Research Project
10246266
Integral membrane protein (IMP) function depends on the membrane environment, both through the activity of specific lipids as allosteric modulators and via bulk membrane properties like viscosity, rigidity, and order. Recent lipidomic analyses have established the compositional details of membrane complexity, and provided clear evidence that disparate cell types ? and even the same cell type in different individuals ? present unique, grossly different membrane environments. Our preliminary observations suggest that these distinct membrane environments influence the conformation and activity of G protein-coupled receptors (GPCRs), and likely modulate their response to targeted therapeutics. Moreover, our recent observations reveal that membrane phenotypes are remarkably susceptible to exogenous perturbations, suggesting that extrinsic factors like diet or lipid synthesis inhibitors could synergize with GPCR-targeted therapeutics. It is therefore critical to define the spectrum of regulatory mechanisms imparted on proteins by lipid bilayers, with this knowledge especially impactful for high value targets, such as GPCRs. Our long-term goal is to exploit tissue-specific differences in membrane environments to predict and tune the efficacy and specificity of therapeutics targeting GPCRs. The objective of this proposal is to leverage advances in lipidomics and GPCR reconstitution to bridge the membrane complexity of live cells with the controlled environments provided by model systems. By combining genetics, cell biology, lipidomics, GPCR reconstitution into proteoliposomes, and molecular simulations we aim to determine the mechanisms by which membrane composition determines ligand binding and signaling characteristics for the adenosine A2A receptor (A2AR). Our central hypothesis is that ligand off-rates and G protein coupling are affected by local membrane order, which is a function of both cholesterol concentration and overall lipid composition. Across model systems, our preliminary observations suggest that cholesterol concentration is a key modulator of GPCR activity, with effects on both ligand binding thermodynamics and signaling kinetics. We will decipher whether the effects of cholesterol are due to specific interactions with the receptor or effects on membrane physical properties by independently varying and measuring these properties in simulations, model membranes, and live cells. Lipidomic analysis of A2AR-relevant target cell types ? neurons, leukocytes, cardiomyocytes, and epithelial cells ? will inform molecular simulations of complex mixtures, and reconstitution of A2AR into membranes obtained from these same cells will determine the extent of signaling variation across cell types. Finally, we will test whether the effects of lipidomic perturbations ? including cholesterol depletion / loading, fatty acid feeding, and drugs that modulate fatty acid and cholesterol synthesis ? recapitulate A2AR functional dependence observed in model systems. This final goal will test a prototype therapy based on lipidomic perturbation, to locally and specifically enhance A2AR therapeutics..
