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Determination of the Contact Energies between a Regulator of G Protein Signaling and G Protein Subunits and Phospholipase Cβ1

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Department of Physiology & Biophysics, State University of New York at Stony Brook, Stony Brook, New York 11794-8661, and Department of Physiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75235
Cite this: Biochemistry 2001, 40, 2, 414–421
Publication Date (Web):December 15, 2000
Copyright © 2001 American Chemical Society

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    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 Cβ (PLC-β) signaling cascade which may allow for rapid deactivation of intracellular Ca2+ release and activation of protein kinase C. Specifically, using fluorescence methods, we have determined the interaction energies between the RGS4, PLC-β, G-βγ, and both deactivated (GDP-bound) and activated (GTPγS-bound) Gαq. We find that RGS4 not only binds to activated Gαq, as predicted, but also to Gβγ and PLCβ1. 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 PLCβ1 or a lipid regulator, phosphatidylinositol-3,4,5 trisphosphate. Ternary complexes between Gαq, Gβγ and phospholipase Cβ1 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 Gαq 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.

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     This work was supported by NIH GM53132.

     State University of New York at Stony Brook.


     Present Address:  National Institute of Standards and Technology, Biotechnology Division, Gaithersburg, MD 20899.

     University of Texas Southwestern Medical Center.


     To whom correspondence should be addressed. E-mail:  [email protected]. Phone:  (631) 444-3071. Fax:  (631) 444-3432.

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