helices in biological membranes (14, 23) and has been used to investigate the homomeric interactions of the glycophorin A (GpA) TM domain (14), other naturally occurring TM helices such as ErbB4 (15) and VacA (24), and randomized recombinant peptides (12)

Summary

Introduction

By interacting with macromolecular extracellular ligands, integrins mediate essential cell-cell and cell-matrix interactions (1). There is a correlation between their activation state and the relative positions of the cytoplasmic (CYTO)[1] domains of their ␣ and ␤ subunits (2–5). These integrins are inactive when their CYTO domains are in proximity and are active when the domains are far apart (6). On the basis of in vacuo molecular modeling, Gottschalk et al (8) proposed a model in which the ␣ and ␤ subunit TM domains interact extensively in both the active and inactive states, with the interaction pattern changing during activation. Other models based on electron microscopy and protein engineering propose that TM domains interact only in the inactive state and move far apart upon activation (4, 6)

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