Abstract
Dimerization is a key regulatory mechanism in activation of transmembrane (TM) receptors during signal transduction. This process involves a coordinated interplay between extracellular (EX), TM, and cytoplasmic (CYTO) regions to form a specific interface required for both ligand binding and intracellular signaling to occur. While several transcriptional activator-based methods exist for investigating TM interactions in bacterial membranes, expression of TM chimera in these methods occurs in a reverse orientation, and are limited to only TM domains for proper membrane trafficking and integration. We therefore developed a new, AraC-based transcriptional reporter assay (AraTM) that expresses EX-TM-CYTO chimera in their native orientation, thereby enabling membrane trafficking to occur independent of the TM chimera used as well as permitting analysis of EX-TM-CYTO interactions in biological membranes. Using integrin α(IIb) TM-CYTO as a model, we observe a large increase in homodimerization for the constitutively active TM mutant L980A relative to wild-type in the TM-CYTO construct (A963-E1008). We also characterized the receptor for advanced glycation endproducts (RAGE), whose homooligomeric state is critical in ligand recognition, and find the specific juxtamembrane region within the CYTO (A375-P394) mediates homodimerization, and is dominant over effects observed when the extracellular C2 domain is included. Furthermore, we find good agreement between our AraTM measurements in bacterial membranes and BRET measurements made on corresponding RAGE constructs expressed in transfected HEK293 cells. Overall, the AraTM assay provides a new approach to identify specific interactions between receptor EX-TM-CYTO domains in biological membranes that are important in regulation of signal transduction.
Highlights
Current bacterial methods to measure TM homodimerization are limited to only TM domains expressed in an inverted orientation
We characterized the receptor for advanced glycation endproducts (RAGE), whose homooligomeric state is critical in ligand recognition, and find the specific juxtamembrane region within the CYTO (A375-P394) mediates homodimerization, and is dominant over effects observed when the extracellular C2 domain is included
To ensure that EX-TM-CYTO fusions are properly integrated into the bacterial inner membrane, we generated chimeras that contain an N-terminal fusion to full-length maltose-binding protein (MBP), including its native signal peptide sequence
Summary
Current bacterial methods to measure TM homodimerization are limited to only TM domains expressed in an inverted orientation. Recent work has illustrated the importance of specific interfaces involving transmembrane (TM) regions of single- and multi-pass membrane proteins in stabilizing receptor signaling complexes, as well as cooperative interactions between TM, cytoplasmic (CYTO) and extracellular (EX) regions that regulate ligand-dependent signal transduction across cell membranes [2]. Two limitations of current LexA- and ToxR-based methods are that they can only be applied to TM domains, and they place TM domains in an inverted orientation in the bacterial inner membrane To address these limitations, we developed a new approach based on the Escherichia coli AraC transcription factor to investigate EX-TM-CYTO receptor domain homodimerization (AraTM). Our results demonstrate the sensitivity of the AraTM method to specific point mutations or domain deletions within EX-TMCYTO constructs, and illustrates the utility of AraTM as a complementary approach to other mammalian cell-based assays to investigate the key domains and motifs responsible for mediating receptor dimerization
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