Sequence analysis of human RGS7 and interaction with polycystin in yeast. (A) RGS7 shares a common domain structure with the mammalian RGS6, RGS9, RGS11, and EGL-10. Depicted in detail is the region unique to RGS7, containing two PEST sequences and a potential coiled-coil structure. The similarity and identity of the RGS7 GGL domain versus that of RGS6, 9, 11, and EGL-10 were 81% and 55%, 60% and 37%, 58% and 33%, and 59% and 40%, respectively. (B) Mutational analysis of RGS7–polycystin interactions in yeast. The presence of coiled-coil structures is depicted as a probability plot by using coils Version 2.1 from the Swiss Institute for Experimental Cancer Research, window 28. Interactions between RGS7 and polycystin were tested in the yeast two-hybrid system, with interaction (+) indicated by β-galactosidase production and leucine prototrophy. Interaction between RGS7 and polycystin is mediated by a region of RGS7 that contains a putative coiled-coil structure (shaded region). (C) Deletion of the C-terminal 70 aa of polycystin (Pkd1.3) abolishes binding to RGS7. Pkd1.1 represents the C-terminal 226 aa of polycystin. The minimal interacting domain, Pkd1.5, caused leucine prototrophy without β-galactosidase production, indicative of a weak interaction. The putative binding domain within polycystin contains a coiled-coil structure, L⁴²¹⁵–R⁴²⁴⁹. N.D., interaction not tested.

RGS7 associates with polycystin in vitro and in vivo. (A) In vitro binding of RGS7 to C-terminal polycystin. The amount of [³⁵S]methionine-labeled RGS7 that bound to the last 112 aa of polycystin fused to GST (GST–Pkd1.4) or to GST–FIT, an unrelated control peptide of equal length, was compared with 2.5% and 5% ³⁵S-labeled RGS7 input protein. The interaction between polycystin and [³⁵S]methionine-labeled RGS7 was blocked by increasing concentrations of unlabeled RGS7. (B and C) In vitro interaction of RGS7 and RGS7.8 with GST–Pkd1.4 and GST–Pkd1.6, respectively. The GST fusion proteins were immobilized to glutathione–Sepharose and incubated with a mixture of equal amounts of RGS7 and RGS7.8 (5% input proteins). RGS7 binds to GST–Pkd1.4, but not to GST–Pkd1.6; no binding to either GST fusion was detectable for RGS7.8 (lacking the PKD1-interacting domain) compared with 5% input proteins. (D) In vivo coimmunoprecipitation of F.RGS7 with C-terminal polycystin. The fragment of polycystin containing the last 112 aa (Pkd1.4) was fused to sIg.7, a construct containing the leader sequence of human CD5 followed by the CH₂ and CH₃ domain of human IgG1 and the transmembrane region of human CD7. The control plasmid, sIg.7-CD40 contains the cytoplasmic domain of CD40 following the end of the transmembrane region of CD7. For interaction between polycystin and RGS7, 293T cells were transiently transfected with F.RGS7 and either sIg.7-CD40 (control) or sIg.7-Pkd1.4. Lysates were immunoprecipitated with protein G and blotted with anti-Flag M2 mAb. To demonstrate that RGS7 binds the polycystin-PKD2 complex, 293T cells were transiently transfected with F.RGS7 and sIg.7-PKD2, an IgG-fusion protein containing the C-terminal cytoplasmic domain of PKD2 (27), and either CD16.7 or CD16.7-Pkd1.4 (27). Lysates were immunoprecipitated with protein G and blotted with anti-Flag M2 mAb. (E) The coiled-coil RGS protein axin binds GSK-3β, but does not interact with polycystin. sIg.7-CD40, sIg.7-Pkd1.4, or HA-tagged GSK-3β were transfected with myc-tagged axin and immunoprecipitated with protein G or a combination of anti-HA mAb and protein G. HA–GSK-3β, but neither sIg.7-CD40 nor sIg.7-Pkd1.4, precipitated axin. (F) EGL-10, a C. elegans homologue lacking a coiled-coil structure, does not interact with the C terminus of polycystin. However, a Flag-tagged chimera of EGL-10 containing the polycystin-binding domain of RGS7 (F.ERE), coprecipitated with sIg.7-Pkd1.4 but not with sIg.7-CD40.

