Determination of the contact energies between a regulator of G protein signaling and G protein subunits and phospholipase C beta 1.

Cell signaling proteins may form functional complexes that are capable of rapid signal turnover. These contacts may be stabilized by either scaffolding proteins or multiple interactions between members of the complex. In this study, we have determined the affinities between a regulator of G protein signaling protein, RGS4, and three members of the G protein-phospholipase Cbeta (PLC-beta) signaling cascade which may allow for rapid deactivation of intracellular Ca(2+) release and activation of protein kinase C. Specifically, using fluorescence methods, we have determined the interaction energies between the RGS4, PLC-beta, G-betagamma, and both deactivated (GDP-bound) and activated (GTPgammaS-bound) Galpha(q). We find that RGS4 not only binds to activated Galpha(q), as predicted, but also to Gbetagamma and PLCbeta(1). These interactions occur through protein-protein contacts since the intrinsic membrane affinity of RGS4 was found to be very weak in the absence of the protein partner PLCbeta(1) or a lipid regulator, phosphatidylinositol-3,4,5 trisphosphate. Ternary complexes between Galpha(q), Gbetagamma and phospholipase Cbeta(1) will form, but only at relatively high protein concentrations. We propose that these interactions allow RGS4 to remain anchored to the signaling complex even in the quiescent state and allow rapid transfer to activated Galpha(q) to shut down the signal. Comparison of the relative affinities between these interacting proteins will ultimately allow us to determine whether certain complexes can form and where signals will be directed.

The pleckstrin homology domain of phospholipase C-beta(2) links the binding of gbetagamma to activation of the catalytic core.

Pleckstrin homology (PH) domains are membrane tethering devices found in many signal transducing proteins. These domains also couple to the betagamma subunits of GTP binding proteins (G proteins), but whether this association transmits allosteric information to the catalytic core is unclear. To address this question, we constructed protein chimeras in which the PH domain of phospholipase C-beta(2) (PLC-beta(2)), which is regulated by Gbetagamma, replaces the PH domain of PLC-delta(1) which binds to, but is not regulated by, Gbetagamma. We found that attachment of the PH domain of PLC-beta(2) onto PLC-delta(1) not only causes the membrane-binding properties of PLC-delta(1) to become similar to those of PLC-beta(2), but also results in a Gbetagamma-regulated enzyme. Thus, PH domains are more than simple tethering devices and mediate regulatory signals to the host protein.

Selective interaction of the C2 domains of phospholipase C-beta1 and -beta2 with activated Galphaq subunits: an alternative function for C2-signaling modules.

Phospholipase C (PLC)-beta1 and PLC-beta2 are regulated by the Gq family of heterotrimeric G proteins and contain C2 domains. These domains are Ca2+-binding modules that serve as membrane-attachment motifs in a number of signal transduction proteins. To determine the role that C2 domains play in PLC-beta1 and PLC-beta2 function, we measured the binding of the isolated C2 domains to membrane bilayers. We found, unexpectedly, that these modules do not bind to membranes but they associate strongly and specifically to activated [guanosine 5'-[gamma-thio]triphosphate (GTP[gammaS])-bound] Galphaq subunits. The C2 domain of PLC-beta1 effectively suppressed the activation of the intact isozyme by Galphaq(GTP[gammaS]), indicating that the C2-Galphaq interaction may be physiologically relevant. C2 affinity for Galphaq(GTP[gammaS]) was reduced when Galphaq was deactivated to the GDP-bound state. Binding to activated Galphai1 subunits or to Gbetagamma subunits was not detected. Also, Galphaq(GTP[gammaS]) failed to associate with the C2 domain of PLC-delta, an isozyme that is not activated by Galphaq. These results indicate that the C2 domains of PLC-beta1 and PLC-beta2 provide a surface to which Galphaq subunits can dock, leading to activation of the native protein.