Electrostatics and Lateral Domains in Membranes

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9729538 McLaughlin The working hypothesis is that physical factors (e.g. electrostatics, surface pressure) can lead to the self assembly of specific lipids and proteins into lateral domains in cell membranes and that these domains are important for cell signaling. The specific objectives are to understand (1) the molecular mechanisms by which basic peptides form lateral domains in phospholipid vesicles; (ii) how proteins present at high concentrations in the cell membrane, e.g. caveolin, self assemble into domains; and (iii) how other cell signaling proteins, e.g. Src, and lipids, e.g. phosphatidylinositol 4,5,-bisphosphate (PIP2), are sequestered in these domains. Theory and experiment will be developed interactively to provide fundamental biophysical information about this complicated but important problem in signal transduction. Reconstituted systems comprising components of the calcium/phospholipid second messenger system (e.g. PIP2, PKC, Src, G proteins, phospholipase C) and peptides corresponding to the scaffolding region of caveolin (residues 82-101) will be studied using fluorescence, surface pressure, and other techniques. Theoretical calculations will be performed using realistic molecular models of peptides, proteins, and membranes. Four factors that may contribute to domain formation will be examined: self-aggregation of the proteins or peptides; penetration of the polar head group region by W, F, and Y residues; the entropic long rod effect described by Onsager; and electrostatics. Theories that account for each factor's relative contribution to domain formation will be formulated. Preliminary results and calculations show that even simple basic peptides such Lys5 and the scaffolding domain of caveolin can sequester PIP2 in lateral domains via nonspecific electrostatic interactions, which may provide a molecular explanation for the sequestration of this lipid in caveolae. This biophysics project has direct biological relevance because there is good evidence that biologic al membranes contain several different types of lateral domains that are important in signal transduction. For example, the plasma membrane lateral domains called caveolae contain high concentrations of caveolin and signaling molecules such as heterotrimeric G proteins, Src family kinases, PKC, and PIP2; the latter is an important lipid that is both the source of two second messengers and acts to anchor several proteins to membranes. Much of the PIP2 that is hydrolyzed in response to receptor stimulation is localized in the caveolae, suggesting they may be a primary site of agonist-stimulated PIP2 turnover. ***