Effect of polycystin on RGS7 protein levels. (A) RGS7 protein expression is augmented by the presence of polycystin or proteasome inhibitors. 293T cells were transfected with F.RGS7 and cotransfected with CD16.7-Pkd1.4 or controls (CD16.7 and CD16.7-Pkd1.6). After 24 hours, cells were incubated for 3 hours in medium alone (lanes 1–3), or with N-Acetyl-Leu-Leu-Norleucinal (ALLN) (40 μmol) or MG132 (40 μmol). Equal amounts of protein were separated by using SDS/PAGE, and Flag-tagged proteins were detected by Western blot analysis. (B) RGS7 protein half-life is increased in the presence of polycystin or a proteasome inhibitor. 293T cells were cotransfected with F.RGS7 and CD16.7.Pkd1.4 or CD16.7 (control). After 24 hours, cells were incubated 3 hours in medium alone or with MG132 (40 μmol). Cells were harvested at the time points indicated after the addition of cycloheximide (40 μg/ml), and whole cell lysates were immunoblotted with anti-Flag M2 to detect F.RGS7. Equal transfection efficiency and amount of loaded protein was monitored by a Flag-tagged green fluorescent protein construct (data shown for F.RGS7 + CD16.7). (C) Ubiquitination of RGS7 is increased in the presence of a proteasome inhibitor. HEK 293T cells were transfected with F.RGS7 and HA–ubiquitin as indicated. Flag/His-tagged RGS7 was purified by nickel-NTA chromatography from lysates of transfected cells, run on a SDS/PAGE gel, and transferred to nitrocellulose membrane. RGS7-ubiquitin conjugates were detected with anti-HA antibodies and F.RGS7 detected with the anti-Flag M2 antibody.

Colocalization of RGS7 with polycystin in 293T cells. Cells were transiently transfected with expression plasmids encoding F.RGS7.1 (a–d and g–i), F.RGS7.4 (e), F.RGS7.8 (f), CD16.7 (b), and CD16.7-Pkd1.4 (d and g–i). Shown are confocal images (Bio-Rad) after labeling of RGS7 with anti-Flag M2 and FITC-conjugated goat anti-mouse IgG (a–f). Nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI). Cells were transfected with RGS7.1 alone (a) or with the control plasmid CD16.7 (b) and were virtually negative other than the DAPI-labeled nuclei. Addition of ALLN at 40 μmol for 6 h resulted in significant augmentation of RGS7 protein levels and a punctate staining pattern (c). Cells cotransfected with F.RGS7.1 and CD16.7-Pkd1.4 showed a marked augmentation of F.RGS7.1 protein levels and a membrane-associated cage-like staining pattern (d). Deletion of the RGS domain (F.RGS7.4) did not elevate expression levels of RGS7 (e). Deletion of the N-terminal domain, including the PKD1-binding domain, resulted in increased and diffuse cytoplasmic expression of the RGS7 truncation (F.RGS7.8) (f). (g–i) Colocalization of RGS7 and polycystin in 293T cells transfected with F.RGS7.1 and CD16.7-Pkd1.4. CD16.7-Pkd1.4 was labeled with FITC-conjugated anti-CD16 mAb (g). F.RGS7 was labeled with anti-Flag M2 followed by rhodamine-conjugated anti-mouse IgG (h). An overlay shows the colocalization of RGS7 and polycystin expression (i). Identical results were obtained with full-length RGS7, indicating that the N-terminal domain does not affect the polycystin-mediated relocalization of RGS7 (data not shown).