The long term objective is to understand how physical factors (electrostatics, diffusion, reduction of dimensionality) produce a flow of information through calcium/phosphoinositide second messenger systems. Phosphatidylinositol 4,5-bisphosphate, PIP2, is the source of three second messengers and is involved in a wide range of membrane-related phenomena, such as the activation of ion channels or enzymes, endocytosis, and exocytosis. How does PIP2 do so much? The working hypothesis is that unstructured clusters of basic/hydrophobic residues on MARCKS and other proteins act as reversible PIP2 buffers: they produce a local positive electrostatic potential that sequesters the polyvalent acidic lipid. Upon a local increase in the level of Ca2+, calcium/calmodulin (Ca/CaM) binds to the cluster, releasing the PIP2.
Six specific aims will explore this hypothesis. First, a simple coloring scheme and peptide binding measurements will be used to identify basic/hydrophobic clusters on medically important proteins. This approach has allowed the PI to identify regions of interest on receptor tyrosine kinases (RTK), G protein coupled receptors, and scaffolding proteins that are important in cancer and as drug targets. PCS, FRET, fluorescence stop flow, and other measurements will be used to determine peptide affinity for membranes, Ca/CaM, and PIP2. The biological consequences of these interactions will be investigated in collaborations with cell biologists. The recent identification of three such clusters on gravin illustrates the power of the combined biophysical/cell biology approach. Second, to use fluorescence measurements to determine the kinetics and life time of Ca/CaM binding to reconstituted peptides corresponding to the basic/hydrophobic juxtamembrane + transmembrane regions of intrinsic membrane proteins. Third, to determine how unstructured basic clusters provide sufficient specificity for PIP2 to target proteins such as the sperm factor PLCzeta to the plasma membrane. Fourth, to test the postulate that GAP-43/neuromodulin acts as a reversible PIP2 sink in the axonal growth cones of neurons. Fifth, to show that the basic/hydrophobic peptides diffuse more rapidly than lipids when bound to membranes that lack PIP2, preventing sequestration of other lipids such as phosphatidylserine and cholesterol. Sixth, to test a model for how basic/hydrophobic regions of proteins (e.g. MARCKS, gravin, RTKs) may nucleate the formation of cholesterol-enriched """"""""rafts"""""""". ? ? ?
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