Abstract
Modification of membrane receptor makeup is one of the most efficient ways to control input-output signals but is usually achieved by expressing DNA or RNA-encoded proteins or by using other genome-editing methods, which can be technically challenging and produce unwanted side effects. Here we develop and validate a nanodelivery approach to transfer in vitro synthesized, functional membrane receptors into the plasma membrane of living cells. Using β2-adrenergic receptor (β2AR), a prototypical G-protein coupled receptor, as an example, we demonstrated efficient incorporation of a full-length β2AR into a variety of mammalian cells, which imparts pharmacologic control over cellular signaling and affects cellular phenotype in an ex-vivo wound-healing model. Our approach for nanodelivery of functional membrane receptors expands the current toolkit for DNA and RNA-free manipulation of cellular function. We expect this approach to be readily applicable to the synthesis and nanodelivery of other types of GPCRs and membrane receptors, opening new doors for therapeutic development at the intersection between synthetic biology and nanomedicine.
Highlights
Membrane proteins play important roles in all aspects of cellular signaling and are the interface through which a cell responds to extracellular cues
To obtain pure and soluble G-protein–coupled receptors (GPCRs) while maintaining proper folding and transitions between conformational states, individual receptor molecules need to be in a native-like environment, which can be provided with the use nanolipoprotein particles (NLPs), known as nanodiscs[3,4]
Using the well-studied β2 adrenergic receptor (β2AR) as a prototypical GPCR17, we validated the utility of this platform by demonstrating that membrane-delivered β2AR responds to ligand binding and triggers cyclic AMP (cAMP) production to rescue the wound healing defects of β1/β2AR double knockout primary cells
Summary
The β2AR signaling has been directly linked to fibroblast migration during wound-healing processes[25,26]. We nanodelivered β2AR onto mouse embryonic fibroblasts (MEFs) from our dKO mice and assayed their wound healing properties using an in vitro scratch assay. Agonist stimulation in addition to β2AR nanodelivery led to a further increase in gap closure, reaching 66% and 95% at the 24 and 48 hr time points, respectively (Fig. 6b, p = 0.0002 after 24 hr and p = 0.008 after 48 hr, n = 3). These results validate the feasibility of GPCR nanodelivery and prove that this is a suitable strategy to selectively modulate cellular signaling and phenotype
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