Although the properties of the cell plasma membrane lipid bilayer are broadly understood to affect integral membrane proteins, details of these interactions are poorly understood. This is particularly the case for the large family of G protein-coupled receptors (GPCRs). Here, we examine the lipid dependence of the human serotonin 5-HT1A receptor, a GPCR that is central to neuronal function. We incorporate the protein in synthetic bilayers of controlled composition together with a fluorescent reporting system that detects GPCR-catalyzed activation of G protein to measure receptor-catalyzed oligonucleotide exchange. Our results show that increased membrane order induced by sterols and sphingomyelin increases receptor-catalyzed oligonucleotide exchange. Increasing membrane elastic curvature stress also increases this exchange. These results reveal the broad dependence that the 5-HT1A receptor has on plasma membrane properties, demonstrating that membrane lipid composition is a biochemical control parameter and highlighting the possibility that compositional changes related to aging, diet, or disease could impact cell signaling functions.

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We demonstrate successful incorporation of the G protein coupled receptor 5-HT1A into giant unilamellar vesicles using an agarose rehydration method. With direct observation using fluorescence techniques, we report preferential segregation of 5-HT1A into the cholesterol-poor liquid disordered phase of the membrane, contradicting previous reports of lipid raft segregation. Furthermore, altering the concentration of cholesterol and sphingomyelin in ternary mixtures does not alter 5-HT1A segregation into the liquid disordered phase.

While current research is centered on observing biophysical properties and phenomena in giant unilamellar vesicles (GUVs), little is known about fabrication parameters that control GUV formation. Using different lipids and rehydration buffers, we directly observe varying dynamics of hydrogel-assisted GUV formation via fluorescence microscopy. We observe the effects of buffer ionic strength, osmolarity, agarose density, and pH on the formation of GUVs using neutral and charged lipids. We find that increasing rehydration buffer ionic strength correlates with increased vesicle size and rate of GUV formation. Increasing buffer acidity increased the rate of GUV formation, while more basic environments slowed the rate. For buffers containing 500 mM sucrose, GUV formation was overall inhibited and only tubules formed. Observations of GUV formation dynamics elucidate parametric effects of charge, ionic strength, pH, and osmolarity, demonstrating the versatility of this biomimetic platform.

Giant Unilamellar Vesicles (GUVs) prepared from phospholipids are becoming popular membrane model systems for use in biophysical studies. The quality, size and yield of GUVs depend on the preparation method used to obtain them. In this study, hydrogels consisting of dextran polymers crosslinked by poly(ethylene glycol) (DexPEG) were used as hydrophilic frameworks for the preparation of vesicle suspensions under physiological ionic strength conditions. A comparative study was conducted using hydrogels with varied physicochemical properties to evaluate their performance for GUV production. The prepared GUVs were quantified by flow cytometry using the Coulter Principle to determine yield and size distribution. We find that hydrogels of lower mechanical strength, increased swellability and decreased lipid interaction favour GUV production, while their resulting size is determined by the surface roughness of the hydrogel film. Moreover, we embedded polymersomes into the crosslinked hydrogel network, creating a DexPEG – polymersomes hybrid film. The re-hydration of lipids on those hybrid substrates led to production of GUVs and the efficient encapsulation of polymersomes in the lumen of GUVs.

As cholesterol fraction increases, ligand-bound receptor occupies more vesicle surface area, demonstrating co-localization with the cholesterol-rich phase.

Pharmacological evidence has suggested the presence of 5-hydroxytryptamine (5-HT, serotonin), 5-HT1D receptors on endothelial cells but these receptors have never been identified unambiguously on this type of cells. We now report that human umbilical vein endothelial cells (HUVEC) express 5-HT1D receptors coupled to inhibition of cyclic AMP formation. 5-HT and the 5-HTlo receptor agonists 5-carboxamidotryptamine (5-CT) and sumatriptan were approximately equipotent at inhibiting forskolin-stimulated cyclic AMP accumulation in HUVEC (mean pECs0 7.6-8.2, maximal effect 30% inhibition). The 5-HTIA receptor agonist, 8-OH-DPAT was clearly less potent (pECs0 6.2) and less efficacious. The selective 5-HT~D receptor antagonist, GR127935 (1 riM) markedly inhibited the effect of 5-HT (apparent pKB 10.8). Reverse transeription-polymerase chain reaction analysis showed the mRNA for 5-HTID~ receptors to be expressed in HU-VEC. These results demonstrate the presence of functional 5-HTID receptors and the expression of 5-HTID~ receptor mRNA in HUVEC. They support the involvement of 5-HT~D~ receptors in endothelial-mediated responses to 5-HT.

We successfully incorporated the human serotonin receptor, G protein coupled receptor (GPCR), 5-HT1AR, in micron scale polymeric giant unilamellar protein-vesicles (pGUPs). By utilizing an agarose rehydration technique for protein incorporation, the GPCR is inserted in biased orientation with the C-terminus cytosolic and N-terminus extracellular as found in the cell plasma membrane. The GPCR is fully functional within the polymeric bilayer exhibiting responses to various ligands. The entire population of incorporated GPCRs displaying activity in pGUPs remains fully functional after lyophilization for 120 hours.