Fidelity of G protein b-subunit association by the G protein g-subunit-like domains of RGS6, RGS7, and RGS11

Several regulators of G protein signaling (RGS) proteins contain a G protein g-subunit-like (GGL) domain, which, as we have shown, binds to Gb5 subunits. Here, we extend our original findings by describing another GGLdomain-containing RGS, human RGS6. When RGS6 is coexpressed with different Gb subunits, only RGS6 and Gb5 interact. The expression of mRNA for RGS6 and Gb5 in human tissues overlaps. Predictions of a-helical and coiled-coil character within GGL domains, coupled with measurements of Gb binding by GGL domain mutants, support the contention that Gg-like regions within RGS proteins interact with Gb5 subunits in a fashion comparable to conventional GbyGg pairings. Mutation of the highly conserved Phe-61 residue of Gg2 to tryptophan, the residue present in all GGL domains, increases the stability of the Gb5yGg2 heterodimer, highlighting the importance of this residue to GGLyGb5 association. The ‘‘regulators of G protein signaling’’ (RGS) protein family consists of at least 20 mammalian gene products that act as GTPase activating proteins on the a-subunits of heterotrimeric, signal-transducing G proteins (1–3). By accelerating the inactivation of GTP-bound Ga subunits, RGS proteins serve as negative regulators of G protein-mediated signaling pathways. Additionally, two RGS proteins, p115–RhoGEF and PDZ– RhoGEF, can also act as effectors, coupling GTP-bound Ga12 andyor Ga13 to Rho activation (4, 5). Regions within certain RGS proteins that lie outside the RGS domain interact with other components of G protein signal transduction machinery. For example, the N-terminal PDZ domain of RGS12 associates in vitro with the C termini of G protein-coupled receptors (6), and the N-terminal domain of RGS4 mediates receptor-selective inhibition of G protein-mediated Ca21 signaling in pancreatic acinar cells (7). We recently identified a G protein g subunit-like (GGL) domain within four mammalian RGS family members and identified its role in mediating specific interaction of RGS7 and RGS11 with G protein b5-subunits (8). In a complementary study, a native RGS7yGb5 complex has been isolated from bovine retina (9). Our discovery of RGS7yGb5 and RGS11y Gb5 associations tripled the number of known interacting partners for this outlier Gb subunit, which previously had been known to interact only with Gg2 (10–13). Here, we describe the cloning, expression, and Gb binding selectivity of RGS6, another RGS protein possessing the GGL domain. Residues critical for Gb subunit binding specificity have been identified by mutagenesis of Gg2 and the GGL domains of RGS6, RGS7, and RGS11. MATERIALS AND METHODS Expression Constructs. cDNAs for RGS7, RGS11, and G protein subunits have been described (8). RGS6 was isolated by reverse transcription–PCR by using sense (59-GCG GCC GCA TGG CTC AAG GAT CCG GGG ATC AAA G-39) and antisense (59-TCT AGA CTG GGA TCA GGG CCT CTT AGC GAG-39) primers as described (14) and subcloned with an N-terminal hemagglutinin (HA)-epitope tag into pcDNA3.1 (Invitrogen). G protein b-subunit cDNAs were subcloned with an N-terminal myc-epitope tag into pcDNA3.1 (Invitrogen). Mutagenesis was performed as described (6). The Gg2 (F61W)yRGS fusion was created by adding a 17-aa linker (PRAAASVMDICRIRPWYP; derived from pcDNA3.1 polylinker) C-terminal to amino acid 70 of the Gg2 (F61W) point mutant, followed by the RGS domain of rat RGS12 (amino acids 716–838; GenBank acc. no. U92280). In Vitro TranscriptionyTranslation. Reactions were performed, and translation products were immunoprecipitated and analyzed by SDSyPAGE as described (8), except for variations in detergent conditions. ‘‘Low-detergent’’ immunoprecipitations were performed and washed in buffer D [50 mM NaCly10 mM MgCl2y50 mM Tris, pH 8.0y1 mM EDTAy10 mM 2-mercaptoethanoly20% (vol/vol) glycerolyComplete protease inhibitors; Roche Diagnostics] containing 0.05% C12E10, whereas ‘‘high-detergent’’ immunoprecipitations were performed in buffer D containing 0.1% Triton X-100 and washed in RIPA-500 buffer containing 500 mM NaCl, 1% Triton X-100, 0.5% deoxycholate, and 0.1% SDS. Transient Transfection and Immunoprecipitation. COS-7 cell lysates were prepared and immunoprecipitated by using RIPA-150 buffer as described (8); proteins were separated by SDSyPAGE, electroblotted onto nitrocellulose, and detected with primary antibodies conjugated to horseradish peroxidase (HRP) and chemiluminescence (Amersham Pharmacia). The HRP-conjugated anti-HA mAb 3F10 was obtained from Roche Diagnostics; HRP-conjugated anti-myc mAb was purchased from Invitrogen. Model Generation and Analysis. Published alignments (15) allowed for replacement of residues at the interface of Gb1 and Gg1 with the corresponding residues of Gb5 and the GGL domain of hRGS11 by using the program O (16). Where necessary, alternate allowed rotamers were chosen to limit steric overlap. Conjugate gradient energy minimization of the initial model was done with X-PLOR (17) by using standard energy functions and a coordinate restraint term applied to Ca-positions to compensate for the lack of experimental restraints. Cavity determination was performed by using the program VOIDOO (18). Structures were presented by using INSIGHT (Molecular Simulations, Waltham, MA). Secondary structure and coiled-coil predictions were performed with PREDICTPROTEIN (www.embl-heidelberg.de; ref. 19). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. PNAS is available online at www.pnas.org. Abbreviations: DD, DEP domain deletion; DC, C-terminus deletion; GGL, G protein g-subunit-like; HA, hemagglutinin; HRP, horseradish peroxidase. Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. AF107619 and AF107620). ‡To whom reprint requests should be addressed. e-mail: dsiderov@ med.unc.edu.