Role of caveolae in G protein signaling

Worcester Polytechnic Institute, Worcester MA

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Abstract

? DESCRIPTION (provided by applicant): Muscle cells undergo both healthy and unhealthy adaptive changes in response to environmental cues. A notable example is the hypertrophic growth of cardiac myocytes following infarction. Cardiac hypertrophy is known to be regulated in some way through the G?q family of G proteins which mediates signals from hypertrophic agents such as angiotensin II. G?q activates phospholipase C-? (PLC?) enzymes to produce Ca2+ signals in cells. Long term adaptive G?q responses involve Rho/Rac protein. We have shown that the activation state of G?q is stabilized by caveolae. Caveolae are membrane invaginations that can mechanically accommodate increases in cell volume by flattening. We find that this flattening releases G?q and destabilizes its association with PLC?. Ca2+ signals are then attenuated. Reduced association with G?q allows PLC? to move into the cytosol to bind to C3PO. C3PO is required for efficient RNA silencing of gene by promoting the activity of the RNA-induced silencing complex (RISC). We have found that increasing PLC? reverses RN A silencing of specific genes through its interaction with C3PO, and produces large changes the cellular levels of microRNAs that have been implicated in heart disease. Here, we will test this novel connection between mechanical stress and gene regulation through caveolae / G?q/ PLC? /C3PO. We will first determine the organization, concentration and oligomerization of G?q, its associated angiotensin receptor and PLC? in caveolae domains using a series of fluorescence imaging and biochemical methods. We will then view their release from caveolae domains in real time as caveolae flatten upon the reversible application of osmotic pressure. We will determine the impact of osmotic stress on changes in the microRNA population due to PLC?-C3PO association in real time using RNA beacons to monitor microRNA and fluorescence complementation to monitor protein association. We will test the ability of these microRNAs to promote physiological changes in the cell. Since we find that active siRNA processing affects Ca2+ through G?q, we will determine if the underlying mechanism involves changes in PLC?-C3PO association that occur as the C3PO machinery is engaged.