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.

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.

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.

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.

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.