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

Abstract

Several regulators of G protein signaling (RGS) proteins contain a G protein gamma-subunit-like (GGL) domain, which, as we have shown, binds to Gbeta5 subunits. Here, we extend our original findings by describing another GGL-domain-containing RGS, human RGS6. When RGS6 is coexpressed with different Gbeta subunits, only RGS6 and Gbeta5 interact. The expression of mRNA for RGS6 and Gbeta5 in human tissues overlaps. Predictions of alpha-helical and coiled-coil character within GGL domains, coupled with measurements of Gbeta binding by GGL domain mutants, support the contention that Ggamma-like regions within RGS proteins interact with Gbeta5 subunits in a fashion comparable to conventional Gbeta/Ggamma pairings. Mutation of the highly conserved Phe-61 residue of Ggamma2 to tryptophan, the residue present in all GGL domains, increases the stability of the Gbeta5/Ggamma2 heterodimer, highlighting the importance of this residue to GGL/Gbeta5 association.

Figure 1

G_β binding specificity of RGS6 and RGS7. G_β subunits were cotranslated in reticulocyte lysates with (A) HA-tagged G_γ2 or (B) HA-tagged, truncated RGS6 (ΔDΔC, where ΔD indicates a DEP domain deletion and ΔC indicates a C-terminal deletion; amino acids 255–456) or truncated RGS7 (ΔDΔC; amino acids 202–395, SwissProt P49802) protein. Lysates were immunoprecipitated (IP) in low detergent with anti-HA mAb, and immunoprecipitated proteins and clarified supernatants were visualized separately by SDS/PAGE and autoradiography. (C) Expression vectors for full-length, HA-tagged RGS6 and individual, myc-tagged G_β subunits were transiently cotransfected into COS-7 cells. Cell lysates were immunoprecipitated with anti-HA mAb, and coimmunoprecipitated G_β subunits were detected by immunoblotting (Blot) with anti-myc-HRP or anti-HA-HRP conjugates.

Figure 2

Comparison of RGS6 and G_β5 expression patterns. Northern blots of 20 μg total RNA (A) or 2 μg poly(A+) RNA from various human tissues (B and C) were serially hybridized with a G_β5 cDNA probe (ref. 8), with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe as a control for RNA loading and quality, and with an RGS6 cDNA probe. kb, kilobase.

Figure 3

In vitro G_β binding specificity of GGL domain mutants. (A) Secondary structure predictions for the RGS6 GGL domain and sequence alignment between G_γ2, RGS6, RGS7, RGS9, and RGS11. Identical residues are in black boxes; conserved residues are shown in shaded boxes. For RGS6, probabilities of α-helical character (indexed to a maximum of 9; ref. 19) and coiled-coil interaction (indexed to a maximum of 1.0; ref. 33) are plotted above the primary sequence of the GGL domain (x axis). α-Helices within G_γ2 (ref. 32) are indicated by an α above the sequence. The position and nature of point mutations are denoted above or below the sequence line with arrows. Individual G_β subunits were cotranslated in reticulocyte lysates with wild-type or mutant RGS6 (B), RGS7 (C), and RGS11 proteins (D–F). HA-tagged RGS or G_γ proteins were immunoprecipitated in low detergent (except as noted in E) with anti-HA mAb, and associated G_β proteins were visualized by SDS/PAGE and autoradiography. (E) Immunoprecipitations (IP) of cotranslated G_β5 and wild-type (lane 1) or W274F mutant (lanes 2 and 3) RGS11ΔDΔC proteins were performed in high-detergent (lanes 1 and 3) or low-detergent (lane 2) conditions and visualized separately from clarified supernatants (Sup’nt) as above.

Figure 4

G_β binding specificity of G_γ1 and G_γ2 mutants. HA-tagged G_γ proteins (wild-type or mutated as indicated) were either cotranslated in vitro (A and C) or cotransfected into COS-7 cells (B) with individual G_β subunits, immunoprecipitated (IP) with anti-HA mAb in either low detergent (0.05% C12E10) or high detergent (RIPA), and visualized by SDS/PAGE and autoradiography (A and C) or immunoblotting (B) with indicated antisera (Blot). “Fusion” denotes chimeric protein composed of HA-tagged G_γ2 (F61W) subunit fused to the rat RGS12 RGS domain.

Figure 5

Specificity-determining residues at the interface of G_β1 and G_γ1 compared with equivalent regions of modeled G_β5 and the GGL domain of RGS11. Highlighted in blue are van der Waals surfaces of G_β1 contacting Phe-64 of G_γ1 (A) or similar contacts between G_β5 and Trp-274 of the RGS11 GGL domain (B). Residues colored red in the G_β1/G_γ1 structure differ from equivalent residues in the G_β5/GGL model. Except for the conserved tripeptide motif (NPF or NPW), thin red and green lines trace the Cα-backbone of G_β1/G_γ1 and G_β5/GGL, respectively. Just before the conserved tripeptide motif, the Cα-traces diverge because of the insertion of a single residue in G_γ1 relative to the GGL domain.