RACK1 binds to a signal transfer region of G betagamma and inhibits phospholipase C beta2 activation.
Receptor for Activated C Kinase 1 (RACK1), a novel G betagamma-interacting protein, selectively inhibits the activation of a subclass of G betagamma effectors such as phospholipase C beta2 (PLCbeta2) and adenylyl cyclase II by direct binding to G betagamma (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 betagamma. We found that RACK1 interacts with several different G betagamma isoforms, including G beta1gamma1, Gbeta1gamma2, and Gbeta5gamma2, with similar affinities, suggesting that the conserved residues between G beta1 and G beta5 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 betagamma interaction as monitored by fluorescence spectroscopy. Interestingly, these peptides are located at one side of G beta1 and have little overlap with the G alpha subunit binding interface. Additional experiments indicate that the G betagamma contact residues for RACK1, in particular the positively charged amino acids within residues 44-54 of G beta1, are also involved in the interaction with PLCbeta2 and play a critical role in G betagamma-mediated PLCbeta2 activation. These data thus demonstrate that RACK1 can regulate the activity of a G betagamma effector by competing for its binding to the signal transfer region of G betagamma.
Chen
Songhai
S
Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-6600, USA. Songhai.Chen@Vanderbilt.edu
Lin
Fang
F
Hamm
Heidi E
HE
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000378:metabolism; D017124:Conserved Sequence; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D044387:GTP-Binding Protein beta Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044388:GTP-Binding Protein gamma Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D007527:Isoenzymes / Q000037:antagonists & inhibitors*; D058619:Maltose-Binding Proteins; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009479:Neuropeptides / Q000378:metabolism*; D054799:Phospholipase C beta; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D000075370:Receptors for Activated C Kinase; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D018411:Spodoptera / Q000166:cytology / Q000378:metabolism; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors*
D002352:Carrier Proteins; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D007527:Isoenzymes; D058619:Maltose-Binding Proteins; D009479:Neuropeptides; D020033:Protein Isoforms; C090294:RACK1 protein, mouse; D000075370:Receptors for Activated C Kinase; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D010738:Type C Phospholipases; D054799:Phospholipase C beta; C518034:Plcb1 protein, mouse; C518037:Plcb2 protein, mouse