Influence of membrane heterogeneity on G-protein coupled receptor signaling.

摘要

A highly crowded and heterogenous environment is the emerging picture of the plasma membrane that would explain the efficiency and fidelity of many signal transduction processes. We determined the effect of plasma membrane heterogeneity on the function and diffusion of components from two class A G protein coupled receptor (GPCR) pathways, the bradykinin type 2 (B2R)/Galphaq and the mu-opioid receptor (muOR)/Galpha i pathways, as well as the epidermal growth factor receptor. In the first part of this dissertation, we describe the effect of nanoscale plasma membrane heterogeneity, as exemplified by caveolae domains, on the B 2R and muOR pathways. We find that caveolae domains increase the signaling potential of the B2R/Galphaq pathway but not the muOR/Galpha i pathway. FRET studies suggest that components of the B2R/G alphaq pathway reside closer to caveolae domains compared to those of the muOR/Galphai pathway and this proximity mediated by molecular interactions of Galphaq with Cav1. Diffusion measurements of these membrane proteins by Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Recovery After Photobleaching (FRAP) show a discrepancy in the apparent diffusion coefficients obtained from the two methods which may be due to the geometry of caveolae domains. By FCS, an increased apparent diffusion was found for both B2R and Galphaq in the presence of caveolae which may be due to the confined, anomalous diffusion of the membrane proteins due to Cav1 interactions. These studies make the prediction that Galphaq-coupled receptors localize close to caveolae domains and their signaling properties impacted by the presence of caveolae domains, as compared to Galphai coupled receptors. In the second part of this dissertation, we describe studies on the effect of cell shape on the distribution of the EGFR and B2R. By numerical simulation with experimental validation with fluorescence imaging, we find that cell shape can influence the balance of reaction-diffusion processes of activated membrane receptors, causing a spatial gradient of receptors on the plasma membrane. Studies of membrane heterogeneity on the B2R/Galpha q and EGFR pathways may have implications on the mechanotransduction and mechanosensing of many cells including cardiac, vascular and smooth muscle cells.