Formation of Higher Order Signal Transduction Complexes as Seen by Fluorescence Spectroscopy

Abstract

Fluorescence spectroscopy is the primary method to view the interactions between membrane-bound proteins in real time. We have been using fluorescence homo- and heterotransfer methods to follow the associations and oligomerization of membrane associated proteins. One system we have focused on is the G-protein-phospholipase Cβ signaling system. This pathway is responsible for transducing extracellular signals that bind to heptahelical surface receptors such as ions, hormones and neurotransmitters. Binding of these agents activate heterotrimeric G proteins which activates phospholipase Cβ (PLCβ), which in turn causes an increase in intracellular calcium and an activation of protein kinase C. Using a combination of fluorescence, biochemical and molecular biology techniques, we have measured the interaction energies of these proteins taking into account their dependence on the surface area of the lipid membrane. We find that activation of PLCβ is mediated by the pleckstrin homology domain, a structural module found in a wide variety of signal transduction proteins. Activation of the catalytic domain by the PH domain plays an analogous role in proteins that are not activated by G proteins. Using energy transfer, we also find that both PLCβ and G proteins have secondary binding sites for each other and for a protein that regulates G protein signaling (RGS). Our data suggest that in cells these proteins form stable signaling complexes capable of mediating high output, rapid and localized signals. We are currently investigating the role that lipid domain or rafts have in stabilizing these protein interactions.