Mechanosensitive ectonucleotidase CD39 is monomeric on the surface of living cells

The ectonucleotidase CD39 plays a critical role in controlling tumor suppression, cardiovascular, and immune behavior by regulating extracellular nucleotide signaling in numerous tissues. The enzyme's extremely high activity, substrate specificity, and processivity have been shown to be modulated by plasma membrane properties, particularly bilayer elasticity, via rotational motions of its two transmembrane helices, suggesting it may serve as an intimate link between membrane mechanical dynamics and physiological function. An underlying question is: what is the basic unit that does this - does it require collective action of multiple CD39 molecules or does CD39's unusual structure, two transmembrane helices flanking an extracellular active site, contain the elements for sensing and transduction by a single CD39 molecule? This question has never been addressed for CD39 on the surface of live cells. Here we present FRET as well as crosslinking evidence that strongly suggests CD39 is monomeric on living cells. We also uncover an interesting difference in crosslinking behavior in extracted membranes versus live cells, raising the possibility that both the lipid bilayer and the cytoskeleton could potentially contribute to regulation. This work provides a foundation for further investigating how this unique molecule might translate dynamic mechanical information into a complex catalytic profile and ultimately to cancer, vascular, and other systems.