RACK1 binds to a signal transfer region of Gβγ and inhibits phospholipase C β2 activation

Receptor for Activated C Kinase 1 (RACK1), a novel G beta gamma-interacting protein, selectively inhibits the activation of a subclass of G beta gamma effectors such as phospholipase C beta 2 (PLC beta 2) and adenylyl cyclase II by direct binding to G beta gamma (Chen, S., Dell, E. J., Lin, F., Sai, J., and Hamm, H. E. (2004) J. Biol. Chem. 279, 17861 - 17868). Here we have mapped the RACK1 binding sites on G beta gamma. We found that RACK1 interacts with several different G beta gamma isoforms, including G beta(1)gamma(1), G beta(1)gamma(2), and G beta(5)gamma(2), with similar affinities, suggesting that the conserved residues between G beta(1) and G beta(5) may be involved in their binding to RACK1. We have confirmed this hypothesis and shown that several synthetic peptides corresponding to the conserved residues can inhibit the RACK1/G beta gamma interaction as monitored by fluorescence spectroscopy. Interestingly, these peptides are located at one side of G beta(1) and have little overlap with the G alpha subunit binding interface. Additional experiments indicate that the G beta gamma contact residues for RACK1, in particular the positively charged amino acids within residues 44 - 54 of G beta(1), are also involved in the interaction with PLC beta 2 and play a critical role in G beta gamma-mediated PLC beta 2 activation. These data thus demonstrate that RACK1 can regulate the activity of a G beta gamma effector by competing for its binding to the signal transfer region of G beta gamma.