Peptide Inhibitors of Regulator of G Protein Signaling 4 (RGS4): Rational and Combinatorial Approaches.

Regulators of G protein signaling (RGSs) accelerate GTP hydrolysis by Gα subunits and profoundly inhibit signaling by G protein-coupled receptors (GPCRs). The distinct expression patterns and pathophysiologic regulation of RGS proteins suggest that inhibitors may have therapeutic potential. The Mosberg and Neubig labs previously reported the design of a constrained peptide inhibitor of RGS4 (YJ34: Ac-Val-Lys-c[Cys-Thr-Gly-Ile-Cys]-Glu-NH2, S-S) based on the structure of the Gαi switch 1 region, however, its mechanism of action was not established. In Chapter 2, the mechanism and structure-activity relationship (SAR) of YJ34 were investigated. Some features were found to be necessary for YJ34 activity, including a N-terminal acetyl group, a C-terminal amide, a Gly at position 5, Cys at positions 3 and 7 and a disulfide bridge. In Chapter 3, a focused one-bead, one-compound (OBOC) peptide library was screened for RGS4 inhibitors. Each peptide in the library contained the above mentioned features that are thought to be required for the function of YJ34. The other 5 amino acids were randomized among the 19 natural amino acids (except Cys) to afford 2.5 million possible peptide sequences. From this screen, two novel inhibitors of RGS4 in a single turnover GTPase assay were found: 2ad (Ac-Gly-Thr-c[Cys-Phe-Gly-Thr-Cys]-Trp-NH2, S-S) and 2nd (Gly-Thr-c[Cys-Phe-Gly-Thr-Cys]-Trp-NH2, S-S). Chapter 4 describes a third hit, 9nd (Tyr-Trp-[Cys-Lys-Gly-Leu-Cys]-Lys-NH2, S-S), which blocks the RGS4-Gαo interaction in the flow cytometry protein interaction assay (FCPIA). Interestingly, this peptide binds RGS4 covalently in a dithiothreitol (DTT)-sensitive manner, suggesting that a disulfide bridge is formed between RGS4 and peptide 9nd. Although it has RGS4 selectivity (RGS4>RGS8>RGS16>RGS19), it appears that the mechanism of action is through non-selective cysteine modification. There are some interesting observations from the project. First, our focused library yielded binding peptides with both the anticipated and unanticipated mechanisms. Second, RGS4 appears to be more sensitive to cysteine modification than other RGS proteins. Third, in the library hits, there is a correlation between activity in the FCPIA assay and hydrophobicity.Ph.D.PharmacologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/60887/1/myersra_1.pd

Novel Peptide Ligands of RGS4 from a Focused One-Bead, One-Compound Library

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65605/1/j.1747-0285.2008.00687.x.pd

Differential modulation of mu-opioid receptor signaling to adenylyl cyclase by regulators of G protein signaling proteins 4 or 8 and 7 in permeabilised C6 cells is Gα subtype dependent

J. Neurochem . (2009) 112 , 1026–1034.Regulators of G protein signaling (RGS) proteins act as GTPase-accelerating protein to negatively modulate G protein signaling and are defined by a conserved RGS domain with considerable amino acid diversity. To determine the effects of specific, purified RGS proteins on mu-opioid signaling, C6 cells stably expressing a mu-opioid receptor were rendered permeable to proteins by treatment with digitonin. Mu-opioid inhibition of forskolin-stimulated adenylyl cyclase by [ d -Ala 2 , N -Me-Phe 4 ,Gly-ol]-enkephalin (DAMGO), a mu-specific opioid peptide, remained fully intact in permeabilized cells. Purified RGS domain of RGS4 added to permeabilized cells resulted in a twofold loss in DAMGO potency but had no effect in cells expressing RGS-insensitive G proteins. The inhibitory effect of DAMGO was reduced to the same extent by purified RGS4 and RGS8. In contrast, the RGS domain of RGS7 had no effect and inhibited the action of RGS8 as a result of weak physical association with Gαi2 and minimal GTPase-accelerating protein activity in C6 cell membranes. These data suggest that differences in conserved RGS domains of specific RGS proteins contribute to differential regulation of opioid signaling to adenylyl cyclase and that a permeabilized cell model is useful for studying the effects of specific RGS proteins on aspects of G protein-coupled receptor signaling.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78702/1/j.1471-4159.2009.06519.x.pd