RACK1 Binds to a Signal Transfer Region of Gβγ and Inhibits Phospholipase C β2 Activation

Songhai Chen, Fang Lin, Heidi E. Hamm
2005 Journal of Biological Chemistry  
Here we have mapped the RACK1 binding sites on G␤␥. We found that RACK1 interacts with several different G␤␥ isoforms, including G␤ 1 ␥ 1 , G␤ 1 ␥ 2 , and G␤ 5 ␥ 2 , with similar affinities, suggesting that the conserved residues between G␤ 1 and G␤ 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␤␥ interaction as monitored by fluorescence spectroscopy.
more » ... ingly, these peptides are located at one side of G␤ 1 and have little overlap with the G␣ subunit binding interface. Additional experiments indicate that the G␤␥ contact residues for RACK1, in particular the positively charged amino acids within residues 44 -54 of G␤ 1 , are also involved in the interaction with PLC␤2 and play a critical role in G␤␥-mediated PLC␤2 activation. These data thus demonstrate that RACK1 can regulate the activity of a G␤␥ effector by competing for its binding to the signal transfer region of G␤␥. G␤␥ subunits liberated from heterotrimeric G proteins following the activation of G protein-coupled receptors play critical roles in many cellular processes (1, 2). They regulate a variety of effector molecules ranging from enzymes such as phospholipase C ␤ (PLC␤) 3 and adenylyl cyclases to ion channels. The G␤ subunit has six isoforms, G␤ 1 , G␤ 2 , G␤ 3 , G␤ 4 , and two G␤ 5 splice variants, long and short (2). Although G␤ isoforms 1-4 share more than 85% amino acid identity, G␤ 5 is only about 50% identical to the other isoforms. G␤ subunits are WD40 repeat proteins whose structures are characterized by the presence of an N-terminal ␣ helix and a C-terminal toroidal structure made up of seven-bladed ␤ propellers with four ␤ strands in each blade (2). This unique toroidal ␤ propeller structure defines multiple surfaces including a top, bottom, and outer surface on G␤ for interactions with receptors, G␣ subunits, and effectors. Mutagenesis analyses and co-crystal studies of G␤␥ with interacting proteins such as phosducin and G protein-coupled receptor kinase 2 (GRK2) have identified multiple protein interaction sites on G␤␥ (1-4). However, residues critical for activation of all known G␤␥ effectors appear to cluster on a surface of G␤␥ that is covered by the G␣ subunit in the heterotrimer (5). This explains why dissociation of G␤␥ from G␣ is required for the activation of all known effectors. In addition to the G␣ contact region, other sites on G␤ also participate in effector binding and activation. These include the N-terminal coiled-coil domain and the outer surface of the ␤ propeller structure, which may define the signaling specificity of G␤␥ for different effectors (6 -8). G␤␥ has a long list of interacting proteins. In addition to being effectors, a number of interacting proteins can also regulate the activity of G␤␥. These include phosducin, phosducin-like proteins, and GRKs (9, 10). Binding of these proteins to G␤␥ results in inhibition of all known effectors as their binding sites on G␤␥ are shared by the G␣ subunits. Recently, we have identified a novel G␤␥-interacting protein, RACK1, that can also modulate G␤␥ functions (11-13). However, unlike other G␤␥ regulatory proteins, RACK1 only affects the activation of a subset of G␤␥ effectors, such as PLC␤2 and ACII, but has no effect on other G␤␥ functions (12). Given the fact that the G␣ binding interface contains molecular determinants for all known G␤␥ effectors, these findings suggest that RACK1 may bind to a domain of G␤␥ outside the G␣ contact region. Here we have used a combination of peptide and fluorescence spectroscopic approaches to define the RACK1 binding sites on G␤␥. Moreover, we have evaluated the contribution of the RACK1 contact residues to G␤␥-mediated PLC␤2 activation. The results of this study provide evidence that RACK1 binds to a unique region of G␤␥ critical for its signal transfer function.
doi:10.1074/jbc.m505422200 pmid:16051595 fatcat:ibioh25uhbhajfi7o4ml55l7ku