Membrane-enabled dimerization of the intrinsically disordered cytoplasmic domain of ADAM10.

Intrinsically disordered protein regions are widely distributed in the cytoplasmic domains of many transmembrane receptors. The cytoplasmic domain of a disintegrin and metalloprotease (ADAM)10, a transmembrane metalloprotease mediating ectodomain shedding of diverse membrane proteins, was recently suggested to mediate the homodimerization of ADAM10. Here we show that a recombinant cytoplasmic domain of ADAM10 (A10Cp) is unstructured as judged by its susceptibility to limited trypsin digestion and its circular dichroism spectrum. In comparison, recombinant transmembrane-cytoplasmic domain of ADAM10 (A10TmCp) reconstituted in dodecylphosphocholine (DPC) micelles exhibits much greater resistance to trypsin digestion, with its cytoplasmic domain taking on a significant ordered structure. FRET analysis demonstrates that, although A10Cp remains monomeric, A10TmCp forms a tight homodimer (K(d) ∼ 7 nM) in DPC micelles. Phospholipid-conjugated A10Cp dose-dependently inhibits formation of A10TmCp homodimer, whereas A10Cp achieves only limited inhibition. Placing the transmembrane and cytoplasmic domains of ADAM10, but not the transmembrane domain alone, in their native orientation in the inner membrane of Escherichia coli produces specific and strong dimerization signal in the AraC-based transcriptional reporter assay. A chimeric construct containing the otherwise monomeric transmembrane domain of L-selectin and the cytoplasmic domain of ADAM10 produces a similar dimerization signal. Overall, these results demonstrate that a transmembrane domain imparts a stable structure to the adjacent and intrinsically disordered cytoplasmic domain of ADAM10 to form a homodimer in the membrane. This finding advances our understanding of the regulatory mechanism of ADAMs and has general implications for membrane-protein interactions in the process of transmembrane signaling.

A novel assay for assessing juxtamembrane and transmembrane domain interactions important for receptor heterodimerization.

Understanding the basis of specificity in receptor homodimerization versus heterodimerization is essential in determining the role receptor plays in signal transduction. Specificity in each of the interfaces formed during signal transduction involves cooperative interactions between receptor extracellular, transmembrane (TM), and cytoplasmic domains. While methods exist for studying receptor heterodimerization in cell membranes, they are limited to either TM domains expressed in an inverted orientation or capture only heterodimerization in a single assay. To address this limitation, we have developed an assay (DN-AraTM) that enables simultaneous measurement of homodimerization and heterodimerization of type I receptor domains in their native orientation, including both soluble and TM domains. Using integrin αIIb and RAGE (receptor for advanced glycation end products) as model type I receptor systems, we demonstrate both specificity and sensitivity of our approach, which will provide a novel tool to identify specific domain interactions that are important in regulating signal transduction.

High-yield membrane protein expression from E. coli using an engineered outer membrane protein F fusion.

Obtaining high yields of membrane proteins necessary to perform detailed structural study is difficult due to poor solubility and variability in yields from heterologous expression systems. To address this issue, an Escherichia coli-based membrane protein overexpression system utilizing an engineered bacterial outer membrane protein F (pOmpF) fusion has been developed. Full-length human receptor activity-modifying protein 1 (RAMP1) was expressed using pOmpF, solubilized in FC15 and purified to homogeneity. Using circular dichroism and fluorescence spectroscopy, purified full-length RAMP1 is composed of approximately 90% α-helix, and retains its solubility and structure in FC15 over a wide range of temperatures (20-60°C). Thus, our approach provides a useful, complementary approach to achieve high-yield, full-length membrane protein overexpression for biophysical studies.

Identifying key juxtamembrane interactions in cell membranes using AraC-based transcriptional reporter assay (AraTM).

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.